Bioconjugate Strategies for Antisense Therapeutic Delivery to ...

Bioconjugate Strategies for Antisense Therapeutic Delivery to Glioblastoma Stem Cells

by

Amy Elizabeth Arnold

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Department of Chemistry University of Toronto

© Copyright by Amy Elizabeth Arnold 2020

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Bioconjugate Strategies for Antisense Therapeutic Delivery to

Glioblastoma Stem Cells

Amy Elizabeth Arnold

Doctor of Philosophy

Department of Chemistry University of Toronto

2020

Abstract

Antisense therapeutics, including antisense oligonucleotides (AONs) and small interfering

ribonucleic acids (siRNAs), are powerful tools for regulating genes, making them a promising

therapy for diseases such as cancer where oncogenic genes are over-expressed. The delivery of

antisense therapeutics to target cells presents a significant challenge due to the many barriers a

nucleic acid must face in order to reach the cytoplasm where it exerts its effects. In this thesis, I

explored multiple strategies for delivery of AONs and siRNAs, focusing on targeting the desired

cell population, inducing endocytosis, and facilitating endosomal escape. This was done within

the context of glioblastoma (GBM), and specifically the glioblastoma stem cells (GSCs), an

aggressive subpopulation of GBM cells that are involved in resistance, migration, and

recurrence. Antisense oligonucleotides against a relevant GBM gene were conjugated to an

antibody engineered to target CD44, a cell surface receptor which is highly expressed on GSCs.

Using this system, we demonstrated functional targeting, endocytosis, and gene knockdown in

the GSCs, leading to a morphological change in the cells. This represented the first time an

antibody-oligonucleotide conjugate was used to target the GSC population. We were challenged

with a lack of endosomal escape when using the antibody delivery platform, so we next looked at

using a protein with a native endosomal escape mechanism to facilitate oligonucleotide delivery.

For the second strategy, I conjugated attenuated diphtheria toxin (aDT), a protein which escapes

the endolysosomal pathway, to siRNAs against relevant gene targets involved in GSC

proliferation and invasion. Using this aDT-siRNA conjugate, we could downregulate genes of

interest in the glioblastoma stem cells, leading to significant changes in cell viability and the

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invasive capacity of these cells. This is the first diphtheria toxin-based siRNA delivery vehicle

and represents a platform technology for siRNA- and AON-based therapies.

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Acknowledgments I would first and foremost like to thank my supervisor, Prof. Molly Shoichet, for five years’

worth of mentorship, support, and advice, and for all the doors that have been opened because of

my time in the Shoichet lab. I learned more than I ever expected, not just about science, but also

about how to communicate my science in a clear way, and how to effectively collaborate with

(many) colleagues. Thank you for being a true champion for women in science and for all the

time you devote to helping your students succeed – I am truly grateful.

Thank you to all of my collaborators, especially to Prof. Masad Damha, Prof. Roman Melnyk,

and Dr. Greg Beilhartz. Prof. Damha – you were instrumental in helping me succeed during my

PhD; although it finished at a different place than it started, you were there with advice at every

turn. Prof. Melnyk and Greg – you brought fresh perspectives and a new direction to my work; I

really appreciate it.

I would like to thank my committee who have continually encouraged me to learn new skills and

think creatively about my work. Prof. Jason Moffat – thank you for asking the tough questions

and inspiring me to see problems from a biologist’s perspective. Prof. Mitch Winnik – thanks for

encouraging me to see the exciting aspects of my work whenever I encountered roadblocks.

Thank you to all of the members of the Shoichet lab that have helped me along the way,

including my earliest mentors Jenn and Chris, as well as Vianney, Marian, Ahil, Ana, and Alex –

you are all fantastic mentors and great friends. Sonja, Laura B, Erics, and Carter – you guys

made up the best lab family ever. Above all, I am grateful to the other member of team

glioblastoma, Laura S. Thank you helping me through late nights and tough times and also

celebrating the good stuff from birthdays and weddings to afternoon trips to Jimmy’s.

I would like to acknowledge all of my family and friends whose endless encouragement and

support was invaluable throughout my PhD. Thank you especially to all of my aunts and uncles

who offered a home-away-from-home while I studied in Toronto. Mom and Dad – even though I

didn’t become a “real” doctor, you guys have always been there for me, and I truly believe that

every success I have had is because of you.

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Finally, and most importantly, I would like to thank my husband, Jarret. From near or from far,

you never stopped believing in me. This thesis is dedicated to you.

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Table of Contents Acknowledgments .......................................................................................................................... iv

Table of Contents ........................................................................................................................... vi

List of Tables ................................................................................................................................. xi

List of Figures ............................................................................................................................... xii

Introduction .................................................................................................................................1

1.1 Rationale ..............................................................................................................................1

1.1.1 Hypothesis and objectives ........................................................................................2

1.2 Glioblastoma stem cells .......................................................................................................3

1.2.1 Glioblastoma ............................................................................................................3

1.2.2 Cancer stem cells .....................................................................................................4

1.2.3 Glioblastoma stem cells ...........................................................................................6

1.2.4 Therapeutic targets associated with GSC proliferation and invasion ......................8

1.3 Antisense therapeutics .......................................................................................................10

1.3.1 Antisense oligonucleotide mechanism ...................................................................11

1.3.2 Small interfering ribonucleic acid mechanism ......................................................12

1.3.3 Stability in the extracellular environment ..............................................................13

1.4 Nanoparticles as delivery vehicles .....................................................................................15

1.4.1 Polymeric formulations ..........................................................................................15

1.4.2 Lipid-based formulations .......................................................................................17

1.4.3 Virus-inspired formulations: cell penetrating and membranelytic peptides ..........19

1.5 Protein conjugates as delivery vehicles .............................................................................20

1.5.1 Antibodies for drug and oligonucleotide delivery .................................................21

1.5.2 Engineered toxins for biomolecule delivery ..........................................................27

Antibody-antisense oligonucleotide conjugate downregulates a key gene in glioblastoma stem cells ...................................................................................................................................32

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2.1 Abstract ..............................................................................................................................32

2.2 Introduction ........................................................................................................................33

2.3 Results ................................................................................................................................35

2.3.1 Antisense oligonucleotide activity .........................................................................35

2.3.2 Co-expression in GSCs of DRR and antigens for CD44, EphA2, and EGFR antibodies ...............................................................................................................37

2.3.3 Internalization of CD44 and EphA2 mAbs ............................................................37

2.3.4 Synthesis of mAb-dsDRR and mAb-dsScrambled conjugates ..............................39

2.3.5 DRR knockdown and cellular uptake of mAb-dsDRR conjugates ........................40

2.3.6 Cellular morphology following DRR knockdown .................................................44

2.4 Discussion and conclusions ...............................................................................................46

2.5 Materials and methods .......................................................................................................47

2.5.1 Cell lines ................................................................................................................47

2.5.2 Antibodies ..............................................................................................................48

2.5.3 AON synthesis .......................................................................................................48

2.5.4 AON duplex formation ..........................................................................................49

2.5.5 Preparation of mAb-dsDRR and mAb-dsScrambled conjugates ...........................49

2.5.6 DRR knockdown assays - lipofectamine2000 transfection protocol .....................49

2.5.7 DRR knockdown assays - treatment with mAb conjugate ....................................49

2.5.8 mAb internalization assay ......................................................................................50

2.5.9 Immunocytochemistry (CD44, EphA2, and DRR) ................................................50

2.5.10 Cellular uptake .......................................................................................................51

2.5.11 Lysosomal accumulation .......................................................................................51

2.5.12 Cellular morphology following DRR knockdown .................................................51

2.6 Acknowledgments ..............................................................................................................51

Attenuated diphtheria toxin mediates siRNA delivery .............................................................52

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3.1 Abstract ..............................................................................................................................52

3.2 Introduction ........................................................................................................................53

3.3 Results ................................................................................................................................55

3.3.1 Conjugation of siRNA to aDT ...............................................................................55

3.3.2 Glioblastoma stem cells (GSCs) express heparin-binding epidermal growth factor (HBEGF) .....................................................................................................57

3.3.3 aDT-siRNA conjugate downregulates ITGB1 and reduces cellular invasion .......58

3.3.4 aDT-siRNA conjugate downregulates eIF-3b and reduces cell viability ..............60

3.4 Discussion and conclusions ...............................................................................................62

3.5 Materials and methods .......................................................................................................64

3.5.1 Cell lines ................................................................................................................64

3.5.2 Attenuated diphtheria toxin ....................................................................................64

3.5.3 siRNAs ...................................................................................................................64

3.5.4 PCR primers ...........................................................................................................65

3.5.5 Immunocytochemistry (HBEGF) ...........................................................................65

3.5.6 Preparation of aDT-siRNA conjugate ....................................................................65

3.5.7 Gene knockdown assays - treatment with aDT-siRNA conjugate .........................65

3.5.8 Gene knockdown assays - positive control treatment with Lipofectamine RNAiMAX .............................................................................................................66

3.5.9 Invasion assay ........................................................................................................66

3.5.10 Adhesion assay .......................................................................................................66

3.5.11 Cell viability assays following treatment with aDT-eIF-3b ..................................66

3.6 Acknowledgements ............................................................................................................67

Thesis discussion .......................................................................................................................67

4.1 The interplay of stability and potency in antisense therapeutics .......................................68

4.2 The importance of endosomal escape in oligonucleotide delivery ....................................70

4.3 Measuring functional effects of oligonucleotide delivery .................................................71

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4.3.1 Functional effects of DRR knockdown ..................................................................72

4.3.2 Functional effects of eIF-3b knockdown ...............................................................72

4.3.3 Functional effects of ITGB1 knockdown ..............................................................73

4.4 Targeting the glioblastoma stem cell population ...............................................................74

4.4.1 Targeting cell surface receptors .............................................................................74

4.4.2 Targeting key genetic alterations for desirable phenotypes ...................................76

4.4.3 Optimizing the delivery of protein conjugates in vivo ..........................................77

Conclusions ...............................................................................................................................78

5.1 Completion of objectives ...................................................................................................79

Recommendations for future work ...........................................................................................80

6.1 Investigation into aDT-siRNA trafficking mechanism ......................................................80

6.1.1 Inhibition of translocation ......................................................................................81

6.1.2 IC50 shifts of modified DT .....................................................................................82

6.1.3 Split-reporter assays ...............................................................................................82

6.2 Optimization of specificity, potency, and endosomal escape ............................................83

6.2.1 Re-targeting the receptor binding domain .............................................................83

6.2.2 Stabilization of the siRNA .....................................................................................84

6.2.3 Exploring RNA analogues: morpholinos and peptide nucleic acids .....................85

6.2.4 Phenotypic assays as a first-line screening tool for oligonucleotide sequences ....86

6.3 Simultaneous delivery of siRNA and other therapeutics ...................................................86

6.3.1 Combination with native toxin domains ................................................................86

6.3.2 Combination with therapeutic proteins ..................................................................87

6.3.3 Combination with chemotherapeutic drugs ...........................................................88

Abbreviations .................................................................................................................................89

Appendix A: Supporting information for “Antibody-antisense oligonucleotide conjugate downregulates a key gene in glioblastoma stem cells” .............................................................93

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Appendix B: Supporting information for “Attenuated diphtheria toxin mediates siRNA delivery” ....................................................................................................................................98

Appendix C: Effect of sugar 2’,4’-modifications on gene silencing activity of small interfering RNA duplexes .......................................................................................................102

Appendix D: Additional characterization of AONs and siRNAs against DRR ...........................117

Appendix E: In vivo biodistribution of a CD44 antibody conjugated to a fluorescent dye .........123

Appendix F: Polymeric nanomicelles for targeted therapeutic delivery to glioblastoma ............128

References ....................................................................................................................................134

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List of Tables Table 1. Common cell penetrating sequences. .............................................................................. 19

Table 2. Rates of selected bioorthogonal click chemistry reactions.183 ........................................ 25

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List of Figures Figure 1.1. Cancer stem cells resist treatment and promote recurrence. The bulk of the tumor is

susceptible to treatment including radiotherapy and chemotherapy, but some CSCs survive,

which can repopulate the tumor even in small numbers. ................................................................ 4

Figure 1.2. AON mechanism of action. The AON hybridizes to complementary strands of

mRNA and induces the activity of RNAseH, which recognizes the DNA/RNA hybrid and

degrades the RNA into its components (nucleoside monophosphates). ....................................... 11

Figure 1.3. siRNA mechanism of action. Dicer cleaves long, double-stranded RNA into short

fragments of siRNA. siRNA is then loaded into the RISC complex, binds to the complementary

mRNA, and RISC degrades the mRNA. ....................................................................................... 13

Figure 1.4. siRNA carriers protect it from nuclease degradation. (A) Free siRNA (blue double

helix) is rapidly degraded by nucleases (orange semi-circle) and (B) cleared by lymphatic

drainage (pale blue ovals). (C) Nanoparticle or protein carriers may protect siRNA from

nucleases and (D) reduce clearance. ............................................................................................. 14

Figure 1.5 Common modifications to oligonucleotides include modifications to both: (A) the

phosphodiester linkage and (B) the 2’ sugar. ................................................................................ 15

Figure 1.6. Reactions of native amino acids for site-specific protein conjugation. A) Thiols can

be modified through reactions with maleimides. B) Tyrosine can be reacted using diazonium

salts. C) Tryptophan can be modified through metallopeptide-catalyzed reactions. .................... 23

Figure 1.7. Cellular entry mechanisms of three “AB” toxins: diphtheria toxin, anthrax toxin, and

Pseudomonas exotoxin A. The R domain of DT binds to heparin-binding epidermal growth

factor (HBEGF) and induces internalization to the early endosomes where the connection

between the T and A domain is cleaved by furin, and the A domain translocates out of the early

endosome through a flexible α-helical pore. The protective antigen (T) domain of AT is cleaved

by extracellular furin, and oligomerizes on the cell surface, binding to the tumor endothelial

marker-8 (TEM-8) or capillary morphogenesis gene-2 (CMG-2) surface receptors. This leads to

association with the A domain and subsequent internalization into the early endosomes where A

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domain translocations through a rigid β-barrel pore. Finally, the R domain of PE binds to the cell

surface receptor Low density lipoprotein receptor-related protein 1 (LRP1), and the connection

between the T and A domain is cleaved by furin in the early endosomes. PE is then trafficked to

the endoplasmic reticulum where it undergoes retrograde translocation. ..................................... 29

Figure 2.1. Downregulated in renal cell carcinoma (DRR) expression is reduced following

transfection of DRR+ U-251 MG cells with DRR antisense oligonucleotides. (A) Oligonucleotide

sequences used: all antisense strands comprise a phosphorothioate backbone while all sense

strands are synthesized with a phosphodiester backbone. (B) DRR expression following

treatment with single stranded DRR AON sequences normalized to untreated control. Data were

analyzed using one-way ANOVA followed by Dunnett’s post-hoc test compared to Scrambled

group (data is shown as mean+SD, n=3, *p<0.05, **p<0.01). (C) DRR expression following

treatment with double stranded anti-DRR oligonucleotide sequences normalized to untreated

control. Data were analyzed using unpaired t-test with Welch’s correction (data is shown as

mean+SD, n≥4, ***p<0.001). (D) Representative western blot showing single stranded and

double stranded antisense oligonucleotide DRR knockdown. ...................................................... 36

Figure 2.2. Patient-derived GSCs strongly co-express antigens CD44 and EphA2 with DRR and

weakly co-express EGFR with DRR. Representative confocal images are shown. Antigens

CD44, EphA2, and EGFR (green); DRR (red); cell nucleus (Hoechst, blue). All scale bars are 50

µM. ................................................................................................................................................ 37

Figure 2.3. EphA2 mAbs and CD44 mAbs are internalized upon binding to cells. (A-C) Flow

cytometry analysis of cell surface receptor internalization following 45 min incubation at 37 °C

with antibodies (A) CD44 mAb and (B) EphA2 mAb compared to (C) non-specific CTL mAb.

Cells without antibodies added (cells only) and cells incubated with antibodies, but held at 4 °C

(no internalization) curves are shown as a comparison to those cells that were allowed to

internalize the mAb for 45 min at 37 °C (45 min internalization). (D) Quantification of

internalized receptor following 45 min incubation period for CD44 mAb and EphA2 mAb

antibodies compared to CTL. Data were analyzed using one-way-ANOVA followed by

Dunnett’s post hoc test compared to CTL group (data is shown as mean+SD, n=3, *p<0.05,

***p<0.001). ................................................................................................................................. 38

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Figure 2.4. CD44 mAb and EphA2 mAb can be efficiently conjugated to dsDRR using click

chemistry. (A) Scheme of antibody modification with dsDRR. (B) 10% PAGE analysis of CD44

mAb-dsDRR conjugation: Lane 1: dsDRR only; Lane 2: CD44 mAb conjugated to dsDRR via

NHS-PEG4-N3 crosslinker; Lane 3: dsDRR and CD44 mAb combined without crosslinker

present. (C) 10% PAGE analysis of EphA2 mAb-dsDRR conjugation: Lane 1: dsDRR only;

Lane 2: EphA2 mAb conjugated to dsDRR via NHS-PEG4-N3 crosslinker; Lane 3: dsDRR and

EphA2 mAb combined without crosslinker present. .................................................................... 40

Figure 2.5. DRR expression of patient-derived GSCs after treatment with CD44 mAb-dsDRR or

EphA2 mAb-dsRR (150 nM) normalized to untreated control. (A) Quantification of DRR

expression following CD44 mAb-dsDRR treatment. Data were analyzed using one-way-

ANOVA followed by Dunnett’s post hoc test compared to CD44 mAb-dsScrambled (data is

shown as mean+SD, n≥4, *p<0.05). (B) Representative western blot showing DRR knockdown

following treatment with CD44 mAb-dsDRR. (C) Quantification of DRR expression following

EphA2 mAb-dsDRR treatment. Data were analyzed using one-way-ANOVA followed by

Dunnett’s post hoc test compared to EphA2 mAb-dsScrambled (data is shown as mean+SD,

n≥3). (D) Representative western blot showing DRR expression following treatment with EphA2

mAb-dsDRR. ................................................................................................................................ 41

Figure 2.6. Antisense oligonucleotides conjugated to CD44 mAb are taken up by GSCs and

trafficked into the endolysosomal pathway. (A) Uptake of CD44 mAb-dsDRR (white arrows)

compared to (B) EphA2 mAb-dsDRR and (C) CTL-dsDRR after 3 h incubation at 37 °C. Control

(CTL) is a non-specific human IgG. (D) Colocalization (white arrows) of CD44 mAb-dsDRR

with the lysosomal compartments following a 2 h pulse and 1 h chase. Cell membrane (wheat

germ agglutinin (WGA), magenta); cell nucleus (Hoechst, blue); AON (Cy3, green); lysosome

(Dextran647, red). Representative z-stack images shown. All scale bars are 50 µM. .................. 43

Figure 2.7. GSCs treated with CD44 mAb-dsDRR have a rounder shape, fewer projections, and

centralized focal adhesions relative to the spindle-shaped cells of the control treatments. (A)

Cells treated with CD44 mAb-dsScrambled. (B) Cells treated with CD44 mAb-dsDRR. (C) Cells

treated with CD44 mAb alone. (D) Cells treated with dsDRR alone. Representative z-stack

images shown. All scale bars are 50 µM. Cell nucleus (Hoechst, blue); Actin (Phalloidin Alexa

Fluor 488, green). (B) Change in cellular morphology is quantified as actin area per cell

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normalized to a no treatment control. Data were analyzed using one-way-ANOVA compared to

CD44 mAb-dsScrambled with Dunnett’s post hoc correction (data is shown as mean+SD, n≥3,

***p<0.001). ................................................................................................................................. 45

Figure 3.1. Using attenuated diphtheria toxin for siRNA delivery. A) Attenuated AB toxins, such

as attenuated diphtheria toxin (aDT), consist of three main components: a receptor binding

domain (R) that binds to a receptor on the cell surface; a translocation domain (T) that allows for

endosomal escape; and a mutated active domain (a) in order for the protein to retain its

trafficking functions but is no longer toxic to cells. Cargo, such as siRNA, can be attached to this

“a” domain. B) siRNA delivered using attenuated DT occurs in five main steps: 1) binding to the

HB-EGF precursor cell surface receptor; 2) endocytosis of the aDT-siRNA cargo; 3)

translocation through the endosomal membrane, inserting the “a” domain and cargo into the

cytoplasm; 4) cleavage of the “a” domain from the rest of the protein; and 5) release of the

siRNA into the cytoplasm where it downregulates the relevant gene. ......................................... 54

Figure 3.2. siRNAs can be conjugated to attenuated diphtheria toxin. A) Attenuated diphtheria

toxin was engineered to contain a free cysteine as a functional handle (aDT-SH), protected by a

SUMO tag and purified using a histidine (His) tag. B) Attenuated diphtheria toxin was reacted

with a PEG crosslinker containing both maleimide and DBCO functional groups to obtain

DBCO-modified attenuated diphtheria toxin (aDT-DBCO). C) The presence of the DBCO

modification on attenuated diphtheria toxin was confirmed by reading the absorbance at 280 nm

(aDT) and 309 nm (DBCO). Curves shown are aDT before modification (blue) and after DBCO

modification (red). D) Azide-modified siRNA was reacted with the DBCO-functionalized

attenuated diphtheria toxin to obtain the aDT-siRNA conjugate. E) Modification of the aDT with

the siRNA was confirmed via PAGE stained with Coomassie blue to localize the diphtheria toxin

protein. Lane 1 shows the aDT-DBCO starting material (MW ~ 72 kDa) and lane 2 shows the

aDT-siRNA conjugate (MW~90 kDa) alone with some unreacted starting material. F)

Purification of the excess siRNA was confirmed via PAGE stained with GelRed to localize the

siRNA. Lane 3 shows the aDT-siRNA conjugate along with unreacted siRNA; lane 4 shows the

aDT-siRNA conjugate after nickel column purification, with only a small amount of excess

siRNA left over. ............................................................................................................................ 57

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Figure 3.3. GSCs express HB-EGF, the native receptor for diphtheria toxin. Representative

confocal images are shown for HB-EGF (anti-HBEGF antibody, green) and nucleus (Hoechst,

blue). The secondary antibody only control confirms lack of non-specific binding. All scale bars

are 50 µm. ..................................................................................................................................... 58

Figure 3.4. aDT-siRNA downregulates ITGB1 expression in GSCs and reduces cellular invasion.

A) aDT-ITGB1 (light red bars) downregulates ITGB1 mRNA expression compared to negative

controls: aDT conjugated to a non-targeting siRNA (aDT-NT, black bars) and ITGB1 siRNA

only without lipofectamine (blue bars) at 24 h post treatment. Positive control is transfected

ITGB1-siRNA with lipofectamine (dark red bars). Data is shown as n=3, mean±SD, normalized

to an untreated control. Data was analyzed using one-way-ANOVA followed by Tukey’s

correction on the logarithmic data (* p<0.05, ** p<0.01). B) Cells were plated in a 3D hydrogel

assay on the surface of pre-formed hydrogels and treated with aDT-ITGB1 conjugates at the

beginning of the experiment. Invasion depth was measured after 5 days. C) aDT-ITGB1 reduces

invasion compared to controls (no treatment and aDT-NT) in a 3D hydrogel model.

Representative images shown. 15 µm red beads label the top of the hydrogel; blue cell nuclei are

labeled using Hoechst. All scale bars are 150 µm. D) Invasion depth was quantified as a

percentage of the untreated control. Data was analyzed using one-way-ANOVA followed by

Tukey’s correction (* p<0.05, ** p<0.01). E) aDT-ITGB1 did not reduce number of adhered

cells in a 3D hydrogel model. Representative images are shown. All scale bars are 150 µm. F)

Number of adherent cells was quantified by counting number of cell nuclei; no significant

difference was observed, demonstrating that differences in invasion were due to ITGB1

downregulation. Data was analyzed using one-way-ANOVA followed by Tukey’s correction. . 59

Figure 3.5. aDT-siRNA downregulates eIF-3b expression in GSCs and reduces cell viability. A)

aDT-eIF-3b (light red bars) downregulates eIF-3b mRNA expression compared to negative

controls: aDT conjugated to a non-targeting siRNA (aDT-NT, black bars) and eIF-3b siRNA

only without lipofectamine (blue bars) at 24 h post treatment. Positive control is transfected

siRNA with lipofectamine, dark red bars. Data is shown as n=3, mean±SD, normalized to an

untreated control. Data was analyzed using one-way-ANOVA followed by Tukey’s correction on

the logarithmic data (* p<0.05). B) aDT-eIF-3b (red bars) reduces cell viability of GSCs at 48 h

post treatment compared to aDT-NT (black bars) at 100 nM. Data is shown as n=3, mean+SD,

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normalized to an untreated control. Data was analyzed using one-way-ANOVA followed by

Tukey’s correction (* p<0.05). ..................................................................................................... 62

Figure 6.1. Structures of nucleic acid analogues including morpholinos and peptide nucleic acids.

....................................................................................................................................................... 85

1

Introduction

Portions of this chapter are derived from the following manuscript:

Arnold, A. E.; Czupiel, P.; Shoichet, M.S. Engineered Polymeric Nanoparticles to Guide the

Cellular Internalization and Trafficking of Small Interfering Ribonucleic Acids. J. Control.

Release 2017, 259, 3–15.

A.E.A. and P.C. contributed equally to the research and writing of this article. M.S.S. edited the

manuscript.

1.1 Rationale

Glioblastoma (GBM) is a highly aggressive and invasive cancer, with the current standard of

care only offering marginal improvements in an already limited survival time. In particular, the

glioblastoma stem cell (GSC) population is hypothesized to be responsible for the resistance of

the cancer to chemotherapy, and these cells also invade deeply into the brain tissue, avoiding

surgical resection and promoting recurrence of the cancer. The basis of many cancers is gene

misregulation: due to underlying mutations, many genes become either under- or over-expressed,

leading to aberrant behaviour of the cells, malignancy, and metastasis.

Antisense technologies, which comprise oligonucleotides such as small interfering ribonucleic

acids (siRNAs) and antisense oligonucleotides (AONs), are powerful tools for the regulation of

gene expression, and offer an ideal therapeutic strategy for diseases caused by genetic mutations,

such as cancer. Cancer cells overexpress oncogenic genes, providing potential targets for gene

knockdown.1-2 siRNAs are short strands of ribonucleic acid (RNA) typically composed of 21-30

base pairs with overhanging 3’ ends that can induce sequence-specific gene silencing at low

(picomolar) concentrations when transfected into cells.3 AONs are short strands of

deoxyribonucleic acid (DNA) with sequence homology to the messenger RNA (mRNA) of

interest that, while typically less potent than siRNAs, can still effect potent gene silencing when

transfected into cells in a similar manner.4

Delivery materials for antisense therapeutics have traditionally been large, positively charged

nanomaterials. However, these materials often are challenged with poor biodistribution, with

2

most ending up in the clearance organs (liver, kidney, or spleen) or the lungs. For this reason,

many of these formulations target cancers or other diseases originating in these organs. In order

to engineer platform technologies that could be used for GSC therapeutics, we wanted to develop

small protein bioconjugates that would target specific receptors on the GSC cells and evade

clearance due to their small size and lack of positive charge. In this thesis, two modalities of

antisense therapeutic delivery are explored: the first using antibodies, which are targeting

proteins from the adaptive immune system that are highly stable in circulation; and the second

using attenuated toxins, which have the additional advantage of sophisticated trafficking

mechanisms once they penetrate cells. Proof-of-concept in vitro studies were carried out to

demonstrate the potential applications of these bioconjugates and their promise as platform

technologies for antisense therapeutic delivery to GSCs and potentially many other diseases.

1.1.1 Hypothesis and objectives

Hypothesis: Protein-conjugated oligonucleotides will selectively and effectively knockdown key

genes in brain cancer cells, including patient-derived models of GBM, and thereby reduce either

cellular proliferation or invasion in vitro.

1. Knockdown an essential GSC gene in vitro using a targeted delivery platform.

i. Validate knockdown using AONs or small interfering ribonucleic acids.

ii. Conjugate AONs to antibodies specific to GSCs.

iii. Evaluate uptake, gene knockdown, and changes in cellular morphology in vitro.

2. Achieve gene knockdown with enhanced endosomal escape.

i. Evaluate suitability of diphtheria toxin as a delivery vehicle for GSC treatment.

ii. Conjugate siRNAs to attenuated diphtheria toxin (aDT).

iii. Evaluate reduction of target mRNA following aDT-siRNA treatment in vitro.

iv. Evaluate functional effects of target knockdown including reduction in cell viability

reduction of cellular invasion in a 3D model.

3

1.2 Glioblastoma stem cells

This section will give an overview of GBM, the cancer stem cell concept, and ultimately focus

on GSCs as a targetable and therapeutically relevant cell population.

1.2.1 Glioblastoma

GBM is one of the deadliest brain cancers, with a median survival time of just over a year with

the current standard of care, which includes aggressive chemotherapy, radiation, and surgical

resection.5 The origin of GBM is unclear, although in at least some cases it develops from low-

grade astrocytomas. Recent studies have shown that the cells of origin could be astrocytes,

oligodendrocyte precursor cells, or neural stem/progenitor cells.6 Most GBMs demonstrate a

diffuse infiltrative growth pattern, meaning the cancer cells interweave with the existing healthy

brain tissue, with single cells or groups of cells diffusing outward into the brain tissue from the

main tumor mass.7 Cells in the invasive front of the GBM tend to take advantage of existing

‘supply lines’ for oxygen and nutrients rather than building their own.7

As many as 50% of GBMs have significant mutations in the Phosphoinositide 3-kinase/Protein

kinase B/Phosphatase and tensin homolog (PI3K/Akt/PTEN) pathway, which plays a significant

role in the cell cycle and cell motility and can have downstream effects including increasing

proliferation and invasion and avoiding apoptosis.8 However, many strategies to combat GBM

by inhibiting this pathway have led to disappointing results in clinical trials.9-11 Other important

oncogenic changes that are frequently observed in GBMs are mutations of isocitrate

dehydrogenase (IDH), which plays a key role in the production of NADPH, and methylation of

O6-methylguanine-DNA methyl-transferase (MGMT), a ubiquitously expressed enzyme

involved in DNA repair. These changes are important as both prognostic indicators and potential

therapeutic targets.12-14

The standard-of-care chemotherapy drug for GBM treatment is temozolomide (TMZ), which is

used because it crosses the blood brain barrier. Unfortunately, TMZ is only a modestly effective

chemotherapeutic and its clinical use is mainly palliative. The average increase in survival time

for patients treated with resection and radiotherapy concurrently with TMZ compared to patients

who received resection and radiotherapy is only 2.5 months.15 Treatment with TMZ also has

significant, dose-limiting toxic side effects which leads to adverse events in 15-20% of patients,

4

including thrombocytopenia, lymphopenia, neutropenia, and myelodysplastic syndrome (a

disturbance of the bone marrow).16 For these reasons, there is a significant unmet need to

develop new therapies for GBMs, and especially the cancer stem cell population, which may be

responsible for many of the aggressive characteristics of GBM (vide infra).

1.2.2 Cancer stem cells

Stem cells are immature cells within the body that have the capacity for self-renewal, meaning

they can divide to generate new stem cells, as well as multi- or pluripotency, which means they

have the capacity to differentiate into multiple adult cell types.17 Although there are many genes

involved in the regulation of stemness, the transcription factors Oct4, Sox2, and Nanog are

essential transcription factors that maintain the self-renewal and pluripotency of pluripotent stem

cells. An increasing body of evidence shows that there is a sub-population of cells within many

cancers that exhibit characteristics of both cancer cells and stem cells, which means they express

markers of self-renewal and pluripotency while also containing oncogenic mutations. The cancer

stem cell (CSC) hypothesis suggests that because these cells have characteristics including self-

renewal, pluripotency, and tumorgenicity, they have the capacity to evade radio- and chemo-

therapy, initiate metastasis, and promote recurrence of the cancer (Figure 1.1).

Figure 1.1. Cancer stem cells resist treatment and promote recurrence. The bulk of the

tumor is susceptible to treatment including radiotherapy and chemotherapy, but some

CSCs survive, which can repopulate the tumor even in small numbers.

The first identification of a tumor-initiating subpopulation of cells was in 1994, when researchers

implanted an enriched fraction of CD34+/CD38- acute myeloid Leukemia cells into an immune-

compromised mouse and observed that the cells were more proliferative and tumorigenic than

the parent population.18 In 1997, Dick et al. studied multiple subtypes of AML and identified

tumor-initiating cells that could differentiate in vitro in all samples regardless of subtype.19

Discoveries of CSC populations in breast cancer20 and brain cancer, specifically

5

medulloblastoma and GBM,21 followed in 2003. Strikingly, as few as 100 cells were required to

initiate the formation of a solid tumor in mouse models in both breast and brain cancers. CSCs

have since been identified in a variety of cancers including colon, pancreas, lung, prostate, and

melanoma.22 There are conflicting theories about the origin of these cells: they may arise from

normal stem cells that gain oncogenic mutations, or from somatic cells that acquire stem-like

characteristics and develop malignant behavior.22 Notably, CSC markers are not ubiquitous - in

each different tissue type, or even subtype, the cells are characterized by expression of diverse

markers,23 which means their identification and isolation presents a significant challenge.24

CSCs play important roles in both resistance to chemo- and radio- therapy25 and cancer invasion

and metastasis. Meirelles et al. found that ovarian CSC populations are resistant to cisplatin and,

even more surprisingly, stimulated by doxorubicin (both common chemotherapy drugs).26 Li et

al. showed that the proportion of CSCs in human breast cancer biopsies increased from 4.7% to

13.6% after a common chemotherapy regimen while the percentage of epithelial cells remained

constant, indicating a higher resistance to chemotherapy in the CSC population.27 Interestingly,

Zielske et al. studied two different patient-derived breast cancer xenografts, and observed that

one CSC population was enhanced following radiotherapy, agreeing with earlier reports, while

the other CSC population was depleted by radiotherapy treatment, perhaps hinting towards a

high degree of variance in CSCs between different patients.28 CSCs are also important in cancer

migration processes. Kaplan et al. demonstrated that bone-marrow derived haematopoietic

progenitor cells hone to pre-metastatic sites and provide a permissive niche prior to the arrival of

tumor cells.29 Pandit et al. discovered that highly metastatic cancer cell lines express higher

proportions of CSC markers than non-metastatic cancer cell lines,30 and Wakamatsu et al. found

a higher proportion of CSC markers in diffuse lymph node metastases than in the primary tumor

site, suggesting that the metastases were initiated by the CSCs.31

Several approaches have been taken to try to specifically target the CSC population. One

approach is to target specific receptors on the CSC surface; since many markers are shared

between CSCs and normal stem cells, there are only a few targetable receptors that also

differentiate between the two populations. CD44 and CD133 are two common CSC markers that

have been used to target the CSC population. Jin et al. eradicated leukemia stem cells using an

anti-CD44 antibody and observed reduced leukemic repopulation.32 Perez et al. have developed

an anti-CD44 antibody that selectively targets the CSC population in head and neck squamous

6

cell carcinoma33 and two clinical trials using this strategy have been completed (NCT01358903

and NCT01641250). Zhao et al. inhibited tumor growth by developing a bispecific antibody for

both T-cells and CD133+ cells, encouraging the T cells to attack the CSC population.34 Another

approach is to target specific pathways that are active in CSCs. Several small molecule mTOR

inhibitors, including Everolimus and Metformin, have been explored to specifically target the

breast CSC population, which leads to significantly slower tumor growth.35-36 In GBM,

JAK/STAT3 inhibitors were used to target the GSC population resulting in prolonged survival in

a mouse model of GBM.37 Taken together, these studies demonstrate that specifically targeting

the CSC population can lead to significant benefit in tumor growth and patient survival, and thus

is a promising approach that is widely applicable to many cancers.

1.2.3 Glioblastoma stem cells

Brain tumor initiating cells were first identified by Singh et al. in 2003, primarily in samples of

aggressive medulloblastomas.21 In 2004 the same group discovered that GBM stem cells, which

comprised 20-30% of the total patient tumor samples, could be transplanted into mouse brains at

low cell numbers and recapitulate diversity of the original patient tumor, including neuron,

oligodendrocyte, and astrocyte lineages.38 These cells have characteristics including self-

renewal, pluripotency, neurosphere formation, proliferation, invasion, and high motility.39 The

major challenges that GSCs present are their highly infiltrative nature and their extreme

resistance to conventional treatments, which make them an important target for treatment of

GBM.39

GSCs are described by many terms, including cancer/tumor/glioma/brain tumor stem cell, stem-

like tumor cell, cancer-/tumor-/glioma-brain tumor-initiating cell, and glioma neural stem cell.40

While these terms are often coined in an attempt to delineate small variations in cell markers and

functional characteristics, for the purposes of this thesis they will all fall under the umbrella of

‘glioblastoma stem cells/GSCs’ that have been identified by one key functional assay:

propagation of a tumor upon transplantation in a rodent brain followed by differentiation into

multiple cell types, recapitulating the diversity of the parental tumor.

For specific targeting of the GSC population, it is important to pick markers that will be

expressed on the GSC cell surface. GSC samples are often thought of as a hierarchy, with a very

small fraction of the cells that are actually self-renewing, nearly quiescent stem cells maintaining

7

a larger population of progenitor cells and perhaps even differentiated progeny.41 This feature of

GSC populations makes them difficult to target, since many different markers will be expressed

at different stages of the cell cycle and even among different stem/progenitor cells within the

same population; so there is not one ubiquitous ‘GSC marker’. However, some common surface

markers have been identified which are discussed below.

1.2.3.1 Surface markers of GSCs

As previously mentioned, CD133 and CD44 are cell surface markers that are shared among

many CSC populations in different tissues and are two of the most common markers for GSCs.

Some additional surface markers that have been explored for the isolation, characterization, and

targeting of the GSC population are EphA2/EphA3,42-43 EGFR,44 CD15/SSEA1, A2B5,

L1CAM/CD171, ALDH1A3.39

CD133 (cluster of differentiation 133) was the first cell surface marker used to isolate GSCs.21, 38

Also known as prominin-1, CD133 is an antigen that is located on projections of the plasma

membrane and is typically expressed on hematopoietic stem and progenitor cells.45 Increased

CD133+ cells in in brain tumors correlates with a more aggressive tumor46 and a poorer

prognosis,47 and recurrent tumors often have higher proportions of CD133 cells,48 implying an

important role for CD133 in cancer recurrence and invasion. Singh et al. found that CD133+ cells

could initiate GBMs whereas CD133- cells could not.38 However, subsequent studies have shown

that CD133- cells can also give rise to GBMs with robust CD133 expression,49-50 implying that

not all GSCs express CD133.

CD44 (cluster of differentiation 44) is a cell membrane glycoprotein that serves as a hyaluronic

acid receptor.51 CD44 is involved in key pathways associated with GSCs including invasion and

proliferation.52 In neural stem cells, CD44 is often co-expressed with the stem cell markers nestin

and Sox2.53 CD44+ cells can generate new tumors that recapitulate the parental tumor in mouse

models, but CD44- cells cannot do the same.54 Importantly, inhibition of CD44 can also slow the

progression of GBM,55 indicating its key role in tumorgenesis and its potential as a therapeutic

target for GBM. GSCs often express a splice variant of CD44, CD44v6, which has been shown

to drive proliferation of GSCs.56

8

Some less common, but still important, proposed markers for GSC populations are

EphA2/EphA3, EGFR, CD15/SSEA1, and L1CAM/CD171. Binda et al. identified EphA2 as a

driver of self-renewal and tumorgenicity in GSCs,42 and Qazi et al. demonstrated that co-

targeting of EphA2 and EphA3 could be a promising approach for GSC targeting and GBM

treatment.43 EGFR is expressed in over half of newly diagnosed GBMs and the variant EGFRvIII

is amplified in GSCs in vivo; however, this amplification is often lost in in vitro culture.44 CD15,

also known as stage-specific embryonic antigen 1 (SSEA1) is a marker for central nervous

system stem cells and is also an enrichment marker for GSCs.57-58 L1CAM is a cell adhesion

molecule expressed in neurons and is involved in growth and migration during nervous system

development, and has been shown to be essential for the survival of CD133+ GSCs.59

1.2.4 Therapeutic targets associated with GSC proliferation and invasion

Many signaling pathways are activated to promote GSC self-renewal, proliferation, and

progression, including receptor tyrosine kinases, Akt, MAPK, Wnt, Notch, Hedgehog, and

JAK/STAT pathways.60 These complex, often inter-connected signaling pathways offer a

multitude of potential therapeutics for gene regulation in the GSC population. For example,

Esposito et al. demonstrated that knockdown of STAT3 reduced cell proliferation and migration

in vitro, leading to a decrease in tumor growth and angiogenesis in vivo.61 Berezovsky et al.

found that knockdown of Sox2, which can be downstream of both Akt and JAK/STAT signaling

pathways, reduced self-renewal and sphere forming properties in GSCs.62 This thesis will focus

on three selected genes for downregulation in GSCs, which are involved with GBM

proliferation, invasion, or both: downregulated in renal cell carcinoma (DRR), Eukaryotic

Translation Initiation Factor 3b (eIF-3b), and integrin b1 (ITGB1).

1.2.4.1 DRR

DRR, also known as Family with Sequence Similarity 107A (FAM107A), was first associated

with cancer as a tumor suppressor gene in renal cell carcinoma.63 In 2010, Petrecca et al.

identified DRR as a driver of GBM invasion that acts as a crosslinker between the actin and

microtubule cytoskeletons.64 They proposed the following theory of cancer progression relating

to DRR: low grade, infiltrative gliomas express high levels of DRR, and display an extremely

invasive phenotype. Over time, additional mutations in some cells lead to the loss of DRR and

9

invasion is reduced while proliferation is dramatically increased, as observed in higher grade

gliomas. This is supported by earlier work that shows invasion and proliferation as temporally

separate events in malignant gliomas.65

In 2014, Petrecca et al. expanded on their earlier work and demonstrated that DRR induces Akt

activation by recruiting kinases to focal adhesions.66 They also discovered a relationship between

the GSC population, and by knocking down DRR in the GSCs, they observed a corresponding

reduction in activated Akt levels and a reduction in invasion in vitro and in vivo. Therefore, DRR

is a promising target for reducing the invasion of GSCs.

1.2.4.2 eIF-3b

eIF-3b is a subunit of the 13-unit eukaryotic translation initiation factor 3 complex (eIF 3) which

is essential for assembling the machinery for protein synthesis within the cell.67 The composition

of eIF 3 is conserved within most eukaryotes.68 One of the first studies that proved eIF-3b played

a role in oncogenesis related eIF-3b expression to tumor grade, stage, and patient survival in

bladder and prostate tumor tissue samples.69 The same study demonstrated that robust eIF-3b

expression is required for proliferation of the tumor and colonization of the cancer cells in a

secondary site such as the lungs.69 Zang et al. studied the role of eIF-3b in clear cell renal cell

carcinoma (ccRCC) and observed that high levels of eIF-3b in patient tumor samples correlated

to a more aggressive tumor and reduced patient survival.70 Knockdown of eIF-3b in ccRCC

impaired the activation of the Akt pathway, inhibiting cell proliferation and inducing apoptosis.

Furthermore, the migration of the cells via epithelial-to-mesenchymal transition was reduced

following eIF-3b knockdown.70 Liang et al. discovered that eIF-3b also plays a key role in the

proliferation of GBM, and by knocking down eIF-3b the proliferation of the GBM cells was

significantly reduced,71 indicating the potential of eIF-3b as a therapeutic target in GBM.

1.2.4.3 ITGB1

Integrins are a large family of cell-adhesion molecules that are displayed on the surface of the

cell as heterodimers. In mammalian cells, there are 18 a integrins and 8 b integrins, with 24

identified dimers of a and b subunits.72 ITGB1 can form 12 different heterodimers to interact

with a variety of extracellular matrix materials, including fibronectin and collagen.73 Binding of

extracellular ligands to ITGB1 can initiate a signal cascade involving signaling partners such as

10

focal adhesion kinase (FAK) and Akt that initiates actin assembly or disassembly, an essential

step in the formation and dissolution of focal adhesions during cell migration.73 ITGB1 has been

shown to enhance cancer cell invasion and migration in breast cancer, pancreatic cancer, and

GBM.74-76 Interestingly, in GBM, knockdown of ITGB1 increased the sensitivity of cells to anti-

angiogenic therapy in addition to reducing the invasiveness of the cancer cells.76 While studies

connecting ITGB1 and GSCs have been limited, ITGB1 is overexpressed in at least some

subtypes of GSCs,77 and ITGB1 is involved in many invasion processes, so it is an interesting

target for preventing the invasion of the GSCs.

1.3 Antisense therapeutics

Antisense therapeutics are powerful tools for controlling gene expression. They were first

identified more than forty years ago: in 1978, the first example of an AON was reported as an

inhibitor of viral replication. Gene silencing by double-stranded siRNAs was first discovered in

C. Elegans in 1998,78 and naturally occurring siRNAs were reported in 1999 in plants.79

Synthetic siRNAs were used to effect gene knockdown in mammalian cells two years later.80

“Naked” AON and siRNA therapeutics have been successful in clinical trials: siRNAs for ocular

diseases when locally delivered at high concentrations, despite limitations including

inflammation and increased ocular pressure;81 and AONs for applications in muscular

dystrophy82 and spinal muscular atrophy.83 Because AONs and siRNAs can target and

downregulate specific genes within diseased cells, they are promising treatments for diseases

caused by genetic mutations such as cancer.84-85 However, efficient and targeted delivery of

nucleic acid therapeutics presents a significant clinical challenge.

Following intravenous injection in order to reach diseased (ie. cancerous) tissue, oligonucleotide

formulations must (1) evade the immune system, (2) avoid interactions with non-target cells, (3)

avoid premature renal clearance, and (4) reach target tissues. There are further issues once they

reach their site of action: the fragile nucleic acid material must avoid degradation by extracellular

nucleases, and overcome poor cell uptake and trafficking into the lysosomal compartments

where the RNA or DNA strands are quickly degraded.86-87 These challenges often require that

antisense therapeutics are combined with specialized delivery materials in order to be effective.

Some of the carriers that are currently used to carry these oligonucleotides into cells will be

presented in sections 1.4 and 1.5. This section will focus on the mechanism and stability of

11

AONs and siRNAs.

1.3.1 Antisense oligonucleotide mechanism

First generation AONs were strands of DNA, 8-50 nucleotides in length, which can bind to

complementary strands of mRNA and induce the activity of RNAseH, an enzyme that recognizes

the DNA/RNA hybrid and degrades the mRNA. (Figure 1.2).88 More recent studies have focused

mostly on chemically modified AONs, with modifications such as the ones shown in Figure 1.5,

which enhance the stability of the AON while reducing off-target effects.89 The AONs also

commonly include a phosphorothioate backbone or are completely replaced with other

backbones, such as morpholine rings, which further increase their resistance to nuclease and

protease degradation.4 The current clinically approved AONs are examples of these second-

generation motifs, including eteplirsen, an AON against Duchenne muscular dystrophy which is

administered as a “naked” AON intravenously,82 and nusinersen, an AON for treatment of spinal

muscular atrophy that is injected directly into the cerebral spinal fluid of the spinal column.83

Figure 1.2. AON mechanism of action. The AON hybridizes to complementary strands of

mRNA and induces the activity of RNAseH, which recognizes the DNA/RNA hybrid and

degrades the RNA into its components (nucleoside monophosphates).

12

1.3.2 Small interfering ribonucleic acid mechanism

siRNAs are 20-30 nucleotide long, double stranded, noncoding RNA that can degrade mRNA in

a sequence-specific manner.90 They are typically more potent, but somewhat less stable, than

their AON counterparts.90 Unlike AONs, siRNAs cannot be delivered without a carrier to

mediate delivery.91 In 2018, the FDA approved the first siRNA-based drug, patisiran, for the

treatment of transthyretin-mediated amyloidosis.92 While this drug uses a lipid nanoparticle

system for delivery, next-generation delivery systems that use smaller siRNA-biomolecule

conjugates are already in the clinical pipeline.92

As shown in Figure 1.3, one of the first steps in the siRNA mechanism is cleavage by an enzyme

called Dicer, which cuts long, double-stranded RNA into short siRNA fragments. The RNA-

induced silencing complex (RISC), which is a multi-protein assembly, then forms and

incorporates the strand of the siRNA with the more stable 5’-end. The complementary mRNA is

identified by sequence alignment to the siRNA and the catalytic protein of the RISC complex,

Argonaute 2, cleaves the complementary mRNA.90

13

Figure 1.3. siRNA mechanism of action. Dicer cleaves long, double-stranded RNA into

short fragments of siRNA. siRNA is then loaded into the RISC complex, binds to the

complementary mRNA, and RISC degrades the mRNA.

1.3.3 Stability in the extracellular environment

In order to increase the delivery efficiency of oligonucleotide payloads, they are often conjugated

or complexed to carriers that protect them from nucleases and rapid clearance (Figure 1.4).

While AONs are relatively stable to nuclease degradation,93 they are less potent than their siRNA

counterpart, and stability is a major challenge to siRNA delivery. Within 15 minutes of injection

in mice, more than 90% of standard 21-mer siRNAs are degraded by serum nucleases or lost via

renal or lymphatic clearance,94 underlining the importance of the delivery vehicle. Polymeric

nanoparticles can increase the stability of siRNAs against degradation: Raja et al. demonstrated

that crosslinked chitosan nanoparticles increased the stability of siRNAs against serum during a

15 day storage at 4°C95 while Zhu et al. increased the half-life of siRNA in the blood to

approximately 8 hours by encapsulating it within a PLGA-based delivery vehicle, resulting in

better tumor accumulation.96 Conjugation to a globular protein, such as an antibody, can also

protect nucleic acid materials from degradation: Bäumer et al. conjugated an siRNA to an anti-

EGFR antibody and saw a significant improvement in stability against serum.97

14

Figure 1.4. siRNA carriers protect it from nuclease degradation. (A) Free siRNA (blue

double helix) is rapidly degraded by nucleases (orange semi-circle) and (B) cleared by

lymphatic drainage (pale blue ovals). (C) Nanoparticle or protein carriers may protect

siRNA from nucleases and (D) reduce clearance.

Oligonucleotides can also be chemically modified in order to increase their stability against

nucleases. These modifications include any change to the native DNA or RNA structure,

typically employed on the phosphodiester bond or sugar ring (Figure 1.5). These modifications

enhance stability and potency, provide longer knockdown duration, reduced off-target effects,

and lower immunostimulatory effects.89, 98-100 Modified oligonucleotides are now commonly

used in research.101-103 As shown in Figure 1.5, some of the most common modifications of

oligonucleotides include modifications to the backbone or nucleosides. For example, backbone

modifications include phosphorothioate104 and boranophosphonate105 linkages, which increase

nuclease resistance, while nucleoside modifications include 2’-O-methyl,106-107 2’-deoxy-2’-

fluoro,108 and locked nucleic acids,109 which increase stability and target binding affinity.

Chemical modification of oligonucleotides and the effect on potency have been extensively

reviewed by Deleavey et al.89 Figure 1.5 is showing the RNA sugar but many of the same

modifications can be used with DNA for AON applications.

15

Figure 1.5 Common modifications to oligonucleotides include modifications to both: (A) the

phosphodiester linkage and (B) the 2’ sugar.

1.4 Nanoparticles as delivery vehicles

This section will provide a review of some current gene delivery strategies in the field. A

significant amount of the recent research has focused on siRNA delivery using nanoparticle

formulations, so many of the references herein will involve the delivery of siRNA using

nanoparticle formulations such as cationic polymers, liposomes, or viral-derived materials.

Examples of more recent AON and siRNA delivery using smaller antibody or toxin conjugates

can be found in section 1.5.

1.4.1 Polymeric formulations

Cationic polymers are often used to facilitate siRNA penetration of the cell110 because they

interact with the anionic proteoglycans of the cell membrane, facilitating endocytosis and

endosomal escape.111-112 One classically used cationic polymer for siRNA delivery is

polyethyleneimine (PEI).113 Highly branched and high molecular weight PEI (>20 kDa) is toxic,

so low molecular weight PEI (<2 kDa) is often used.114 In one example, Lee et al. used low

molecular weight PEI for the delivery of ‘polymerized’ siRNA - that is, chains of repeating

O

O OHPO O

BaseOO

O OHPO S

BaseOO

O OHPO BH3

BaseO

O

O OPO O

BaseOO

O FPO O

BaseOO

O OPO O

BaseO

A

BUnmodified RNA Phosphorothioate RNA Boranophosphonate RNA

2'-O-methyl RNA 2'-deoxy-2'-fluoro RNA Locked Nucleic Acid

16

siRNA segments connected by disulphide bonds. By using this PEI delivery system, they were

able to achieve significant knockdown in vitro of red fluorescent protein (RFP) in RFP+

melanoma cells.115 Despite efficient transfection, any cell will non-specifically take up PEI and

other positively charged polymers. Since most nano-scale formulations naturally accumulate in

the liver,116-117 many strategies deliver therapeutics against diseases of the liver.117-118 In order to

target other tissues, the positively charged polymer must be shielded until it reaches the tumor

site.

To temporarily shield their positive surface charge, cationic nanoparticles are modified with

sheddable poly(ethylene glycol) (PEG) coronas using various stimulus-responsive coupling

strategies. For example, Li et al. developed a polymeric nanoparticle that is responsive to matrix

metalloproteinase 7 (MMP-7), an enzyme that is overexpressed by breast cancer cells and found

at high concentrations in the tumor microenvironment.119 The nanoparticle corona is composed

of a PEG block linked by an MMP-7 cleavable peptide to a cationic block. When the

nanoparticle reaches the tumor microenvironment, extracellular MMPs cleave the peptide,

shedding the PEG layer and exposing the cationic layer, raising the zeta-potential of the

nanoparticle from +5.8 to +14.4 mV and increasing cellular internalization 2.5-fold.

Nanoparticles pre-treated with MMP-7 resulted in significant knockdown in vitro; however, this

system was not studied in vivo, so it is still unclear whether this strategy will result in improved

biodistribution.119 Despite the shielded cationic charge, significant toxicity was observed at high

nanoparticle:siRNA ratios, underlining the importance of nanoparticle safety to their utility.

Targeting ligands can be attached to polymeric delivery vehicles to increase the specificity of

cellular uptake. These specifically bind receptors overexpressed on cancer cell membranes,

facilitating receptor-mediated endocytosis of the nanoparticle.120 Interestingly, the MMP-7

responsive nanoparticle, previously discussed,119 was conjugated to folate ligands.121 In this case,

PEG cleavage was triggered by MMP-7 at the tumor site, exposing folate-conjugated

nanoparticles for receptor-mediated endocytosis. In vitro experiments, including MMP-7 pre-

treatment and folate ligand competition assays, revealed that knockdown was dependent on both

MMP-7 activity and folate receptor binding. Under optimal conditions, the formulation achieved

significant luciferase protein knockdown with no detectible cytotoxicity in a luciferase positive

breast cancer cell line.121

17

Antibodies can also be conjugated to nanoparticle formulations for targeted siRNA delivery,

triggering internalization via a receptor-mediated endocytosis pathway.122 Palanca-Wessels et al.

synthesized a nanoparticle in which siRNA was encapsulated and to which anti-human epidermal

growth factor receptor 2 (HER2) antibodies were conjugated for cellular internalization.123

Delivery of siRNAs against a variety of chemotherapy resistance-associated mRNAs resulted in

significant target gene knockdown in vitro and in vivo in a mouse model of ovarian cancer.

However, the authors noted a slight immune response in some of the streptavidin-containing

control groups.123-124

1.4.2 Lipid-based formulations

One of the most common delivery methods for siRNAs and AONs is lipid-based formulations,

which incorporate bulky alkyl chain ‘tail’ groups with small, hydrophilic ‘head’ groups that often

incorporate at least some cationic components in order to enhance cellular uptake and endosomal

escape.125 Additional components that are often included in lipid-based formulations are

cholesterol, which can associate with lipid bilayers;126-127 PEGylated lipids, which prevent

aggregation and off-target uptake;128 and targeting ligands to enhance uptake in the target

tissue.129

Lipid-based materials were one of the first materials investigated for siRNA delivery, and

include formulations such as liposomes, micelles, microemulsions, and solid lipid

nanoparticles.84 One of the most well-known examples of a lipid nanoparticle formulation of

siRNA is Patisiran, a liposomal formulation of siRNA to treat hereditary transthyretin

amyloidosis that marks the first siRNA drug to achieve FDA approval.130

To optimize delivery of nucleic acids, the pKa of an ionizable lipid should be low enough that it

is relatively neutral during circulation (to avoid off-target uptake and minimize toxicity), but

high enough that it is protonated in the early or late endosomes.131 Endosomal escape is induced

by the protonation of the lipid head group and subsequent association with the negatively

charged plasma membrane.132 Jayaraman et al. conducted a study of 53 different ionizable lipids

and their effects on siRNA delivery and gene silencing in vivo. They determined that the

silencing efficacy reached an optimized point around an average pKa of 6.44.133

18

The optimum amount of cholesterol incorporated in the lipid formulation depends on the system,

but some studies have shown that the rate of drug release depends on having at least a minimum

amount of cholesterol incorporated into the lipid bilayers.134 Zhang et al. achieved optimal

siRNA delivery using a cholesterol:lipid ratio of 1:1 or 1:2 (30-50% cholesterol) in a liposomal

formulation.135

Interestingly, Zhang et al. also found that PEGylation of liposomes decreases transfection

efficiency, perhaps due to steric hinderance of the bulky PEG preventing the lipids from

interacting with cell membranes.135 Bao et al. further investigated the effect of PEGylation on

lipid nanoparticle systems and discovered that an optimal level of siRNA-mediated gene

silencing was obtained at approximately 0.5 mol% PEG, which was enough to limit toxic side

effects of the lipid nanoparticle while maintaining a high level of gene silencing.128

Targeting ligands may also be added to lipid formulations to enhance their on-target effects. For

example, Akinc et al. investigated the use of the lipoprotein ApoE, which targets liver cells, for

delivery of siRNAs to hepatocytes in vivo.136 They confirmed the effect of their formulation by

delivering the ApoE-targeted formulation in ApoE-knockout mice and observing a strong degree

of gene silencing, as opposed to wild type mice where they observed less than 20% silencing at

the same dose.136 Other targeting moieties that have been investigated for lipid formulations are

Vitamin A, which is shuttled to hepatic cells,137 and N-acetylgalactosamine (GalNAc), a small

molecule that has been investigated for delivery to the liver because of its ability to bind to the

asialoglycoprotein receptor displayed on the surface of hepatocytes.136 Importantly, most

targeted liposomal formulations have involved binding receptors in the liver, which is probably

because most lipid-based formulations naturally accumulate in the liver and it is a major

challenge to achieve biodistribution of these formulations to other organs.117

Although most of these examples involve siRNA delivery, liposomal formulations have also

been investigated for the delivery of AONs. Wyrozumska et al. used a cationic lipid formulation

incorporating the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as well as

cholesterol and PEGylated lipids to deliver AONs against a gene BCL-2 that is overexpressed in

leukemia cells, and observed improved biodistribution of the PEGylated carriers and a prolonged

survival in the mouse model of leukemia.138

19

1.4.3 Virus-inspired formulations: cell penetrating and membranelytic

peptides

Cell penetrating peptides (CPPs) have been exploited to bring oligonucleotide cargo into cells.

The CPPs are typically < 40 amino acids, cationic, and viral-derived.139 There have been many

reviews focused on the characteristics and mechanisms of CPPs139-141 and while there are

numerous CPP sequences (Table 1), they usually lack specificity as they will cross any cell

membrane. While the internalization pathways of most CPPs are not well-defined, internalization

is initialized via interactions of the cationic CPPs with the phospholipids of the cell

membrane.142

Table 1. Common cell penetrating sequences.

Name Sequence Origin Chargec

TAT (48-60)143-

144

GRKKRRQRRRPPQ Derived from HIV type 1 +8

Penetratin145 RQIKIWFQNRRMKWKK Antennapedia homeodomain +7

TP10 146-147 GWTLNS/AGYLLGKINL

KALAALAKKILa

Neuropeptide galanin-

mastoparan fusion

+4

VP22148 NAKTRRHERRRKLAIER Herpes simplex virus +7

Polyarginine149 Rna, n=8-9 Engineered for positive

charge

+8 or +9

Pep-1150 KETWWETWWTEWSQP

KKKRKVb

Fusion of NLS from simian

Virus 40 and reverse

transcriptase of HIV-1

+3

CADY151 GLWRALWRLLRSLWRL

LWRAb

Derived from PPTG1 peptide,

addition of W and charged

amino acids

+5

20

a C-terminal amide. b C-terminal cysteamide. c pH 7.4.

To achieve greater specificity of CPPs, one of three strategies is typically employed: (1)

triggering CPP deprotection at the tumor site; (2) local delivery of the CPP to the tumor site; or

(3) conjugation to cell-targeting ligands. Using the first strategy, Sun et al. synthesized a

polyarginine CPP, used for siRNA complexation and siRNA release, sandwiched between a

hydrophobic poly(caprolactone) (PCL) block and a hydrophilic PEG block.152 The PEG corona

was conjugated to the CPP through 2-propionic-3-methylmaleic anhydride linkers, which are

cleavable under the acidic environment of the tumor site, deshielding the CPP and facilitating

cellular uptake in vitro and in vivo. For the second strategy of local CPP delivery, Kanazawa et

al. used an intranasal delivery route to carry siRNA directly to the brain in a mouse model of

brain cancer using a CPP-nanoparticle formulation.153 PCL nanoparticles were conjugated to a

TAT CPP and hydrophilic PEG. The authors were able to use the TAT peptide for siRNA

complexation and delivery. For the third strategy, conjugating CPPs to cell targeting peptides can

increase their specificity. Fang et al. conjugated the TAT CPP to A1, a peptide with high affinity

for vascular endothelial growth factor receptor-1 (VEGFR1) and demonstrated selective delivery

to tumor cells overexpressing VEGFR.154 Similarly, R9 can be fused to a cyclic arginine-glycine-

aspartic acid (cRGD) peptide for targeting.155

Another use for viral-derived CPPs is endosomal escape of oligonucleotide formulations.

Endosomolytic peptides destabilize membranes when a critical concentration of the peptide is

located near a membrane, followed by interaction with the negatively charged phospholipid

bilayer and pore formation. The resultant pore then destabilizes and disrupts the membrane.156

For example, Cheng et al. synthesized virus-inspired polymers for endosomal release by grafting

‘caged’ melittin, a membranolytic peptide, through a disulfide bridge to a polymer containing a

hydrophilic cationic block for therapeutic loading and a pH-sensitive block for the triggered

display of the melittin peptide.157 The authors demonstrated greater transfection efficiency in

vitro and in vivo relative to commercially available reagents.

1.5 Protein conjugates as delivery vehicles

Recently, there have been several examples of smaller biomolecules conjugated to siRNAs or

AONs for targeted delivery to tissues. One of the most clinically advanced examples is siRNA

21

directly conjugated to GalNAc (a sugar derivative that is taken up by liver cells), a product from

Alnylam that is close to FDA approval.158 These small conjugates have advantages over large,

cationic nanoparticles including reduced immune response, specificity of targeting, and

monodispersity of particle size.84 It has also been demonstrated that the stability of nucleic acid

material against nucleases can be enhanced by conjugation to biomolecules such as globular

proteins.97 This section will provide a review of current delivery strategies for nucleic acids

using proteins including antibodies and toxins, as well as some background on antibody-drug

conjugates (ADCs) and engineered toxins for other therapeutic applications.

1.5.1 Antibodies for drug and oligonucleotide delivery

One of the most common ways of targeting therapeutics to specific cell types is by using

antibodies or antibody fragments (Fabs).159 Antibodies are proteins that are produced by the

immune system to be highly specific for antigens that are displayed on the surface of cells,

making them ideal candidates for targeted drug delivery with limited off-target binding.160

Antibodies can be engineered to bind to specific antigens, expressed using mammalian protein

expression systems, and functionalized via expression as fusion proteins or chemical

conjugation.161 Most of the work exploring antibodies for drug delivery has been as conjugates

between antibodies and small molecule drugs. This section will include a brief overview of

bioconjugation strategies that are applicable to antibodies as well as other types of proteins; some

key examples of antibody-drug conjugates; and some recent work with antibody-oligonucleotide

conjugates.

1.5.1.1 Bioconjugation strategies

There are abundant options available for conjugating a protein including (but not limited to): (1)

lysine modification via NHS chemistry; (2) reacting a native or engineered cysteine, tyrosine,

tryptophan, or N-terminal residue; (3) introduction of a non-native amino acid; or (4) use of a

peptide-tagging sequence.162 The selection of the appropriate conjugation chemistry requires

consideration of site-specificity, native amino acid availability, and stability of an engineered

protein.

(1) Lysine or N-terminal amine modification

22

One of the most widely-used bioconjugation strategies is modification of lysine residues. Most

proteins contain many available lysine residues - for example, an antibody usually contains at

least 20 free lysines for modification.163 Typically this is done using reactive N-

hydroxysuccinimide (NHS) esters, although there are numerous other amine-reactive functional

groups that can be chosen.163 NHS esters are relatively unstable in aqueous solutions so must be

quickly used, especially at basic pH,164 but the adduct that forms after reaction is a stable amide

bond with a half-life of at least 7 years in water.165 There are some strategies that can specifically

target the N-terminal amine for modification,166 but in most cases this modification is not site-

specific and several modifications will be introduced in a single protein, so care must be taken to

avoid labeling the active site of the protein of interest.

(2) Reacting cysteine, tyrosine, tryptophan

If site specificity is required, one of the options to achieve limited and specific functionalization

is targeting an amino acid that is less abundant on the native protein, or engineering a protein

with a unique residue for functionalization, such as a cysteine, tyrosine, or tryptophan. Cysteines

are a common option for site-specific modification because they are readily reactive but their

abundance in protein structures is low.167 Available cysteines can be modified using reactive

groups such as maleimides or activated thiols (

Figure 1.6A).168 Proteins, such as antibodies, can be reduced to yield free thiols for

modification;169 however, caution must be taken to avoid destabilizing the protein, inducing

HSN

O

O

+R R N

O

O

S

Protein

HONO2

N2

+R O

NH

R

HNRO2C

N2

Ph+

Dirhodium Peptide Cat.HN

RO2C

Ph

A

B

C

23

aggregation, or reducing essential disulfide bridges of the protein. Other amino acids with

relatively low abundance that can be modified include tyrosine, which can be reacted with

diazonium salts;170 and tryptophan, which can be modified through metallopeptide catalyzed

reactions (

Figure 1.6B,C).171

Figure 1.6. Reactions of native amino acids for site-specific protein conjugation. A) Thiols

can be modified through reactions with maleimides. B) Tyrosine can be reacted using

HSN

O

O

+R R N

O

O

S

Protein

HONO2

N2

+R O

NH

R

HNRO2C

N2

Ph+


RO2C

Ph

A

B

C

HSN

O

O

+R R N

O

O

S

Protein

HONO2

N2

+R O

NH

R

HNRO2C

N2

Ph+


RO2C

Ph

A

B

C

24

diazonium salts. C) Tryptophan can be modified through metallopeptide-catalyzed

reactions.

(3) Introduction of non-native amino acid

When engineering proteins, it is possible to introduce a non-native amino acid through the use of

a unique codon and introduction of the corresponding transfer RNA/synthetase pair.172 This

strategy can be used to introduce site-specific functional handles for bioorthogonal conjugation.

For example, moieties such as ketones,173 azides,174 alkynes,175 and anilines176 have been added

to proteins in this manner, and used for applications including labeling of live cell surfaces177 and

photocrosslinking.178 This strategy can yield highly specific conjugations for diverse

applications, but engineering the expression of a protein is a non-trivial process and care must be

taken to avoid destabilizing or deactivating the protein of interest.

(4) Use of a peptide-tagging sequence

Another site-specific method for introducing modifications to proteins is through the use of a

peptide-tagging sequence that an enzyme will recognize and label with the desired functional

group following protein expression. This method is particularly effective if the protein of interest

is present in a complex mixture but specific labeling is required.162 Some options for enzymatic

tagging via a peptide sequence include: biotin ligase, an enzyme that recognizes a peptide

sequence to biotinylate a specific site;179 transglutaminase, an enzyme which recognizes a

substrate sequence, and catalyzes a transamination reaction to introduce the desired

functionalization;180 lipoic acid ligase, which has been used to introduce non-native functionality

such as alkyl azides at the site of an acceptor peptide;181 and sortase, which recognizes one

peptide sequence on the protein of interest and another on the group to be conjugated and

mediates ligation.182

In addition to considerations of the site of protein conjugation, there is also the selection of linker

chemistry. One option that yields highly efficient reactions, amenable to aqueous conditions,

with limited or no by-products is conjugation via click chemistry.183 The relative rates of several

bioorthogonal click chemistry reactions are shown below. The selection of the chemistry requires

considerations including hydrophobicity of the reactive groups, available functional sites, and the

required reaction rate.

25

Table 2. Rates of selected bioorthogonal click chemistry reactions.183

Reaction Rate (Approx) (M-1s-1)

Azide-strained alkyne 10-1 – 10-2 (0.067)

Copper-mediated azide-alkyne 10-200

Maleimide-furan 10-5

Tetrazine-norbornene 106

(Oxime or hydrazide)-(aldehyde or ketone) 2-20

1.5.1.2 Antibody-drug conjugates

ADCs are conjugates between antibodies that bind to cell surface antigens and chemotherapy

drugs. An antibody could be an ideal delivery vehicle for drugs due to its favorable

pharmacokinetic profile, with a typical elimination half-life of 18-21 days for IgGs.184 The

promise of an ADC is a highly specific and selective delivery combined with the potency of the

conjugated drug,185 but ADCs have encountered some limitations in clinical trials due to the

relatively low potency of chemotherapeutics.186 Four antibody-drug conjugates have reached

FDA approval: three for hematologic cancers and one to treat breast cancer.187 The first ADC to

reach clinical approval was gemtuzumab ozogamicin (GO), a CD33-antibody delivering

calicheamicin, a DNA-damaging drug.188 Post-approval, GO encountered some roadblocks due

to off-target toxicity, but was later re-introduced to the clinic following optimization of

pharmacokinetic and safety profiles.188 More recently, a HER2-antibody (trastuzumab) carrying

emtansine, a cytotoxic chemotherapeutic, obtained FDA approval for breast cancer treatment;189

one of the most significant findings from this ADC was that significant efficacy was only

observed if the patients were carefully selected for high HER2-expression in the tumor tissue.189

In pre-clinical and clinical studies, antibodies-drug conjugates have been explored for numerous

other solid tumors including GBM.190 The majority of antibody-drug conjugates for GBM

treatment have been using an antibody against either EGFR, a receptor tyrosine kinase which is

26

expressed in the majority of primary GBMs, or the variant EGFRvIII, which is expressed in 25-

50% of GBM patients.191-193 Interestingly, there is some evidence that EGFRvIII may be

expressed in the GSC population.194 Many initial studies that focused on targeting GBM with

unmodified EGFR or EGFRvIII antibodies have yielded disappointing results.190, 195 Recently,

ABT-414, comprising an EGFR monoclonal antibody conjugated to a cytotoxic payload

monomethyl auristatin F (MMAF) underwent a three-arm clinical trial and resulted in a

progression-free survival (PFS) of 30% at six months, which could be a significant improvement

over the standard of care which generally yields a PSF of 10-20%.196 A clinical trial to compare

ABT-414 directly to ABT-414 combined with TMZ or TMZ alone is currently underway for

EGFR amplified GBM patients (NCT01800695).

1.5.1.3 Antibody-oligonucleotide conjugates

Since ADC efficacy has encountered limitations due to the low potency of many

chemotherapeutic drugs, as well as off-target cytotoxicity, a potential way forward is to

conjugate antibodies to highly specific and potent antisense therapeutics such as AONs or

siRNAs.

One of the first examples of an antibody-siRNA conjugate was via avidin-biotin conjugation:

Xia et al. conjugated streptavidin with an antibody against the human insulin receptor (HIR) and

combined this system with a biotinylated siRNA against luciferase, observing gene silencing at

low nanomolar concentrations.197 In vivo, they were able to observe luciferase silencing in

multiple organs including the brain.198 Following a similar biotin-avidin approach, Shankland et

al. used an antibody against podocytes in the kidney and conjugated the antibody to a positively

charged protamine molecule via neutravidin-biotin conjugation. siRNA against two relevant

targets for kidney disease, nephrin and transient receptor potential channel 6 (TRPC6), was

complexed to the positively charged protamine for delivery and significant gene silencing was

observed in vivo.199 Bäumer et al. conjugated the same protamine molecule directly to an anti-

EGFR antibody for complexation with an anti-KRAS siRNA and demonstrated that the therapy

induced apoptosis and slowed tumor growth in an in vivo model of colon cancer.200

Some potential limitations of antibody-siRNA conjugates were explored by Siebel et al., who

have developed a platform technology for the production of antibody-siRNA conjugates via

cysteine conjugation and generated a variety of these bioconjugates with different siRNA and

27

antibody targets.201 This led to several key findings about antibody siRNA conjugates: first, they

used both cleavable and non-cleavable linkers and observed no difference between the silencing

activity between the two types of linkers, indicating that release from the protein is not essential

for siRNA activity following delivery. Second, they discovered that only some combinations of

antibodies and cell lines could lead to efficient siRNA delivery and gene silencing, so the

antibody-siRNA platform was not universally applicable. Third, they observed a significant

amount of overlap between the delivered antibody-siRNA conjugate and the endolysosomal

pathway, and hypothesized that an enhanced endosomal escape mechanism could lead to a

higher degree of gene silencing. They used this information to develop an ‘optimized’ antibody-

siRNA conjugate for delivery of a lethal siRNA to prostate cancer xenografts and observed a

significant reduction in tumor growth.201

Despite some limitations of antibody-oligonucleotide conjugates, antibodies continue to be

explored for delivery of both AONs and siRNAs. Sugo et al. developed an antibody-siRNA

conjugate targeted to cardiac and skeletal muscles and were able to downregulate myostatin in

vivo, which translated into a recovery of movement ability.202 Satake et al. developed an

antibody-AON conjugate where the antibody was targeted to a leukemia target, CD22, for the

AON-mediated knockdown of a novel target MAX dimerization protein 3 (MXD3) and observed

a significant improvement in survival time in a mouse model of leukemia.203 In one of the most

recent examples, Wang et al. developed an antibody-siRNA conjugate for the treatment of

malignant melanoma, targeting the oncogene livin, and observed significant silencing in vivo.204

These diverse examples of antibody-siRNA and -AON conjugates and their efficacy in different

tissues exemplify the promise of antibody conjugates as truly targeted therapies where delivery is

not limited to the clearance organs. I explored this strategy for AON delivery as described in

more detail in Chapter 2.

1.5.2 Engineered toxins for biomolecule delivery

“AB” toxins, including toxins such as diphtheria toxin (DT), anthrax toxin (AT), and

Pseudomonas exotoxin A (PE), comprise two main subunits: The “A” domain, which is the

active toxin domain that is highly lethal to cells, and the “B” domain, made up of a receptor

binding component (R) and a translocation component (T).205 These toxins are efficient at

binding to the target cells, inducing endocytosis, and inserting the toxin domain into the

28

cytoplasm.206 These characteristics, combined with the recent expansion of protein engineering

techniques, make the AB toxin class an intriguing candidate as both targeted cytotoxic agents

and as vehicles for delivery of other drugs.

DT, AT, and PE all have slightly different cellular entry mechanisms (Figure 1.7) but they all

bind to an extracellular receptor and are eventually translocated into the cytosol. All three toxins

bind to a target receptor, induce endocytosis, and undergo cleavage by an enzyme called furin in

the endosomes; however, the protein domains of DT and PE remain linked by an essential

disulfide bond.207-208 PE induces trafficking to the endoplasmic reticulum where the disulfide

linkage is broken by protein disulfide isomerase (PDI) before it undergoes retrograde

translocation into the cytosol.209 Translocation of DT and AT occurs at the reduced pH of the

early endosome, and while the DT creates a flexible α-helical pore that is highly permissive to

different types of cargo,210 AT creates a rigid β-barrel pore that may require the complete

unfolding and refolding of the cargo.211 The disulfide bond of DT is cleaved after translocation

by intracellular enzymes.206 Each of these toxins has been exploited and re-engineered for the

targeted killing of diseased cells, or as attenuated delivery vehicles for diverse cargo, usually of

other therapeutic proteins. This section will provide some key examples of drug delivery using

these AB toxins.

29

Figure 1.7. Cellular entry mechanisms of three “AB” toxins: diphtheria toxin, anthrax

toxin, and Pseudomonas exotoxin A. The R domain of DT binds to heparin-binding

epidermal growth factor (HBEGF) and induces internalization to the early endosomes

where the connection between the T and A domain is cleaved by furin, and the A domain

translocates out of the early endosome through a flexible α-helical pore. The protective

antigen (T) domain of AT is cleaved by extracellular furin, and oligomerizes on the cell

surface, binding to the tumor endothelial marker-8 (TEM-8) or capillary morphogenesis

gene-2 (CMG-2) surface receptors. This leads to association with the A domain and

subsequent internalization into the early endosomes where A domain translocations

through a rigid β-barrel pore. Finally, the R domain of PE binds to the cell surface

receptor Low density lipoprotein receptor-related protein 1 (LRP1), and the connection

30

between the T and A domain is cleaved by furin in the early endosomes. PE is then

trafficked to the endoplasmic reticulum where it undergoes retrograde translocation.

1.5.2.1 Targeted toxins as cytotoxic agents

One therapeutic strategy that utilizes toxins is by targeting the native toxic domain to a specific

cell-surface receptor. The modification of AT presents a significant challenge as alteration or

removal of the R domain can destabilize the protein.212 However, DT and PE are both amenable

to alteration or removal of the R domain and re-engineering of the protein structure to target

different cell surface receptors.

There are several examples of PE-based immunotoxins in the clinic, including moxetumomab

pasudotox, a PE fragment fused to an antibody fragment against CD22 for treatment of

lymphoma, which recently demonstrated positive results in a phase I clinical trial.213 Similarly, a

PE immunotoxin targeting CD25 for lymphoma and leukemia treatment recently underwent an

initial clinical trial and achieved complete remission in at least one leukemia patient.214 There is

at least one clinically approved therapeutic based on the delivery of the DT toxic domain:

denileukin difitox (Ontak) which is a DT variant targeted to the IL-2 receptor that has shown

significant benefit to cutaneous T-cell lymphoma patients.215 In clinical studies, Resimmune,

which combines the A and T domains of the DT with antibody fragments against CD3, has

shown promising effects in a clinical trial for lymphoma patients, including four complete

remissions (16% of patients).216 Interestingly, DT variants have also been investigated for GBM

treatment. This is an exciting strategy because DT has the potential to naturally cross the blood

brain barrier.217 The DT A and T domains were fused with an antibody fragment targeting EGFR

and EGFRvIII for targeting GBM and a robust anti-tumor efficacy was observed in an orthotopic

in vivo model of malignant glioma, prompting FDA approval of a phase I/II clinical study that is

currently underway.218 Taken together, these examples highlight the capacity of DT and PE for

targeted therapeutic delivery tailored to virtually any desired cell surface receptor.

1.5.2.2 Attenuated toxins for therapeutic delivery

31

More recently, some researchers have shifted to taking advantage of the receptor binding and

translocation components of AB toxins to deliver desired cargo into cells, while the toxic domain

is attenuated or completely removed.210, 219 This has most commonly been done with AT, and

one of the first examples was from Arora et al. where they removed the native AT toxic domain

and replaced it with the toxic domains of shiga and diphtheria toxin to show that the modified

protein still had cytotoxic effects on cells.220 Delivery of more diverse cargo soon followed, with

Ballard et al. using attenuated AT to delivery cytotoxic T-cell epitopes into mammalian cells,

indicating that the disarmed toxin fragment could deliver cargo other than AB toxin domains.221

More recently, non-canonical cargo including antibody mimics, polypeptides, and

chemotherapeutic drugs have been delivered into cells using the attenuated AT as a delivery

vehicle. Liao et al. conjugated small antibody mimics including monobodies and affibodies to

the attenuated AT for the perturbation of intracellular protein-protein interactions.222 Using

functional readouts including intracellular protein disruption and apoptosis, they confirmed that

delivery was more effective than a comparable vehicle such as a cell penetrating peptide.222

Shortly thereafter, Rabideau et al. demonstrated that AT could deliver linear peptides and small

molecule drugs including doxorubicin and MMAF to the cytosol of cells and that these remained

intact and functional following translocation.223 However, AT was not able to deliver cyclic

peptides or larger, polycyclic drugs such as docetaxel, perhaps due to the structural rigidity of the

AT translocation pore.223 Surprisingly, despite this limitation Dyer et al. were able to deliver

AONs and siRNAs to cells using disarmed AT and observed an impressive knockdown

efficiency of a proof-of-concept target, Syntaxin5; however, they have yet to evaluate the

functional effects of AT-mediated knockdown in vitro or in vivo.224

Some evidence indicates that DT might have a more permissive translocation pore. Auger et al.

demonstrated that DT could deliver intact, folded proteins directly into the cytosol by

conjugating DT to a fluorescent protein mCherry, which has high conformational stability and is

not likely to unfold.210, 225 In comparison, AT is not able to deliver mCherry into cells, likely

because it does not unfold prior to translocation.226 Auger et al. also optimized the linker length

between the cargo and the DT and determined that the appended cargo was invisible to the

translocation machinery with the correctly engineered construct.210 This strategy has been used

by Park et al. to deliver a therapeutically relevant protein, human purine nucleoside

phosphorylase (PNP), to rescue function in PNP-depleted cells.227 This is a novel treatment

32

modality for patients with inherited PNP mutations and could be extended for the delivery of

other essential enzymes involved in disease. Another potential advantage of DT as a delivery

vehicle is its reported ability to cross the blood-brain barrier (BBB) via HBEGF-mediated

transcytosis.228 As proof-of-principle, Gaillard et al. demonstrated that an attenuated DT

conjugated to horseradish peroxidase (HRP) could reach the brain tissue following intravenous

injection, whereas free HRP could not.217 Together, these studies imply that DT has a relatively

permissive translocation pore and can specifically target and cross the blood brain barrier, so it

has great potential as a drug delivery vehicle to brain disease including GBM. I explored

attenuated diphtheria toxin (aDT) for delivery of siRNA as described in Chapter 3.

Antibody-antisense oligonucleotide conjugate downregulates a key gene in glioblastoma stem cells

This chapter was published in Molecular Therapy - Nucleic Acids:

Arnold, A. E.; Malek-Adamian, E.; Le, P. U.; Meng, A.; Martínez-Montero, S.; Petrecca, K.;

Damha, M. J.; Shoichet, M. S. Antibody-Antisense Oligonucleotide Conjugate Downregulates a

Key Gene in Glioblastoma Stem Cells. Mol Ther Nucleic Acids 2018, 11, 518–527.

A.E.A. conceived and performed experiments and wrote the first and subsequent drafts of the

manuscript. E.M.-A. synthesized and characterized all oligonucleotide materials and provided

major revisions of the first draft. P.U.L. provided cell lines, advice, and feedback. A.M.

performed flow cytometry experiments. S.M.-M. conceptualized fundamental work on the

project and provided feedback on the manuscript. M.J.D., K.P., and M.S.S. secured funding,

supervised and guided the research, and participated in writing the manuscript.

2.1 Abstract Glioblastoma stem cells (GSCs) are invasive, treatment-resistant brain cancer cells that express

downregulated in renal cell carcinoma (DRR), also called FAM107A, a genetic driver of GSC

invasion. We developed antibody-antisense oligonucleotide conjugates to target and reduce

DRR/FAM107A expression. Specifically, we used antibodies against antigens expressed on the

glioblastoma stem cells, such as CD44 and EphA2, conjugated to chemically modified antisense

oligonucleotides (AONs) against DRR/FAM107A, which were designed as chimeras of DNA

and 2ʹ-deoxy-2ʹ-fluoro-beta-D-arabinonucleic acid (FANA) for increased nuclease stability and

33

mRNA affinity. We demonstrate that these therapeutic conjugates successfully internalize,

accumulate, and reduce DRR/FAM107A expression in patient-derived GSCs. This is the first

example of an antibody-antisense strategy against cancer stem cells.

2.2 Introduction Glioblastoma cancer stem cells (GSCs) are hypothesized to account, at least partially, for

treatment failures in aggressive glioblastoma multiformes.229-233 The GSCs are highly

invasive,234-235 resistant to radiation and conventional chemotherapy,236-237 and have the capacity

to initiate new tumor growth.232, 238

Downregulated in renal cell carcinoma (DRR/FAM107A)63 is an established genetic driver of

GSC invasion. It acts by regulating focal adhesion dynamics at the leading edge of migrating

cells64 and by activating Akt signaling, making it an ideal anti-invasion target.64, 66, 239

Antisense oligonucleotides (AONs), which are short (15-21 nucleotide) strands of DNA, are

potent regulators of gene expression.93 There are several clinically approved antisense

oligonucleotide therapeutics, including Eteplirsen, a treatment for muscular dystrophy,240 and

Nusinersen, which is used in the treatment of spinal muscular atrophy.83

Herein, we engineered antisense oligonucleotides (AONs) for DRR/FAM107A knockdown in

patient-derived GSCs. The AON is stabilized against nuclease degradation by substituting some

of the DNA residues for 2ʹ-deoxy-2ʹ-fluoro-beta-D-arabinonucleic acid (FANA) residues,

thereby increasing treatment longevity while maintaining potency.89, 241 We designed a “gapmer”

AON with FANA modifications flanking the DNA core because FANA gapmers bind target

mRNA with high affinity and elicit mRNA degradation while protecting the 3ʹ-end from

exonuclease degradation.242 This construct also includes a phosphorothioate (PS) backbone in

lieu of the naturally-occurring phosphodiester (PO) backbone for added nuclease resistance and

effective RNaseH-mediated cleavage of mRNA.243-244 Given that PS-DNA chemistry may

increase immunostimulation, we incorporated a 2ʹ-deoxy-5-methyl-cytidine at a CpG motif

within the AON sequence to obviate this issue in future studies.245-246

Antibody targeting is a powerful tool to guide therapeutic delivery to specific cell types, both as

pendant groups on larger nanoparticle systems247-250 and as direct antibody-drug conjugates

(ADCs).251-255 In glioblastomas, administration of an anti-EGFR antibody-monomethyl auristatin

34

F ADC has resulted in a survival benefit for EGFR-amplified glioblastoma patients256 whereas

administration of an anti-EGFR antibody alone failed.257 To mediate AON delivery, we chose to

investigate three monoclonal antibodies (mAbs) against three GSC surface markers: CD44, a

neural stem cell marker and a marker of GSCs within the glioblastoma tumor;258-260 EphA2, a

key component of cell-cell signaling that is overexpressed in many cancers and also in GSCs;42

and EGFR, which is amplified in 40-60% of all glioblastomas.261

We developed an antibody-conjugated double-stranded antisense oligonucleotide (dsAON)

therapeutic using click chemistry between an azide-modified antibody and an alkyne-modified

dsAON with a unique architecture: the sense strand is modified at the 5ʹ and 3ʹ ends with

functionalities for click chemistry (dibenzylcyclooctyne, DBCO) and for imaging (Cyanine 3

fluorophore, Cy3), respectively. While the sense strand is covalently conjugated to the antibody,

the antisense strand is conjugated only via hybridization to this sense strand to facilitate its

release once inside the cells. Direct conjugation of large molecules such as antibodies to the

antisense strand can interfere with RNase H recognition of the corresponding AON:mRNA

duplex.262 This approach is an improvement over the few antibody-antisense therapeutic

conjugates reported, with the majority of these being non-covalent, cationic complexation97 or

disulfide linkages directly to the antisense strand, which are unstable and prone to

degradation.263-264 Effective delivery and gene knockdown using antisense oligonucleotides has

been demonstrated with dsDNA systems where the DNA sense strand is left unmodified and as

such susceptible to nuclease-mediated digestion by endogenous cellular enzymes.265 This

degradation is the necessary driving force to release the antisense strand, which is then available

for hybridization with the target mRNA.265

We chose to use antibodies engineered by phage-display in order to target the glioblastoma stem

cells.266 Based on immunocytochemistry data, we identified CD44 and EphA2 as the best

candidates for AON delivery. We then verified that these antibodies were internalized upon

binding prior to conjugating them to the dsAONs via click chemistry. We found that the CD44

mAb-dsAON conjugate significantly reduced DRR expression, which correlated with a change

in cellular morphology. Thus, we can reduce expression of a key GSC target, DRR/FAM107A,

using antibody-dsAON conjugates and this provides a framework for antibody-AON conjugate

testing against GSCs in vivo.

35

2.3 Results

2.3.1 Antisense oligonucleotide activity

We tested four modification patterns of the DRR/FAM107A AON and compared these to a

scrambled sequence control (Figure 2.1) in order to maximize efficacy and reduce

immunogenicity: (i) Gapmer motif, consisting of flanking FANA modifications with a DNA

core; (ii) Altimer motif, where FANA substitutions were alternated every 3 nucleotides with

DNA;267 (iii) Gapmer-MeC with a 5-methylcytidine modification at the CpG motif (5MeCG) in

order to reduce immunogenicity; and (iv) Altimer-MeC with a similar 5MeCG modification

(Figure 2.1A). We observed significant DRR knockdown following transfection into DRR-

overexpressing (DRR+) U-251 MG glioblastoma cells with all single stranded antisense

oligonucleotides by western blot compared to the scrambled control (Figure 2.1B). Although not

statistically significant, we observed a trend towards greater knockdown when using Gapmer

MeC compared to Altimer MeC. Therefore, all future studies were carried out using the Gapmer

MeC strand.

We hybridized the therapeutically active Gapmer MeC antisense strand to a carrier sense strand

modified with a 3ʹ Cy3 for imaging studies and a 5ʹ DBCO for click chemistry (see melting

curves in Figure A.1). The Cy3 and DBCO functionalities were incorporated into the sense

strand during solid-phase oligonucleotide synthesis. We transfected this double stranded

antisense oligonucleotide into DRR+ GBM cells and demonstrated significant reduction of DRR

expression compared to a scrambled control Figure 2.1C). Representative western blots are

shown (Figure 2.1D).

36

Figure 2.1. Downregulated in renal cell carcinoma (DRR) expression is reduced following

transfection of DRR+ U-251 MG cells with DRR antisense oligonucleotides. (A)

Oligonucleotide sequences used: all antisense strands comprise a phosphorothioate

backbone while all sense strands are synthesized with a phosphodiester backbone. (B) DRR

expression following treatment with single stranded DRR AON sequences normalized to

untreated control. Data were analyzed using one-way ANOVA followed by Dunnett’s post-

hoc test compared to Scrambled group (data is shown as mean+SD, n=3, *p<0.05,

**p<0.01). (C) DRR expression following treatment with double stranded anti-DRR

oligonucleotide sequences normalized to untreated control. Data were analyzed using

unpaired t-test with Welch’s correction (data is shown as mean+SD, n≥4, ***p<0.001). (D)

Representative western blot showing single stranded and double stranded antisense

oligonucleotide DRR knockdown.

37

2.3.2 Co-expression in GSCs of DRR and antigens for CD44, EphA2, and EGFR antibodies

We screened the patient-derived GSCs for co-expression of DRR with potential antigens for

targeted delivery of the antisense oligonucleotides: CD44, EphA2 or EGFR. All of the GSCs

used in this study strongly co-expressed DRR with CD44 and EphA2 in all fields of view, yet

only weakly expressed EGFR (Figure 2.2). Thus, CD44 mAb and EphA2 mAb were pursued for

further study.

Figure 2.2. Patient-derived GSCs strongly co-express antigens CD44 and EphA2 with DRR

and weakly co-express EGFR with DRR. Representative confocal images are shown.

Antigens CD44, EphA2, and EGFR (green); DRR (red); cell nucleus (Hoechst, blue). All

scale bars are 50 µM.

2.3.3 Internalization of CD44 and EphA2 mAbs

We quantified the internalization of the CD44 and EphA2 mAbs at 15, 45, and 90 min using a

flow cytometry-based assay with DRR+ U-251 MG cells. We first incubated the cells with the

CD44 (Figure 2.3A), EphA2 (Figure 2.3B), or non-specific CTL mAb (Figure 2.3C) at 4 °C,

incubated this sample for the given time period at 37 °C in order for internalization to occur, and

38

then incubated the cells with a fluorescently tagged secondary antibody at 4 °C (see orange

curves in Figure 2.3A-C). We compared these to a control sample held at 4 °C for the entire

experiment, which would prevent internalization (see blue curves in Figure 2.3A-C). Cells in the

absence of antibody staining exhibited a low background fluorescence (see red curves in Figure

2.3A-C). We used median fluorescent intensity (MFI) to quantify internalization. Cells allowed

to internalize the antibody at 37 °C (orange curves) demonstrated a reduced MFI compared to

cells held at 4 °C (blue curves) due to a lower amount of cell surface receptor, indicating

internalization. The differences between the 15, 45, and 90 min time points for each mAb were

not statistically significant (Figure A.2). However, as we observed a trend towards greater

internalization at 45 min with both mAbs, the 45 min time point is shown for comparison in

Figure 2.3. At 45 min, we observed internalization of the EphA2 mAb and the CD44 mAb, and

they were both internalized significantly more than the CTL antibody (Figure 2.3D).

Figure 2.3. EphA2 mAbs and CD44 mAbs are internalized upon binding to cells. (A-C)

Flow cytometry analysis of cell surface receptor internalization following 45 min

incubation at 37 °C with antibodies (A) CD44 mAb and (B) EphA2 mAb compared to (C)

non-specific CTL mAb. Cells without antibodies added (cells only) and cells incubated with

antibodies, but held at 4 °C (no internalization) curves are shown as a comparison to those

cells that were allowed to internalize the mAb for 45 min at 37 °C (45 min internalization).

39

(D) Quantification of internalized receptor following 45 min incubation period for CD44

mAb and EphA2 mAb antibodies compared to CTL. Data were analyzed using one-way-

ANOVA followed by Dunnett’s post hoc test compared to CTL group (data is shown as

mean+SD, n=3, *p<0.05, ***p<0.001).

2.3.4 Synthesis of mAb-dsDRR and mAb-dsScrambled conjugates

For antibody-mediated delivery of DRR, we first modified each of CD44 and EphA2 antibodies

with an azide functional group by covalently bonding NHS-PEG4-N3 to one of the lysines

therein. The azide modification enabled click conjugation of the DBCO-modified sense

oligonucleotide to the antibody following hybridization with either the Gapmer MeC (dsDRR) or

a scrambled sequence (dsScrambled) (Figure 2.4A). We determined conjugation efficiency by

gel electrophoresis followed by ImageJ quantification to be 60% for dsDRR conjugation to both

CD44 mAb (Figure 2.4B) and EphA2 mAb (Figure 2.4C). We observed the appearance of two

bands for both the CD44 mAb-dsDRR and the EphA2 mAb-dsDRR conjugates (lane 2 of Figure

2.4B,C), which we expect is due to the conjugation of either one or two antisense oligonucleotide

strands per antibody. To confirm the absence of non-specific aggregation or adsorption, the

dsDRR was mixed together with either the CD44 or EphA2 mAb (without prior azide

modification of the antibodies) and no evidence of conjugation was detected (lane 3 of Figure

2.4B,C).

To further verify the conjugation of the antisense oligonucleotides to the antibodies, we reacted

azide-modified mAb with the DRR sense strand and this conjugate was analyzed by MALDI-

TOF mass spectrometry (Figure A.3). Experimental results closely matched the expected

conjugate mass (Mobt = 160.0 kDa vs. Mth = 160.2 kDa).

40

Figure 2.4. CD44 mAb and EphA2 mAb can be efficiently conjugated to dsDRR using click

chemistry. (A) Scheme of antibody modification with dsDRR. (B) 10% PAGE analysis of

CD44 mAb-dsDRR conjugation: Lane 1: dsDRR only; Lane 2: CD44 mAb conjugated to

dsDRR via NHS-PEG4-N3 crosslinker; Lane 3: dsDRR and CD44 mAb combined without

crosslinker present. (C) 10% PAGE analysis of EphA2 mAb-dsDRR conjugation: Lane 1:

dsDRR only; Lane 2: EphA2 mAb conjugated to dsDRR via NHS-PEG4-N3 crosslinker;

Lane 3: dsDRR and EphA2 mAb combined without crosslinker present.

2.3.5 DRR knockdown and cellular uptake of mAb-dsDRR conjugates

To determine the knockdown efficiency of the EphA2 mAb-dsDRR and CD44 mAb-dsDRR

conjugates, we incubated patient-derived GSCs with each formulation for 72 h at 150 nM and

compared to mAb-dsScrambled, dsDRR alone, and antibody alone controls. We quantified DRR

protein expression using western blot analysis normalized first to α-tubulin and then to a no

treatment control. The CD44 mAb-dsDRR conjugate significantly reduced DRR expression in

41

patient-derived GSCs compared to the CD44 mAb-dsScrambled control (Figure 2.5A,B). We

observed no knockdown with the negative controls (dsDRR or CD44 mAb alone).

Unexpectedly, treatment with EphA2 mAb-dsDRR conjugate did not reduce DRR expression

relative to any of the EphA2 mAb-dsScrambled, dsDRR, or EphA2 mAb controls (Figure 2.5C,

D).

Figure 2.5. DRR expression of patient-derived GSCs after treatment with CD44 mAb-

dsDRR or EphA2 mAb-dsRR (150 nM) normalized to untreated control. (A)

Quantification of DRR expression following CD44 mAb-dsDRR treatment. Data were

analyzed using one-way-ANOVA followed by Dunnett’s post hoc test compared to CD44

mAb-dsScrambled (data is shown as mean+SD, n≥4, *p<0.05). (B) Representative western

blot showing DRR knockdown following treatment with CD44 mAb-dsDRR. (C)

Quantification of DRR expression following EphA2 mAb-dsDRR treatment. Data were

analyzed using one-way-ANOVA followed by Dunnett’s post hoc test compared to EphA2

42

mAb-dsScrambled (data is shown as mean+SD, n≥3). (D) Representative western blot

showing DRR expression following treatment with EphA2 mAb-dsDRR.

To better understand this discrepancy between the knockdown observed for CD44 mAb-dsDRR

and EphA2 mAb-dsDRR, we examined cellular uptake with patient-derived GSCs. We analyzed

uptake of the conjugates by confocal microscopy of Cy3-labeled dsDRR after a 3h incubation

with the mAb-dsDRR conjugates at 75 nM and 150 nM (Figure A.4). At 150 nM, we observed

considerable uptake of CD44 mAb-dsDRR (Figure 2.6A) whereas we observed minimal uptake

of both EphA2 mAb-dsDRR (Figure 2.6B) and control, IgG-dsDRR (Figure 2.6C). This suggests

that the lack of knockdown observed for EphA2 mAb-dsDRR is correlated to a reduced cell

uptake of the EphA2 mAb-dsDRR conjugate.

To gain greater insight into the fate of the CD44 mAb-dsDRR conjugate, we incubated the

patient-derived GSCs with the CD44 mAb-dsDRR conjugate and a lysosomal marker for 2 h,

and then incubated the GSCs cells with fresh media for an additional 1 h to allow for complete

internalization. The dsDRR Cy3 signal almost completely co-localized with the lysosomal

marker (Figure 2.6D), suggesting that the majority of the conjugate was trafficked into the

endolysosomal pathway, likely with a small percentage trafficked to the cytoplasm to account for

the gene knockdown observed.

43

Figure 2.6. Antisense oligonucleotides conjugated to CD44 mAb are taken up by GSCs and

trafficked into the endolysosomal pathway. (A) Uptake of CD44 mAb-dsDRR (white

arrows) compared to (B) EphA2 mAb-dsDRR and (C) CTL-dsDRR after 3 h incubation at

37 °C. Control (CTL) is a non-specific human IgG. (D) Colocalization (white arrows) of

CD44 mAb-dsDRR with the lysosomal compartments following a 2 h pulse and 1 h chase.

Cell membrane (wheat germ agglutinin (WGA), magenta); cell nucleus (Hoechst, blue);

44

AON (Cy3, green); lysosome (Dextran647, red). Representative z-stack images shown. All

scale bars are 50 µM.

2.3.6 Cellular morphology following DRR knockdown

DRR knockdown often results in a change in cell phenotype from spindle- to rounder-shaped

cells, reflecting a less invasive morphology.64 While untreated GSCs have a spindle-shaped

morphology, we found that GSCs treated with CD44 mAb-dsDRR exhibit a rounder cell

morphology with fewer, shorter projections and centralized focal adhesions, suggesting reduced

invasive capacity (Figure 2.7A). We quantified this effect by measuring cell area per nucleus,

with actin staining delineating the cell membrane and normalized to an untreated control.

Although the cell area was variable due to the complex cell bundles, we observed a significant

decrease in cell area per nucleus with the CD44 mAb-dsDRR treatment relative to controls

including the CD44 mAb alone, dsDRR alone, and CD44 mAb-dsScrambled (Figure 2.7B).

45

Figure 2.7. GSCs treated with CD44 mAb-dsDRR have a rounder shape, fewer projections,

and centralized focal adhesions relative to the spindle-shaped cells of the control

treatments. (A) Cells treated with CD44 mAb-dsScrambled. (B) Cells treated with CD44

mAb-dsDRR. (C) Cells treated with CD44 mAb alone. (D) Cells treated with dsDRR alone.

Representative z-stack images shown. All scale bars are 50 µM. Cell nucleus (Hoechst,

blue); Actin (Phalloidin Alexa Fluor 488, green). (B) Change in cellular morphology is

quantified as actin area per cell normalized to a no treatment control. Data were analyzed

using one-way-ANOVA compared to CD44 mAb-dsScrambled with Dunnett’s post hoc

correction (data is shown as mean+SD, n≥3, ***p<0.001).

46

2.4 Discussion and conclusions Here we report, for the first time, an antisense oligonucleotide-antibody conjugate constructed

via covalent click conjugation through a non-therapeutic, sense carrier strand and show how each

component of the system is optimized for maximum knockdown of DRR.

The antisense therapeutic strand was optimized for effective knockdown first through fluoro

modification of specific oligonucleotides and then by 5-methylation of the CpG containing

oligonucleotide. FANA modification has been shown to stabilize the especially sensitive

flanking regions of the ASO while an unmodified gap consisting of 9 DNA nucleotides allows

for efficient endonucleolytic ribonuclease H activity.268 We found a trend towards more potent

DRR knockdown with the Gapmer motif compared to the alternating oligonucleotide FANA-

modified Altimer. CpG motifs trigger an innate immune response;245, 269 yet, methylation of the

cytosine residue in strands containing this motif can decrease this immune response, thereby

decreasing off-target effects.246, 270 While the immune response was not examined herein, with an

ultimate view of testing this strategy in vivo, we confirmed the activity of the methylated CpG

therapeutic strand and used it in all of the in vitro studies.

We chose a targeted antibody-mediated approach for antisense oligonucleotide delivery because,

like many polyanions, AONs do not readily cross the cell membrane, especially at low

concentrations.108 To determine the optimal antibody for targeted delivery, first we screened the

GSCs for antigen expression and then for antibody internalization. While CD44, EphA2 and

EGFR are all expressed in many patient-derived glioblastoma stem cells,42, 258, 261, 271 only CD44

and EphA2 were strongly co-expressed with DRR. Notably, EGFR amplification is often lost in

vitro, which correlates with our observations.272 By flow cytometry, it appeared that both CD44

and EphA2 mAbs were internalized by the GSCs; however, the extent of internalization did not

correlate with silencing efficacy. CD44 mAb-dsDRR more effectively knocked down DRR

expression than EphA2 mAb-dsDRR, which was corroborated by greater accumulation of

dsDRR in the GSCs when delivered with CD44 mAb. This may be due to slight differences in

internalization pathway or route of endosomal escape, which are difficult to observe using

established techniques. Our results highlight that the pathway or percentage of receptor

internalization does not necessarily correlate to silencing efficacy, which is consistent with

47

earlier reports;273 however, the increased uptake observed by fluorescence microscopy did

correlate with better knockdown.

To gain insight into the trafficking of the CD44 mAb-dsDRR, we used a pulse-chase experiment

and found that the internalized CD44 mAb-dsDRR colocalized with the lysosomes. While it is

recognized that antibody-oligonucleotide conjugates are typically trafficked into the

endolysosomal pathway,264, 273-276 the DRR knockdown observed demonstrates that some dsDRR

is trafficked into the cytoplasm. While the Cy3 signal associated with the duplex sense strand is

colocalized with the lysosomes, some of the antisense strand may have dissociated from the

sense strand and diffused into the cytoplasm. To further increase the potency of knockdown,

incorporation of moieties to prevent lysosomal accumulation and facilitate early endosomal

escape in order to optimize knockdown are being actively pursued.275

We show the first example of an antibody-antisense therapeutic conjugate used to modulate the

genetic expression of cancer stem cells. With sufficient dsDRR uptake, we observe both gene

knockdown and a change in cell morphology, consistent with a less invasive phenotype: we

observe a clear distinction from spindle-shaped cells without treatment to a more rounded

morphology with CD44 mAb-dsDRR treatment. This lays the foundation for future studies

where the goal will be to knockdown the DRR gene in GSCs in vivo and thereby reduce tissue

invasion and cell metastasis.

2.5 Materials and methods

2.5.1 Cell lines

GBM DRR+ cells (U-251 MG glioblastoma cells with stable transfection of DsredDRR fusion

protein) were cultured as previously described.64 Patient-derived GSCs were provided by the

Petrecca lab at McGill University following consent from the patients and approval by the

hospital ethics committee. GSCs were expanded as neurosphere in complete neurocult-

proliferation media (Neurocult NS-A Proliferation kit (Stem Cell 05751), 20ng/ml recombinant

EGF, 20ng/ml recombinant bFGF, and 2µg/ml heparin).

48

2.5.2 Antibodies

CD44 and EphA2 mAbs were provided by the Toronto Recombinant Antibody Centre

(TRAC).266 EGFR mAb (cetuximab) is a clinically available formulation (Erbitux 2 mg/mL, Eli

Lilly). CTL antibody (IgG from human serum) was purchased from Sigma and used as received

(Cat. No. I4506).

2.5.3 AON synthesis

DNA amidites and gene machine compatible reagents were purchased from ChemGenes and

used as received, and all DBCO-TEG (Cat No 10-1941) and Cyanine 3 CPG (Cat No 20-5913-

41) were purchased from GlenResearch. All antisense oligonucleotides were synthesized on an

Applied Biosystems (ABI) 3400 DNA synthesizer at 1 µmol scale using Uny-linker CPG as solid

support, except for the sense delivery oligonucleotide which utilized Cyanine 3 CPG as the solid

support. The synthesis cycle conditions were as previously described239 with the exception that

0.1 M I2 in 1:2:10 pyridine/water/THF was used for oxidation of antisense oligonucleotides, and

0.02 M I2 in 1:2:10 pyridine/water/THF was used for the Cy3/DBCO modified sense delivery

oligonucleotides. When a phosphorothioate backbone was needed, a 0.10 M solution of

((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiozaoline-3-thione (DDTT) in Py:MeCN

(9:1) was used for the oxidation step instead of the aqueous I2 solution. Deprotection and

cleavage from the solid support was achieved in pure aqueous NH4OH for 48 h at room

temperature. Purifications were performed by HPLC, using a Protein Pak DEAE 5PW analytical

anion-exchange column. A stationary phase of Milli-Q water and a mobile phase of 1 M LiClO4

in water was used for analysis and purification using a gradient of 0−50% over 46 min.

Following purification, excess LiClO4 salts were removed using NAP-25 sephadex size-

exclusion columns. The oligonucleotides were analyzed by LC-MS using a Dionex Ultimate

3000 coupled to a Bruker Maxis Impact QTOF in negative ESI mode. Samples were run through

an Acclaim RSLC 120 C18 column (2.2 uM 120A 2.1 x 50 mm) using a gradient of 98% mobile

phase A (100 mM HFIP and 5 mM TEA in H2O) and 2 % mobile phase B (MeOH) to 40 %

mobile phase A and 60% mobile phase B in 8 minutes. The data was processed and

deconvoluted using the Bruker DataAnalysis software version 4.1 (Table S1).

49

2.5.4 AON duplex formation

Sense and antisense oligonucleotide strands were annealed in annealing buffer (10 mM Tris, pH

7.5–8.0, 50 mM NaCl, 1 mM EDTA) by heating at 95 °C for 2 minutes followed by slow cooling

to room temperature over 1 h. The annealed oligonucleotides were stored at 4 °C until use.

Duplex formation was assessed by native PAGE in Tris/Borate/EDTA buffer followed by

imaging on a Typhoon FLA 9500 biomolecular imager.

2.5.5 Preparation of mAb-dsDRR and mAb-dsScrambled conjugates

mAbs were modified with NHS-PEG4-N3 (Thermo 26130) according to the provided protocol

from Thermo Fisher Scientific. Briefly, a 100 mM stock solution of NHS-PEG4-N3 was prepared

in dimethyl sulfoxide (DMSO, Sigma 472301). 10 eq. of this solution was added to the mAb in

PBS (Sigma D8537) and this reaction was shaken for 1 h at RT. After purification by dialysis for

24 h against PBS, 2 eq. of dsDRR or dsScrambled was added to the N3-modified mAb and this

reaction was allowed to proceed for 3 h at 37 oC. The resulting product was stored at -80 oC until

use or at 4 oC for up to 24 h. Conjugation efficiency was analyzed by 10% PAGE.

2.5.6 DRR knockdown assays - lipofectamine2000 transfection protocol

This protocol was adapted from Anzahaee et al.239 Briefly, DRR+ GBM cells were seeded at

120,000 cells/well in a 6-well plate and allowed to adhere overnight. Antisense oligonucleotides

(AONs) were complexed to lipofectamine2000 (Thermo 11668027, at 3 µL/well) for 20 minutes

in Opti-MEM media (Thermo 31985062) according to the lipofectamine2000 reagent protocol

and added to the cells in DMEM media (11995605) for a final DMEM:Opti-MEM ratio of

1.5:0.5 and AON concentration of 25 nM. At 24 h, an additional 1 mL DMEM media was added.

After a total of 72 hours of incubation at 37 oC, cells were collected and lysed using 0.1% NP-40

(Fluka 74385). Protein expression was assessed via western blot analysis of DRR with beta-

tubulin as a loading control.

2.5.7 DRR knockdown assays - treatment with mAb conjugate

GSCs were seeded at 250,000 cells/well in poly(l-ornithine), PLO (Sigma P4957) and laminin

(Thermo CB-40232) coated 6-well plates and allowed to adhere overnight in Neurocult NS-A

50

proliferation media (Stem Cell 05751). mAb conjugates (or AONs or mAb alone) were added to

500 uL Opti-MEM media and added to the cells for a final Opti-MEM:Neurocult ratio of 1.5:0.5

v/v and mAb-AON concentration of 150 nM. 24 hours later, an additional 1 mL Neurocult media

was added. After a total of 72 hours of incubation at 37 °C, cells were collected and lysed using

0.1% NP-40 (add company info). Protein expression was assessed via western blot analysis of

DRR with alpha-tubulin as a loading control.

2.5.8 mAb internalization assay

This protocol was adapted from Mumper et al.277 Briefly, the internalization of the CD44 and

EphA2 mAbs were determined after measuring surface levels of mAb after various incubation

periods at 37 °C. CD44 mAb at 4 µg/mL in 2% FBS (Sigma F1051) in PBS (FACS buffer) was

added to an equivalent volume of 1x106 cells in FACS buffer for one hour on ice at 4 °C. The

cells were then washed 3X in ice cold FACS buffer and suspended in 200 µL at 37 °C for the

indicated time. A control sample was held at 4 °C throughout the experiment. At the indicated

time point, cells were removed from 37 °C, quenched with 1 mL ice cold FACS buffer, and then

spun to a pellet and resuspended in 2 µg/mL goat anti-human IgG, Alexa Fluor 488 (Thermo A-

11013) in FACS buffer. Cells were washed 2X with ice cold FACS buffer, 1X with ice cold

PBS, and then fixed with 2% PFA (Bioshop PAR070) in PBS and stored at 4 °C until analysis on

a BD Accuri C6 flow cytometer. Internalized receptor was quantified using the median

fluorescence intensity calculated from FlowJo 10 software.

2.5.9 Immunocytochemistry (CD44, EphA2, and DRR)

GSCs were seeded at 20,000 cells/well in a PLO/laminin coated 8-well coverglass plate and

allowed to adhere overnight at 37 °C. The cells were then fixed using 4% PFA. Cells were

permeabilized using 1% Triton X-100 (Sigma T-9284) and then stained with either Epha2 mAb

or CD44 mAb together with an DRR antibody provided by the Petrecca lab at McGill University.

Secondary antibodies were anti-human IgG-Alexa Fluor 488 and goat anti-rabbit IgG-Alexa

Fluor 647 (Thermo A-21245). The cells were then counter-stained with Hoechst 33342 and

images were captured on an Olympus FV1000 confocal microscope.

51

2.5.10 Cellular uptake


allowed to adhere overnight at 37 °C. The cells were then treated with the indicated formulation

for 3 h at 37 °C, washed with PBS, and fixed using 4% PFA. The cells were stained with WGA-

Alexa Fluor 488 or 647 (Thermo W11261 or W32466), counterstained with Hoechst (Invitrogen

H1399), and imaged on an Olympus FV1000 confocal microscope.

2.5.11 Lysosomal accumulation


allowed to adhere overnight at 37 °C. The cells were then treated with the CD44 mAb-dsDRR at

150 nM and the lysosomal marker Dextran Alexa Fluor 647 (Thermo D22914) at 25 µg/mL for 2

h at 37 °C (pulse). The media was then replaced and the cells were incubated for an additional 1

h at 37 °C (chase), then washed with PBS and fixed using 4% PFA. The cells were then stained

with WGA Alexa Fluor 488, counterstained with Hoechst, and images were captured on an

Olympus FV1000 confocal microscope.

2.5.12 Cellular morphology following DRR knockdown


allowed to adhere overnight at 37 °C. The cells were then treated with the indicated formulation

for 72 h at 37 °C, washed with PBS, and fixed using 4% PFA. The cells were then stained with

mouse anti-vinculin antibody (Sigma V9131) and anti-mouse Alexa Fluor 647 (Thermo A-

21236) antibodies, followed by Alexa Fluor 488 Phalloidin (Thermo A-12379) to label actin

filaments. The cells were counter-stained with Hoechst and images were captured on an

Olympus FV1000 confocal microscope.

2.6 Acknowledgments

We are grateful to the Canadian Institute for Health Research (CHRP to MSS, MJD, KP) and the

Natural Sciences & Engineering Council of Canada (CHRP to MSS, MJD, KP and a CGSD to

AEA). We thank Professors Dev Sidhu and Jason Moffat and Dr. Jarrett Adams of the TRAC

facility for thoughtful discussions and providing us with CD44 mAbs and EphA2 mAbs. We

52

thank members of the Shoichet, Damha, and Petrecca labs for thoughtful review of this

manuscript.

Attenuated diphtheria toxin mediates siRNA delivery

This chapter was submitted for publication in Science Advances:

Arnold, A. E.; Smith, L. J.; Beilhartz, G.; Bahlmann, L. C.; Jameson, E.; Melnyk, R.; Shoichet,

M. S. Attenuated diphtheria toxin mediates siRNA delivery. Sci Adv (in submission).

A.E.A. devised and performed experiments and drafted the initial manuscript. L.J.S. synthesized

materials, performed experiments, and revised the manuscript. G.B. and R.M. provided

materials, assisted in conceptualization and experimental design, and revised the manuscript.

L.C.B. contributed to conceptualization and experimental design and revised the manuscript. E.J.

assisted in performing experiments. M.S.S. provided guidance and supervision in

conceptualization and experimental design and assisted in writing the manuscript.

3.1 Abstract

Toxins can act as sophisticated delivery vehicles to cells by binding to cell surface ligands,

inducing endocytosis, and quickly escaping the endolysosomal pathway to release their cargo

directly into the cytoplasm. To take advantage of this mechanism without the inherent toxicity of

toxins, we engineered conjugates between an attenuated diphtheria toxin and siRNAs to achieve

gene downregulation in glioblastoma cells. Since glioblastoma is highly invasive, we delivered

siRNA against integrin-ß1 (ITGB1) - a gene that promotes invasion and metastasis - and siRNA

against eIF-3b - a survival gene. We demonstrated mRNA downregulation of both genes in

patient-derived glioblastoma cells and the relevant associated functional outcomes: knockdown

of ITGB1 led to a significant inhibition in the invasive behavior of the glioblastoma cells using

an innovative 3D hydrogel model; and knockdown of eIF-3b resulted in significant cell death.

This is the first example of diphtheria toxin being used to deliver siRNAs, and the first time a

toxin-based siRNA delivery strategy has been shown to induce relevant genotypic and

phenotypic effects in cancer cells.

53

3.2 Introduction

Pathogens produced by bacteria have evolved over millions of years to achieve highly

sophisticated mechanisms of penetrating cells, responding to intracellular cues to guide their

trafficking, and to deliver their payload to a desired site of action.278 In particular, “AB” type

toxins, such as anthrax toxin (AT) and diphtheria toxin (DT), bind to the cell surface, are

endocytosed, and then escape the endosomal pathway, and translocate into the cytosol.279 These

toxins are made up of three domains: an active (toxic) domain, “A,” that can be mutated to

attenuate its toxicity for delivery applications (ie. “a”); a translocation domain, “T,” that

facilitates escape from the endosomes; and a receptor binding domain, “R,” that binds a receptor

on the cell surface for targeting and inducing endocytosis (Figure 3.1A).205-206 Taking advantage

of the cell entry mechanism of AB toxins through cargo-toxin fusion constructs has been

explored for delivery of diverse protein cargos including peptides, effector proteins, and

antibody-like proteins.210, 219, 222, 227 There has been only one example of oligonucleotide delivery

with attenuated anthrax toxin reported; however, this preliminary study did not demonstrate any

functional effects of gene knockdown.224

Oligonucleotides, such as small interfering ribonucleic acids (siRNAs), are powerful tools to

regulate gene expression in diseased cells, with the potential to return them to a normal

phenotype.280-281 However, therapeutic siRNA delivery represents a significant clinical challenge

due to the many physiological barriers that must be overcome for efficacy,282 as well as the

toxicity and immunogenicity of many currently used delivery vehicles.111, 283 One of the biggest

barriers to siRNA delivery is entrapment in the endolysosomal pathway after endocytosis,

quickly leading to degradation of the fragile, nuclease-prone siRNA.284-285 Strategies that

enhance the endosomal escape of siRNA have recently attracted much attention.286-288 Many of

these strategies utilize liposomal formulations for the delivery of siRNA; however, the

biodistribution of these nanoparticles is largely limited to clearance organs such as the liver and

kidney.289 To overcome biodistribution challenges, targeting proteins such as antibodies have

been explored to deliver siRNAs to specific tissues with some success;97, 264, 276 however, only a

very small percentage of antibody-siRNA conjugates escape the endosomal pathway, limiting

their efficacy.273 AB toxins, such as DT, have the capacity for both endosomal escape and

specific targeting,290 and are thus attractive candidates for siRNA delivery into the cytoplasm

(Figure 3.1B).

54

Figure 3.1. Using attenuated diphtheria toxin for siRNA delivery. A) Attenuated AB toxins,

such as attenuated diphtheria toxin (aDT), consist of three main components: a receptor

binding domain (R) that binds to a receptor on the cell surface; a translocation domain (T)

that allows for endosomal escape; and a mutated active domain (a) in order for the protein

to retain its trafficking functions but is no longer toxic to cells. Cargo, such as siRNA, can

be attached to this “a” domain. B) siRNA delivered using attenuated DT occurs in five

main steps: 1) binding to the HB-EGF precursor cell surface receptor; 2) endocytosis of the

aDT-siRNA cargo; 3) translocation through the endosomal membrane, inserting the “a”

domain and cargo into the cytoplasm; 4) cleavage of the “a” domain from the rest of the

protein; and 5) release of the siRNA into the cytoplasm where it downregulates the relevant

gene.

55

We explored whether attenuated DT could be used as a delivery vehicle for siRNAs against two

cancer gene targets: (1) eIF-3b, a transcription factor that is overexpressed in many cancers and

inhibition of which slows growth and initiates apoptosis;291 and (2) ITGB1, which is involved in

the formation of focal adhesions and activation of actin rearrangement, the inhibition of which

reduces cellular invasion and metastasis.77, 292 We chose to focus on the delivery of siRNA to

glioblastoma (GBM) cells, and specifically glioblastoma stem cells (GSCs), because: (i) DT has

been reported to cross the blood brain barrier, making it an attractive vehicle against intractable

diseases like GBM;293-294 (ii) the diphtheria toxin receptor, heparin-binding epidermal growth

factor (HBEGF), is widely expressed in the central nervous system295 and is overexpressed in at

least a subset of malignant gliomas;296 (iii) there is a growing body of evidence that the

aggressiveness of glioblastoma is, at least in part, caused by the GSC subpopulation;232, 297 and

(iv), the GSCs are rapidly dividing and highly invasive,258, 298 allowing functional readouts for

both cell viability and invasiveness.

We conjugated an engineered, attenuated DT (aDT) to Dicer-substrate siRNAs against eIF-3b

and ITGB1 using azide-alkyne click chemistry. There are many conjugation strategies available

to conjugate cargo to proteins; however, the essential disulfide bond linking the A and T domains

of diphtheria toxin precluded the use of reducing agents during synthesis of the aDT-siRNA

conjugate.299 The click chemistry approach that we used was efficient, bio-orthogonal, produced

no byproducts,183 and maintained the integrity of the rest of the protein by avoiding the use of

reducing agents. We examined the biological effects of these conjugates by measuring mRNA

expression levels and assessed cell viability and invasiveness, and thus demonstrate this platform

strategy in vitro with both gene knockdown and functional assays in primary human

glioblastoma cells.

3.3 Results

3.3.1 Conjugation of siRNA to aDT

For aDT mediated siRNA delivery, we used an engineered, attenuated diphtheria toxin (aDT)

containing a cysteine for chemical modification, a SUMO tag for increased stability and a His

tag for purification (Figure 3.2A). We modified this aDT-cysteine variant with a maleimide-

modified crosslinker containing a dibenzocyclooctyne (DBCO) as a handle for further chemical

modification (Figure 3.2B). Successful synthesis of aDT-DBCO was demonstrated by

56

absorbance, with clear peaks at 280 nm (representing the diphtheria toxin) and 309 nm

(representing the DBCO). The peak at 309 nm was absent in aDT alone (Figure 3.2C). We then

reacted Dicer-substrate siRNA containing an azide functionality at the 3’ end of the sense strand

with the aDT-DBCO to form the attenuated diphtheria toxin-siRNA conjugate (aDT-siRNA,

Figure 3.2D). Previous studies have shown that conjugation of Dicer-substrate siRNAs through

the 3’-end of the sense strand leads to more potent gene suppression than conjugation at the 5’-

end of the sense strand or either terminus of the antisense strand.300 High (5-10) equivalents of

the siRNA were required to obtain an adequate (~50%) conjugation efficiency at 37 °C (Figure

S1). However, we found that we could achieve 55% conjugation of aDT to siRNA with only one

equivalent of siRNA by adapting a method from Kataoka et al.301 wherein the aDT-siRNA

mixture was incubated for 1 h at RT, frozen at -20 °C, and then thawed for 1 h at 4 °C (Figure

3.2E). We purified the aDT-siRNA by conjugating it to nickel beads via the histidine tag on the

aDT and washing away the excess siRNA (Figure 3.2F). The aDT-siRNA demonstrated similar

stability to siRNA against serum nucleases (Figure S2), which would be encountered both in the

extracellular environment as well as inside the endolysosomal pathway.

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Figure 3.2. siRNAs can be conjugated to attenuated diphtheria toxin. A) Attenuated

diphtheria toxin was engineered to contain a free cysteine as a functional handle (aDT-SH),

protected by a SUMO tag and purified using a histidine (His) tag. B) Attenuated diphtheria

toxin was reacted with a PEG crosslinker containing both maleimide and DBCO functional

groups to obtain DBCO-modified attenuated diphtheria toxin (aDT-DBCO). C) The

presence of the DBCO modification on attenuated diphtheria toxin was confirmed by

reading the absorbance at 280 nm (aDT) and 309 nm (DBCO). Curves shown are aDT

before modification (blue) and after DBCO modification (red). D) Azide-modified siRNA

was reacted with the DBCO-functionalized attenuated diphtheria toxin to obtain the aDT-

siRNA conjugate. E) Modification of the aDT with the siRNA was confirmed via PAGE

stained with Coomassie blue to localize the diphtheria toxin protein. Lane 1 shows the aDT-

DBCO starting material (MW ~ 72 kDa) and lane 2 shows the aDT-siRNA conjugate

(MW~90 kDa) alone with some unreacted starting material. F) Purification of the excess

siRNA was confirmed via PAGE stained with GelRed to localize the siRNA. Lane 3 shows

the aDT-siRNA conjugate along with unreacted siRNA; lane 4 shows the aDT-siRNA

conjugate after nickel column purification, with only a small amount of excess siRNA left

over.

3.3.2 Glioblastoma stem cells (GSCs) express heparin-binding epidermal growth factor (HBEGF)

The GSCs used in this study were patient-derived glioblastoma cells grown in stem-cell

conditions, as previously described.302 We verified that aDT was a good candidate for targeting

and internalization in GSCs by both staining the cells for the diphtheria toxin receptor, HBEGF,

which was abundantly evident (green staining in Figure 3.3), and measuring the IC50 of the

native (toxic) diphtheria toxin against these cells to ensure the correct mechanism of action. The

IC50 of the native diphtheria toxin in the GSCs was 6.0 ± 1.0 pM (Figure S3), indicating high

sensitivity to the toxin and a conserved internalization and translocation mechanism therein. All

further experiments were conducted with a non-native, attenuated diphtheria toxin with an

insignificant level of toxicity as previously reported.210

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Figure 3.3. GSCs express HB-EGF, the native receptor for diphtheria toxin. Representative

confocal images are shown for HB-EGF (anti-HBEGF antibody, green) and nucleus

(Hoechst, blue). The secondary antibody only control confirms lack of non-specific binding.

All scale bars are 50 µm.

3.3.3 aDT-siRNA conjugate downregulates ITGB1 and reduces cellular

invasion

We conjugated attenuated aDT to Dicer-substrate siRNA against two relevant gene targets:

ITGB1 (to make aDT-ITGB1) and eIF-3b (to make aDT-eIF-3b). We treated the GSCs with the

aDT-ITGB1 conjugate and observed a significant reduction in the target mRNA compared to

negative controls of siRNA only (without lipofectamine) and aDT conjugated to a non-targeting

siRNA (aDT-NT) at 50 nM (Figure 3.4A). To ascertain whether the reduction in ITGB1

expression would correspond to a phenotype of either reduced invasion or adhesion, we seeded

the cells on top of a crosslinked hyaluronic-acid based 3D hydrogel into which the cells normally

invade (Figure 3.4B).303 Impressively, we observed a striking decrease in invasiveness in aDT-

ITGB1-treated cells compared to untreated or aDT-NT controls (Figure 3.4C, D). To determine

whether cell adhesion influenced these results, we pre-treated the cells cultured in 2D tissue

culture polystyrene flasks with both aDT-ITGB1 and aDT-NT prior to plating them on the 3D

hydrogels. After several wash steps, we observed no significant difference in the number of

adhered cells between any of the treatment groups, demonstrating that cell adhesion did not

impact the reduced cell invasion observed with aDT-ITGB1 treatment (Figure 3.4E, F).

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Figure 3.4. aDT-siRNA downregulates ITGB1 expression in GSCs and reduces cellular

invasion. A) aDT-ITGB1 (light red bars) downregulates ITGB1 mRNA expression

compared to negative controls: aDT conjugated to a non-targeting siRNA (aDT-NT, black

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bars) and ITGB1 siRNA only without lipofectamine (blue bars) at 24 h post treatment.

Positive control is transfected ITGB1-siRNA with lipofectamine (dark red bars). Data is

shown as n=3, mean±SD, normalized to an untreated control. Data was analyzed using one-

way-ANOVA followed by Tukey’s correction on the logarithmic data (* p<0.05, ** p<0.01).

B) Cells were plated in a 3D hydrogel assay on the surface of pre-formed hydrogels and

treated with aDT-ITGB1 conjugates at the beginning of the experiment. Invasion depth

was measured after 5 days. C) aDT-ITGB1 reduces invasion compared to controls (no

treatment and aDT-NT) in a 3D hydrogel model. Representative images shown. 15 µm red

beads label the top of the hydrogel; blue cell nuclei are labeled using Hoechst. All scale bars

are 150 µm. D) Invasion depth was quantified as a percentage of the untreated control.

Data was analyzed using one-way-ANOVA followed by Tukey’s correction (* p<0.05, **

p<0.01). E) aDT-ITGB1 did not reduce number of adhered cells in a 3D hydrogel model.

Representative images are shown. All scale bars are 150 µm. F) Number of adherent cells

was quantified by counting number of cell nuclei; no significant difference was observed,

demonstrating that differences in invasion were due to ITGB1 downregulation. Data was

analyzed using one-way-ANOVA followed by Tukey’s correction.

3.3.4 aDT-siRNA conjugate downregulates eIF-3b and reduces cell viability

aDT-eIF-3b treated glioblastoma cells exhibited significant downregulation in the target mRNA

compared to the relevant negative controls of siRNA only controls (without lipofectamine) and

aDT-NT at 50 nM (Figure 3.5A). We confirmed that the eIF-3b siRNA could reduce cell

viability in the GSCs by complexing eIF-3b siRNA and NT siRNA with a commercially

available transfection reagent and observing a significant difference in cell viability with eIF-3b

siRNA-mediated knockdown (Figure S4). Furthermore, we observed a significant (albeit modest)

reduction of cell viability with an aDT-eIF-3b concentration of 100 nM (Figure 3.5B).

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Figure 3.5. aDT-siRNA downregulates eIF-3b expression in GSCs and reduces cell

viability. A) aDT-eIF-3b (light red bars) downregulates eIF-3b mRNA expression

compared to negative controls: aDT conjugated to a non-targeting siRNA (aDT-NT, black

bars) and eIF-3b siRNA only without lipofectamine (blue bars) at 24 h post treatment.

Positive control is transfected siRNA with lipofectamine, dark red bars. Data is shown as

n=3, mean±SD, normalized to an untreated control. Data was analyzed using one-way-

ANOVA followed by Tukey’s correction on the logarithmic data (* p<0.05). B) aDT-eIF-3b

(red bars) reduces cell viability of GSCs at 48 h post treatment compared to aDT-NT

(black bars) at 100 nM. Data is shown as n=3, mean+SD, normalized to an untreated

control. Data was analyzed using one-way-ANOVA followed by Tukey’s correction (*

p<0.05).

3.4 Discussion and conclusions

We show the first example of siRNA delivery with an attenuated diphtheria toxin. Exploiting the

sophisticated trafficking mechanism of proteins, such as that of attenuated diphtheria toxin, has

the potential to overcome many of the barriers to siRNA delivery. Using our aDT-siRNA

conjugate, we were able to downregulate two distinct genetic targets and observe significant

changes in cell proliferation and invasion, supporting the robust nature of aDT-mediated drug

delivery and its potential as a novel treatment strategy for aggressive cancers such as

glioblastoma. Attenuated diphtheria toxin is an advantageous siRNA delivery vehicle for

glioblastoma treatment for two main reasons: (1) targeting via the receptor binding domain and

(2) endosomal escape via the translocation domain. HBEGF proved to be a good target for

siRNA delivery as it is expressed in the GSCs that are used in this study and widely expressed in

brain tissue.295 It is essential to develop strategies that can target the GSCs in addition to the bulk

tumor as GSCs may be responsible for tumor recurrence.304

Endosomal escape is important for the delivery of siRNAs in order to avoid trafficking into the

late endosomes/lysosomes and subsequent degradation by nucleases.305-306 Attenuated diphtheria

toxin has been reported to translocate out of the endolysosomal pathway at a relatively early

endosomal stage (pH ~6.5), protecting its cargo from the harsh degradation environment of the

lysosomes.307-308 The attenuated diphtheria toxin and its cargo translocate through a flexible α-

helical pore,210 making endosomal escape difficult to assay directly because most current

methods measure widespread membrane leakage.309-310 However, the gene knockdown and

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subsequent phenotypic effects of siRNA delivery that we observed confirmed successful aDT-

mediated translocation, as previous work has shown that without translocation and endosomal

escape the aDT cannot deliver cargo.210, 311 We confirmed the robust nature of the aDT-siRNA

system by downregulating two genes of interest in the target cells. In comparison, in systems that

do not have inherent endosomal escape capabilities, such as antibody-siRNA conjugates,

delivery has been shown to be highly target dependent.201 While an anthrax toxin was used for

siRNA delivery previously, knockdown of only a proof-of-concept target was shown without

downstream functional effects.224 Using our aDT conjugate system, we observed phenotypic

changes in both cell invasion and growth.

Diffuse tumor cell infiltration is a hallmark of GBM, and recent findings have suggested GSCs

play important roles in migration and invasion.298, 312 It has been reported that inhibiting GSC

invasion is essential to slowing the progression of GBM.313 ITGB1 is involved in cellular

binding to many extracellular matrix components, including fibronectin,72 and ITGB1

knockdown has been shown to reduce invasion in cancer cells.314 Thus, we hypothesized that

ITGB1 knockdown would reduce the invasive behavior of GSCs. We demonstrated the

effectiveness of the aDT-ITGB1 conjugate in reducing of cell invasion using a previously

validated 3D hydrogel platform,303 confirming a functional effect of siRNA-mediated

knockdown. Moreover, as GSCs have also been shown to drive tumor growth,41 we also were

interested in using our treatment strategy to reduce cellular proliferation. We employed an

siRNA targeted against eIF-3b, which is known to reduce protein synthesis leading to a reduction

in cell growth and viability.315 Using the aDT-eIF-3b conjugate, we successfully reduced eIF-3b

expression and observed a corresponding change in cell viability. Thus, we demonstrated

functional effects of gene knockdown in two different pathways, indicating that aDT-siRNA can

be considered a platform strategy.

For the first time, we demonstrated a robust platform for siRNA delivery to glioblastoma stem

cells using an attenuated diphtheria toxin with an inherent endosomal escape mechanism. In

future work, this conjugate will be used for siRNA delivery in vivo to orthotopic models of

glioblastoma. Further extensions of this work could include incorporation of chemically

stabilized siRNAs89 and combination with chemotherapeutics in order to develop novel

treatments for a broad range of cancers and other diseases.

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3.5 Materials and methods

3.5.1 Cell lines Patient-derived glioma neural stem cells (GNS 411) were a gift from the lab of Dr. Peter Dirks,

with Research Ethics Board approval at the Hospital for Sick Children, Toronto, and the

University of Toronto. These cells are referred to as glioblastoma stem cells (GSCs) throughout

the manuscript. Cells were maintained in an incubator (37 °C, 5% CO2, 95% humidity) grown in

Corning Primaria flasks (Corning 353808) coated with poly-L-ornithine (PLO, Sigma-Aldrich

P4957) and laminin (Sigma-Aldrich 11243217001). GSC growth media contained serum-free

NeuroCult NS-A Basal Media (StemCell Technologies 05750) supplemented with N2, B27,

EGF, FGF, and heparin as previously described for neural stem cells.316

3.5.2 Attenuated diphtheria toxin Attenuated diphtheria toxin (aDT) was expressed as previously described.210 Briefly, aDT was

expressed in E. coli BL21(DE3) cells, induced with 1 mM isopropyl-β-d-1-thiogalactopyranoside

(IPTG) for 4 h at 37 °C using the Champion pet-SUMO expression system (Invitrogen). Cells

were harvested by centrifugation, resuspended in lysis buffer (20 mM Tris-HCl pH 8.0, 0.5 M

NaCl, 20 mM imidazole, benzonase, lysozyme, and protease inhibitor cocktail) and lysed by an

EmulsiFlex C3 microfluidizer (Avestin) at 15,000 psi. The lysates were centrifuged at 18,000

× g for 20 min. The histidine-tagged DT was purified by Nickel affinity chromatography using a

His-Trap FF column (GE-Healthcare).

3.5.3 siRNAs Dicer-substrate siRNAs were purchased as annealed duplexes from Integrated DNA

Technologies. All siRNAs were suspended at a concentration of ~50 nM using nuclease free

duplex buffer (Dharmacon B-002000-UB-100). Concentrations were verified by measuring the

absorbance at 260 nm. Sequences of Dicer-substrate siRNAs used were as follows: (eIF-3b

sense) 5’-rGrG rArUrA rCrGrC rUrUrA rGrCrA rUrCrU rArUrG rArArA CT-Azide-3’; (eIF-3b

antisense) 5’-rArG rUrUrU rCrArU rArGrA rUrGrC rUrArA rGrCrG rUrArU rCrCrA rG-3’;

(ITGB1 sense) 5’-rGrA rCrUrG rUrUrC rUrUrU rGrGrA rUrArC rUrArG rUrArC TT-Azide-3’;

(ITGB1 antisense) rArA rGrUrA rCrUrA rGrUrA rUrCrC rArArA rGrArA rCrArG rUrCrA rC-

3’.

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3.5.4 PCR primers Primers used were purchased from ACGT Corp. for eIF-3b317 (forward: 5’-

TGTGAAAGGTACCTGGTGAC-3’ and reverse: 5’-AATAGGCCAATGGGCTGAG-3’) and

for ITGB1318 (forward: 5’- GAAAACAGCGCATATCTGGAAATT-3’ and reverse: 5’-

CAGCCAATCAGTGATCCACAA-3’).

3.5.5 Immunocytochemistry (HBEGF) GSCs were plated to reach 60-80% confluency on chambered coverglass slides coated with poly-

L-ornithine (PLO) and laminin. Cells were then fixed with 4% PFA and stained using an

antibody specific for HBEGF (Abcam, ab66792) followed by a fluorescently labeled secondary

antibody (Thermo Fisher A-11001). The cells were then counter-stained with Hoechst 33342 and

images were captured on an Olympus FV1000 confocal microscope.

3.5.6 Preparation of aDT-siRNA conjugate Attenuated diphtheria toxin was modified with a dibenzocyclooctyne-PEG4-maleimide

crosslinker (DBCO-PEG4-Mal, Sigma-Aldrich 760676) by adding 4 equivalents of a 10 mM

solution in DMSO of the DBCO-PEG4-Mal crosslinker to a ~10 µM solution of the protein in 20

mM Tris, 150 mM NaCl, 5% glycerol, pH 7.5 and incubating at RT for 30 min. The aDT-DBCO

conjugate was then purified by dialysis against Tris pH 7.5, 1% glycerol for 48 h to remove any

excess crosslinker. The aDT-DBCO conjugate was characterized by absorbance to confirm a

presence of a peak at 309 nm representing the incorporation of the DBCO moiety (75% yield).

The aDT-DBCO conjugate was then mixed with an azide-containing siRNA at a 1:1 ratio,

followed by a 1 h incubation at RT, overnight incubation at -20 °C, and 1 h incubation at 4 °C.

The conjugate was then bound to nickel beads for 1 h at 4 °C, eluted from the beads in 500 mM

imidazole, and buffer exchanged into Tris pH 7.5, 1% glycerol. Successful conjugation was

confirmed by PAGE analysis and conjugation efficiency was quantified using imageJ software

(45% total yield; 80% recovery of DT, 55% siRNA conjugation efficiency).

3.5.7 Gene knockdown assays - treatment with aDT-siRNA conjugate aDT-siRNA was mixed with Opti-MEM for a final concentration of 50 nM and added to 24-well

Primaria plates coated with PLO/laminin. Cells were then added to the plates with the conjugate

at a density of 50,000 cells per well. 24 hours following treatment, cells were collected and

66

lysed. RNA was purified from the cells and gene expression was analyzed via quantitative RT-

PCR (qPCR).

3.5.8 Gene knockdown assays - positive control treatment with Lipofectamine RNAiMAX

Transfection complexes with Lipofectamine RNAiMAX (Thermo Fisher 13778075) were

prepared according to the manufacturer’s protocol in a reverse transfection procedure. Briefly,

siRNA and Lipofectamine were mixed in Opti-MEM serum reduced media (Thermo Fisher

31985062) and added to 24-well Primaria plates (Corning 353847) coated with PLO/laminin.

Cells were then added to the plates with the treatment at a density of 150,000 cells per well. 24

hours following transfection cells were collected and lysed. RNA was purified from the cells and

gene expression was analyzed via qPCR.

3.5.9 Invasion assay Hydrogels were synthesized as previously described by Tam et al.7 Cells were plated on

hydrogels at a density of 3500 cells/hydrogel and allowed to adhere for 24 h. Cells were then

treated with aDT-ITGB1 conjugates at a concentration of approximately 50 nM. 48 h after

treatment, fresh media was added to each well and cells were fixed with 4% PFA 4-5 days after

treatment. Cells were stained with Hoechst to identify cell nuclei and 15 µm fluorescent beads

were added to each well to label the surface of the hydrogel. Hydrogels were imaged using

confocal microscopy and depth of invasion was analyzed using a custom MATLAB script.

3.5.10 Adhesion assay aDT-siRNA was mixed with Opti-MEM for a final concentration of 50 nM and added to 24-well

Primaria plates coated with PLO/laminin. Cells were then added to the plates with the conjugate

at a density of 50,000 cells per well. 24 h after treatment, cells were lifted off the 2D plates using

accutase and re-plated on hydrogels at a density of 15,000 cells per hydrogel. 1 h after re-plating

on the hydrogel, cells were washed 5 times with PBS and adhered cells were fixed with 4% PFA,

stained with Hoechst and imaged using confocal microscopy. The number of adhered cells was

quantified using custom computational software.

3.5.11 Cell viability assays following treatment with aDT-eIF-3b aDT-eIF-3b was mixed with Opti-MEM for the desired final concentration and then added to 96-

well plates coated with PLO/laminin. Cells were then added to the plates with the conjugate at a

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density of 8,000 cells per well. Fresh media was added to each well 24 h following treatment. At

48 h, cell viability was measured via PrestoBlue viability assay and quantified as a percentage of

untreated controls.

3.6 Acknowledgements

We thank the lab of Dr. Peter Dirks (Hospital for Sick Children, Toronto) for providing the

patient-derived glioblastoma stem cells. We thank Professor Masad J. Damha (McGill

University) for thoughtful advice on siRNA chemistry. We thank members of the Shoichet and

Melnyk labs for thoughtful review of this manuscript. We are grateful to the Canadian Institute

for Health Research (CHRP and Foundation to MSS) and the Natural Sciences & Engineering

Council of Canada (CHRP and Discovery to MSS, CGSD to AEA and LCB, PGSD to LJS).

MSS also acknowledges salary support from the Canada Research Chairs program.

Thesis discussion

The promise of antisense therapeutics is the reversal of disease symptoms through controlling

cellular behavior a genetic level. This could include phenotypes including reduced cellular

invasion, increased apoptosis, or lower resistance to chemotherapeutic treatment. However,

despite recent advances in siRNA/AON delivery, including several clinical approvals, many

formulations do not target specific tissues and struggle to escape degradation in the

endolysosomal pathway, limiting their efficacy.

In addition, it is essential to target the right cell population: there is growing recognition in the

field that the successful treatment of cancer requires the obliteration of not just the bulk tumor

but also the CSCs, which are more resistant to conventional treatment methods. In particular,

GBM is a highly recurrent and aggressive tumor even after resection of tumor tissue,

chemotherapy, and radiotherapy, emphasizing the need for a therapy that targets the GSC

population.

This thesis attempts to address two main challenges in the field of cancer treatment: the delivery

of antisense therapeutics and the targeting of GSCs. First, we explored targeted delivery of

AONs to GSCs by using antibodies against specific cell surface receptors of the GSCs, including

CD44 and EphA2. For this work, we selected a relevant target for knockdown, DRR, which has

68

implications in the survival, proliferation, and invasion of GSCs. Appendix F describes an initial

attempt to achieve targeted knockdown of DRR by functionalizing a nanoparticle with antibody

and oligonucleotide pendant groups; however, we quickly realized this approach would not be

successful, and so pivoted to utilizing simpler antibody-oligonucleotide conjugates. Using

antibody bioconjugates as delivery vehicles, we were able to deliver AONs to cells, observe

uptake and knockdown, and see a corresponding change in the morphology of the cells. This

represented the first time that an antibody-oligonucleotide conjugate had been used for delivery

to GSCs. We were limited by a high amount of uptake of the antibody-AON formulation into the

endolysosomal pathway, and wanted to use a delivery vehicle with an inherent endosomal escape

mechanism, which led us to next explore using DT as a delivery vehicle for oligonucleotides. We

conjugated DT to highly potent siRNAs to modulate GSC phenotypes, including siRNAs against

eIF-3b, a target for cell proliferation, and against ITGB1, a target for cell invasion. Using the DT

as a delivery vehicle, we observed efficient knockdown and corresponding changes in

proliferation and invasion phenotypes, demonstrating the potential of DT as a platform

technology for oligonucleotide delivery. In the following discussion we will explore the

importance of these findings to the field of drug delivery.

4.1 The interplay of stability and potency in antisense

therapeutics

Throughout the work presented in this thesis, a variety of AONs and siRNAs were explored for

gene knockdown. Modifications to increase oligonucleotide potency have been extensively

explored,89 and while many of these modifications have been shown to increase potency,239, 319-

320 there is also the possibility that modifications will interfere with RISC loading and mRNA

cleavage by making the duplex too stable or forcing the nucleic acid to adopt a suboptimal

geometry.321-322 In addition, it is widely accepted that AONs are more stable but less potent than

siRNAs,323 but AONs still can be useful for many applications, and continue to progress in the

clinical pipeline.324 Clearly, both stability and potency play important roles in the selection and

overall efficacy of modified oligonucleotides.

In the variety of modification patterns for AONs and siRNAs explored in this thesis, particularly

as described in Chapter 2, Appendix C, and Appendix D, we observed that higher stability of the

oligonucleotides often correlated to lower potency. These differences are most striking in

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comparing the AONs and siRNAs against DRR. DRR siRNAs are extremely potent for

knockdown, especially the unmodified control siRNA, but this unmodified siRNA is the least

stable strand against nuclease-mediated degradation. The siRNAs against eIF-3b and ITGB1 are

also very potent for knockdown of the target genes, but degrade quickly in the presence of

nucleases. In contrast, the AONs against DRR are the most stable out of the oligonucleotides

tested, but less potent than all of the other siRNA sequences against DRR. As described in

Appendix C and Appendix D, we also observed this trend with other modification patterns of the

DRR siRNA: siRNAs with higher degrees of modification were more stable to nuclease

degradation but less potent. This could be due to sub-optimal design of modification chemistries

and patterns, but the data demonstrates the importance of measuring and optimizing both

stability and potency while considering the impact on the desired functional effect.

The correlation between stability and potency that we observed throughout different aspects of

this thesis should be considered for any future work. The trade-off between stability and potency

for the oligonucleotides used is a major limitation; a possible improvement would be to search

for a pattern of chemical modifications that would stabilize the oligonucleotide while also

enhancing potency. For example, this has been observed with alternating 2’-methoxy and 2’-

fluoro modifications within siRNA sequences.325 Interestingly, Patisiran, the first FDA-approved

formulation of siRNA, employs siRNA that contains a combination of 2’-methoxy-modified and

unmodified ribonucleosides; unfortunately, the exact pattern of modifications is protected.326 In

contrast, the siRNAs used as described in Appendix C and D contained mostly 2’-fluoro and

some 4’-methoxy modifications, which seem to have limited the observed potency. It is known

that small differences in chemical modifications of siRNA can have large effects on the loading

of the siRNA into the RISC complex and therefore negatively impact gene silencing;327

therefore, a larger screen should be performed on ITGB1 or eIF-3b siRNAs to identify

sequences, potentially a combination of 2’-fluoro and 2’-methoxy modifications, that maintain or

increase the potency of silencing. In this way, nuclease stability could be improved without

sacrificing, and perhaps even increasing, potency of gene knockdown. Such a strategy could

dramatically increase the efficacy of the delivery systems discussed in this work and will be

discussed further in section 6.2.2.

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4.2 The importance of endosomal escape in oligonucleotide

delivery

Endosomal escape is a key challenge in the field of oligonucleotide delivery. The fragile nucleic

acid material must escape the degradation pathway of the endolysosomes before the acidic pH

and the activity of nucleases and other enzymes render the AONs or siRNAs inactive.120

Numerous strategies have been explored to escape the endosomal pathway, many of them

involving incorporation of cationic components.127, 285, 328 While enhancing endosomal escape

can improve transfection efficiency,329-331 most of these strategies lead to increased cytotoxicity

and lack of targeting capacity, limiting biodistribution to a few clearance organs.332 For delivery

methods that rely on “spontaneous” endosomal escape, such as antibody-siRNA conjugates, it is

estimated that only a fraction of one percent of conjugates are released into the cytoplasm.122

We also observed this challenge in our own work: as described in Chapter 2, when we delivered

a fluorescently labeled Ab-AON conjugate to GSCs, a significant amount of material was

determined to be overlapping with the lysosomes, indicating a high degree of degradation was

likely occurring. This is a significant limitation of the system, and agrees with other recent work

in the field of antibody-siRNA conjugates.122 The knockdown we observed at 150 nM of the Ab-

AON conjugate was significant but did not reach the levels of knockdown observed with 25 nM

of transfected AON using a commercially available lipofectamine system. One can assume that if

a larger fraction of the AON or siRNA was escaping the endosomal pathway, it would lead to a

higher degree of knockdown efficiency.

After observing these limitations with the Ab-AON system, we started exploring aDT as a

delivery system for antisense therapeutics as described in Chapter 3, and the toxin has the

advantage of an inherent endosomal escape mechanism.205 We also moved to using siRNAs

instead of AONs as we wanted to deliver a more potent therapeutic to give a large therapeutic

window for observing significant knockdown. Previous literature has shown that without

translocation and endosomal escape of the aDT, its delivery efficiency is decreased at least 1000-

fold,210 so we knew that if we observed any knockdown by the siRNA, it would indicate that the

translocation of the siRNA had occurred. We were excited to explore the aDT as an

oligonucleotide delivery vehicle because only one other AB toxin, anthrax, has been explored for

AON and siRNA delivery, and while the strategy appeared to be successful, they did not explore

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the downstream functional effects of siRNA delivery.224 Thus, we were the first to explore a

toxin for siRNA delivery and functional gene knockdown.

When we conjugated siRNAs against eIF-3b and ITGB1 to aDT, we could observe

downregulation of both targets, indicating successful delivery and endosomal escape of the aDT-

siRNA conjugate. We were also encouraged that we could see potency of the aDT-siRNA at 50

nM, whereas we did not see any effect of the Ab-AON until the concentration of the conjugate

reached 150 nM. This supports our theory that aDT is a more efficient delivery vehicle than the

antibodies for antisense therapeutic delivery, at least in this example. The potency that we

observed with the aDT-siRNA conjugate did not reach the levels of the transfected siRNA, and

some possible future improvements to the system will be discussed in section 6.2. Nevertheless,

we were excited to see effective gene knockdown using aDT as a delivery vehicle, and the

protein function is dependent on the translocation through the endosomal membrane, indicating

that endosomal escape plays a key role in the diphtheria-toxin mediated siRNA delivery. As

discussed above, endosomal escape is a major challenge in the field of siRNA delivery,120, 122, 305

and we even observed this challenge with the Ab-AON system, where a significant portion of the

Ab-conjugate material colocalized with the lysosomal compartments. The improved delivery that

we observed with the aDT-mediated delivery compared to the Ab-mediated delivery supports our

hypothesis that improved endosomal escape can enhance siRNA delivery, and using aDT as a

delivery vehicle is a significant first step in overcoming this challenge.

A limitation of this work is that we were not able to directly assay the translocation and cytosolic

accumulation of siRNA, which would further support our hypothesis that endosomal escape is

enhancing siRNA delivery. Possible future strategies for examining the trafficking of aDT-

siRNA within the cells will be discussed in section 6.1.

4.3 Measuring functional effects of oligonucleotide delivery

The translation of oligonucleotide therapeutics has been met with many practical challenges, and

efficacy in the clinic remains elusive: with a few exceptions, most trials have concluded at the

Phase I and early Phase II stages due to a lack of efficacy.333 There is a significant number of

recent publications that report only on the gene silencing at the mRNA/protein level, stopping

short of a functional result, or only demonstrate knockdown of a non-endogenous, proof-of-

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concept target such as luciferase or GFP.224, 334-336 An in vivo study to look at outcomes such as

tumor growth and animal survival would be of significant value, but these studies are lengthy

and not cost-effective. In order to determine whether the delivery of AONs and siRNAs that we

achieved using antibodies and aDT as delivery vehicles was sufficient to observe real, functional

effects, we evaluated the phenotype following knockdown using several assay including

assessment of cell shape, measuring cell viability, and monitoring cell invasion following

treatment. The results of these assays increase our confidence that these delivery strategies will

be robust strategies for in vivo gene knockdown and GBM treatment.

4.3.1 Functional effects of DRR knockdown

DRR is a cytoskeletal crosslinker that organizes microtubules and actin at focal adhesions,64 and

also recruits Akt to focal adhesions, activating several downstream pathways.66 Cells with lower

levels of DRR have been shown to lead to functional effects including decreased invasion in a

collagen matrix, a more rounded phenotype, and less invasion following direct injection of the

cells into brain tissue.64, 66

As described in Chapter 2, we observed significant DRR knockdown with a CD44-targeted

antibody as a delivery vehicle for the DRR AON. A simple phenotypic assay to measure whether

the DRR knockdown was having a significant functional effect involved measuring the area of

the cell to determine whether it had a rounded morphology, as has been observed when DRR

expression is reduced, or the more spread-out, spindle-like morphology of the native cell

population. We were able to observe a significant difference in the morphology of the cells using

this assay, indicating that the knockdown measured at the protein level was sufficient to lead to a

phenotypic change. As demonstrated by Petrecca et al., a similar change in the cellular

morphology has been shown to lead to reduced invasion following in vivo implantation of

treated cells.64, 66 Thus, the difference in phenotype that we observed is a promising indicator of a

reduced capacity for invasion in future in vivo studies, which is significant as inhibition of GSC

invasion can lead to a significantly reduction in tumor growth, at least in preclinical studies.313

4.3.2 Functional effects of eIF-3b knockdown

The silencing of the translation initiation factor eIF-3b has been shown to reduce protein

synthesis and impair the activity of the Akt pathway, leading to downstream effects including

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apoptosis and cell death.70 This impact on cell viability has been demonstrated in colon cancer,337

esophageal squamous carcinoma,338 and bladder cancer,69 and interestingly has also been

observed in GBM,71 which is one of the reasons why eIF-3b was chosen as a genetic target for

this work.

As described in Chapter 3, we delivered siRNA against eIF-3b to GSCs using the aDT-siRNA

conjugate. We assessed the functional effects of eIF-3b knockdown in GSCs by measuring the

cell viability after treatment. Importantly, we determined there was a significant effect on cell

viability following aDT-mediated eIF-3b knockdown. Although the effect we observed is

significant, it was modest and other studies have demonstrated more dramatic inhibition

following eIF-3b knockdown; for example, the proliferation of some bladder cancer cell lines is

almost halted following eIF-3b knockdown.69 Although eIF-3b activation has been demonstrated

in GBM,71 it has not been studied in the context of GSCs and it is possible that this cell

population is more resistant to apoptosis initiated by eIF-3b knockdown. To clarify the

relationship between the GSCs and eIF-3b expression, future work could include comparing

expression of eIF-3b in GSC cells compared to bulk tumor cells, as well as measuring more

proximal effects of eIF-3b knockdown, such as the rate of total protein synthesis or the increase

in apoptotic markers.

4.3.3 Functional effects of ITGB1 knockdown

Integrin β1 (ITGB1) is essential to invasion in several cancers, often via dimerization with α5

integrins and association with fibronectin or fibronectin-mimetic peptides.74, 339-340 The

association with integrins to extracellular ligands initiates a signaling cascade that leads to

reorganization of actin fibres,341 and ITGB1 knockdown has been shown to decrease cellular

invasion and/or adhesion in multiple models.314, 342

As described in Chapter 3, we measured cellular invasion and adhesion as functional readouts for

ITGB1 knockdown. We first delivered aDT-ITGB1 to GSCs and determined that there was a

decrease in ITGB1 expression. To examine the effect on invasion, we used a previously

developed 3D hydrogel that has been validated as a drug screening tool for both proliferation and

invasion.303 This hydrogel contained fibronectin-mimetic peptides, which the cells bind to via a

dimer of ITGB1 and integrin alpha 5,343 so we hypothesized that ITGB1 knockdown would

inhibit GSC invasion in the hydrogel. Previous studies have demonstrated reduced ITGB1

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expression can lead to a reduction of invasion in head and neck squamous cell carcinoma cells in

a Transwell assay of invasion, where cells migrate through a permeable membrane from one

chamber to another.314 When we treated the cells with aDT-ITGB1 in the 3D hydrogel model, we

observed a significant decrease in the depth of invasion of the GSCs. We also measured adhesion

to determine whether the effect was due to reduced adhesion or invasion and observed no

significant difference in the initial adhesion of the cells, indicating the observed decrease in cell

depth was due to an effect on cell invasion. There are no existing examples in the literature of a

synthetic hydrogel used to measure the effects of ITGB1 knockdown in 3D, and this represents

the first time the 3D hydrogel model developed in our lab has been used as a screening tool for a

novel therapeutic. Thus, we were excited to see that we could use it to measure phenotypic

effects of siRNA delivery against invasion targets such as ITGB1.

4.4 Targeting the glioblastoma stem cell population

GSCs contribute to the aggressive behavior of GBM, including the invasion, metastasis,

proliferation, and recurrence of the tumor.39-40, 312 Recent evidence has demonstrated that if a

therapeutic can target the GSCs, then in vivo tumor formation arising from GSC implantation is

significantly inhibited.344 In this thesis, we explored several methods of targeting GSCs,

including targeting cell surface receptors that have been identified as markers for GSCs,

targeting genes responsible for the aggressive phenotypes of GSCs, and exploring the optimal

delivery strategy for GSC therapeutics in vivo.

4.4.1 Targeting cell surface receptors

One method of targeting GSCs is to specifically target cell surface receptors that will be

expressed on the GSCs. This strategy requires careful consideration of the off-target effects of

the delivered drug, since many receptors that are expressed on GSCs are also expressed in the

bulk tumor and in neural stem cells.345

As described in Chapter 2, we used antibodies against specific antigens in order to deliver

antisense therapeutics to the GSCs. One of the cell surface receptors that we targeted in this work

was CD44, a potential GSC marker that is also expressed by neural stem cells.346-347 We were

able to successfully deliver AONs against DRR to GSCs and observe significant knockdown of

DRR at the protein level using the CD44-targeted strategy. We confirmed specificity of binding

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by also conjugating the AON to a non-specific IgG and did not observe any internalization of the

AON. We also explored another antibody against EphA2, which is a cell surface marker shown

to play a role in the tumorgenicity of GSCs and is overexpressed in at least some GBM cells with

stem-like characteristics.42-43 Using this antibody for AON delivery we did not detect any

downregulation of the DRR following delivery.

One potential explanation for the difference in delivery efficacy is the internalization pathways

of the different antibodies. There is evidence in the literature that in some cases the CD44

receptor is recycled following internalization into the early endosomes,348-349 which could save

the AON from degradation in the lysosomes, with some Ab-AON conjugate spontaneously

escaping from the early endosomes prior to recycling. In contrast, the EphA2 receptor is likely

shuttled to the lysosomes for degradation following internalization,350 which could lead to the

degradation of any cargo that it is carrying into the cells. We saw some evidence of this

difference in internalization pathways by conducting a flow cytometry experiment, as described

in Appendix A; at every timepoint, the EphA2 antibody was internalized to a greater extent than

the CD44 antibody, suggesting that the EphA2 antibody is internalized and is not recycled back

to the cell membrane.

Despite the success of our CD44-targeted strategy, recent evidence has suggested that CD44 is

not an ideal marker for the GSCs because some cells that are low in CD44 expression have the

most GSC-like traits including sphere formation and stem cell marker expression.351 Another

marker that we could have chosen to target the GSCs is CD133, one of the first markers that was

used to identify the GSC population.38, 347 However, it has also been demonstrated that CD133-

negative cells have GSC characteristics including initiation of tumor formation.49 A successful

strategy for GSC therapeutics will likely need to target several different “GSC markers,” or

deliver a targeted drug to a broader cell population, in order to overcome these challenges.

As described in Chapter 3, we used aDT as a delivery vehicle for siRNA. aDT in its native form

targets the cell surface receptor HBEGF,352 which is widely expressed in many tissues and

especially the brain.295 HBEGF itself may play a role in GBM initiation,296 although it has not

been shown to be overexpressed specifically in the GSC population. We confirmed that HBEGF

was expressed in the GSCs studied as described in Chapter 3 by immunocytochemistry and also

by measuring the IC50 of the native toxin to confirm the correct binding and trafficking of the

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toxin. We did not confirm whether aDT-siRNA preferentially targets the GSC population in

comparison to bulk tumor or other neural cells, which may be a limitation of this targeting

strategy. However, since the delivered therapeutic is siRNA that targets expression of a specific

gene, HBEGF targeting could still be a good strategy for GSC therapy with the selection of the

correct gene for downregulation in order to limit negative off-target effects.

4.4.2 Targeting key genetic alterations for desirable phenotypes

Another way that we targeted the GSC population is by selecting genes for downregulation that

had been shown to be over-expressed in the GSCs, or were associated with specific phenotypes

that we wanted to modulate in the GSCs. Some previous work has targeted genes involved in

stemness, including Sox2 or Nanog,39, 62 but we wanted to avoid targeting these genes as they are

also essential for neural stem cells. We also tried to select genes for downregulation that would

have obvious phenotypic effects, such as reduction in proliferation or invasion, if the

downregulation was effective.

As previously discussed, DRR has been shown to be involved in the migration and invasion of

GSCs by acting as a cytoskeletal crosslinker.64, 66 Petrecca et al. discovered that the knockdown

of DRR using AONs decreased the invasion of patient-derived GSCs in an in vivo model.66 As

described in Chapter 2, we delivered AONs against DRR to GSCs using CD44-targeted

antibodies and observed a reduction of DRR expression which correlated to a significant change

in the morphology of the cells, suggesting a less invasive phenotype. However, as described in

Appendix D, we delivered DRR AONs to DRR-overexpressing cells and, while there was a trend

towards a reduced capacity for invasion, the cell number significantly increased indicating a

greater rate of cell proliferation. This dichotomy that we observed between DRR-mediated

proliferation and invasion, where cells with reduced DRR expression leading to reduced invasion

also have increased proliferation, was originally suggested by Giese et al.65 and also observed by

Petrecca et al. where they observed increased proliferation with reduced DRR expression.64

Although this complicates DRR as an anti-cancer target, since proliferation of cancer cells is not

desirable, there is some evidence that increased proliferation of cancer cells could also sensitize

them to chemotherapeutic treatment,353 and thus this still could be a good strategy for

specifically targeting the GSC population.

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To separate the proliferation and invasion phenotypes, as described in Chapter 3, we explored the

delivery of two different siRNAs, one associated with proliferation and one associated with

invasion, using the aDT as a delivery vehicle. Although there is likely a small population of

nearly-quiescent ‘stem’ cells within the GSC population,40 they give rise to a larger population of

progenitor-like cells that are quickly dividing,347 so proliferation is a common phenotype in GSC

populations.41 EIF-3b has been shown to promote proliferation in GBM and knockdown of eIF-

3b can reduce proliferation.71 Using aDT to deliver eIF-3b siRNA, we observed a significant

reduction in the proliferation of the GSCs. GSCs have also been shown to be highly invasive and

contribute to migration and metastasis of GBM,37, 77, 298, 312 and ITGB1 is involved in many

invasion mechanisms,74-75, 339-341 so we wanted to try to reduce ITGB1 expression and determine

whether this had a significant effect on the invasion of the cells. We conjugated an siRNA

against ITGB1 to aDT and the invasion of the cells treated with this conjugate was significantly

reduced in our 3D hydrogel model. Reduction of invasion in GBM, particularly when combined

with other therapeutic strategies, is a promising strategy for reducing tumor growth and

recurrence rates.354-356 These proof-of-concept experiments have shown that we can use aDT to

modulate specific phenotypes in the GSCs and future work will include expanding this strategy

for the knockdown of other genetic targets, for example genes involved in GSC fitness or TMZ

resistance that have been identified in CRISPR screens.302

4.4.3 Optimizing the delivery of protein conjugates in vivo

As described in Appendix E, we examined the behavior of an Ab-fluorophore conjugate

following injection in vivo. This study incorporated multiple injection paradigms, including

direct injection into the brain tissue, intraventricular injections, and intravenous injections, all in

an attempt to maximize the delivery of the Ab material to the tumor cells. We observed that the

best way to target the GSCs was to inject the Ab-fluorophore directly into the tumor space, and

with this strategy multiple areas of colocalization were observed between the GSCs and the

fluorophore-labeled antibody. We expect that this direct injection strategy would be the best way

to deliver the maximal amount of bioconjugate, whether Ab-AON or aDT-siRNA, to the GSCs

for preliminary in vivo studies and potentially for future clinical applications as well. This agrees

with current literature that suggests local administration of therapeutics to glioblastomas

overcomes several significant barriers, including rapid clearance in circulation and penetrance of

the blood-brain-barrier.357

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Conclusions

In this thesis, I developed two strategies for the delivery of antisense therapeutics to GSCs. First,

AONs against a gene of interest, DRR, were conjugated to antibodies that were specific for

GSCs including CD44 or EphA2. Primary validation steps included the optimization of the

modification pattern of the AON for the best DRR knockdown efficacy, as well as an

internalization assay performed with the antibodies of interest to confirm that they would be

endocytosed following receptor binding. The best DRR AON had a “Gapmer”-motif with

modifications on the flanking ends of the oligonucleotide strand, and we found that both

antibodies were internalized by the GSCs. Next, we conjugated the AONs to the antibodies and

treated the GSCs with the conjugates in vitro. We found that the CD44-conjugated DRR AON

could significantly reduce DRR expression in GSCs, whereas the EphA2-conjugated DRR AON

did not. This satisfied the first objective, to “knockdown DRR/FAM107A in vitro using a

targeted delivery platform.” Additionally, we used the fluorophore-labelled AON to examine

conjugate uptake and colocalization with the endolysosomal pathway, and saw that although the

CD44 Ab-AON conjugate was taken up more than the EphA2 Ab-AON conjugate, supporting

our knockdown results, there was still a high degree of colocalization between the conjugate and

the lysosomes, suggesting a significant amount of endolysosomal trafficking and degradation.

Importantly, this study demonstrated that we could knock out a gene of interest in the GSCs

using a targeted antibody delivery platform.

Next, we explored an alternate delivery platform: aDT, which binds to cell surface receptors but

almost immediately escapes the endolysosomal pathway following endocytosis. We validated

that the GSCs were a suitable cell type for aDT-mediated delivery by staining the stem cells for

the aDT receptor, HBEGF, and by measuring the IC50 of the native toxin to confirm the correct

trafficking mechanism. The patient-derived GSCs used for these studies displayed a high amount

of HBEGF staining and a low IC50, confirming the suitability of the aDT for delivery in these

cells. aDT was then conjugated to two different siRNAs, one against a target for cell

proliferation, eIF-3b, and one against a target for invasion, ITGB1. We successfully reduced

expression of both eIF-3b and ITGB1 using the aDT-siRNA delivery platform and confirmed

functional effects including reduced cell proliferation and invasion. These studies confirmed that

aDT could successfully deliver siRNA to GSCs, representing the first time that aDT had been

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used for siRNA delivery and highlighting the promise of attenuated toxins as a platform delivery

technology for antisense therapeutics to diseases including cancer.

5.1 Completion of objectives

This research was motivated by the following hypothesis:

Protein-conjugated oligonucleotides will selectively and effectively knockdown key genes in

brain cancer cells, including patient-derived models of GBM, and thereby reduce cellular

proliferation or invasion in vitro.

Herein we described the delivery of AONs and siRNAs to GSCs using either antibodies or toxins

as delivery vehicles. Achievements of the objectives originally outlined in Chapter 1 are outlined

below.

1. Knockdown of an essential GSC gene in vitro using a targeted delivery platform.

• Screened several modification patterns of DRR AONs to determine the best

oligonucleotide sequence for DRR knockdown.

• Confirmed cell-surface staining of CD44 and EphA2 receptors, as well as efficient

internalization of CD44 and EphA2 antibodies in GSCs to support their suitability as

AON delivery vehicles to GSCs.

• Conjugated AONs to antibodies and confirmed successful conjugation via PAGE and

mass spectrometry.

• Delivered Ab-AON conjugates to GSCs and induced significant DRR downregulation

using CD44 antibody-mediated delivery.

• Visualized significant internalization of CD44 Ab-AON into GSCs and colocalization

with the endolysosomal pathway.

• Measured a significant difference in cell morphology following DRR knockdown,

potentially indicating a less invasive phenotype.

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These data were presented in Chapter 2 and published in Molecular Therapy-Nucleic Acids.

2. Achieve gene knockdown with enhanced endosomal escape.

• Confirmed HBEGF staining and low IC50 of native DT in GSCs, indicating the

suitability of the DT as a delivery vehicle for GSCs.

• Conjugated siRNAs to aDT using a freeze-thaw method and confirmed successful

conjugation using PAGE.

• Reduced eIF-3b expression in GSCs using aDT-eIF-3b and measured a corresponding

significant difference in cell viability following knockdown.

• Reduced ITGB1 expression in GSCs using aDT-ITGB1 and measured a corresponding

significant difference in cellular invasion in a 3D hydrogel model following knockdown.

These data were published in Chapter 3 and submitted for publication to Science Advances.

Recommendations for future work

In this thesis, novel delivery strategies for antisense therapeutics were developed and used to

target GSCs. Three areas of future work stem from the research conducted in this thesis:

investigation into the aDT-siRNA trafficking mechanism; optimization of specificity, potency,

and endosomal escape; and combination therapies with native toxins, therapeutic proteins, or

chemotherapeutic drugs.

6.1 Investigation into aDT-siRNA trafficking mechanism

Recently, Lacroix et al. have shown that it is difficult to draw any reliable conclusions about

trafficking from fluorescently labeled DNA nanostructures, because nuclease degradation can

separate the fluorophore from the nucleic acid material and give misleading information about its

intracellular location.358 While fluorescent labeling of the AON gave us some qualitative

information about trafficking as described in Chapter 2, we hesitated to fluorescently label the

siRNA bound to the aDT as described in Chapter 3 because it would be much more quickly

degraded than the AON and likely to give inaccurate trafficking information. However, there are

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still several methods that could allow insights into the trafficking mechanism of the aDT-siRNA

conjugate.

6.1.1 Inhibition of translocation

One assay that could prove translocation of the aDT is essential for siRNA delivery is inhibiting

the translocation of the toxin and proving that this eliminates the knockdown efficiency observed

for targets such as eIF-3b and ITGB1. One possibility would be to use a small molecule inhibitor

of endosomal acidification, which would inhibit the translocation of the aDT. Bafilomycin A1 is

a small molecule that has been used for this purpose,210 as it inhibits the ATP-mediated transport

of protons across the endosomal membrane.359 For example, it has been shown to reduce the

efficiency of gene delivery with poly(L-lysine) carriers, which rely on the acidification of the

endosome to escape the endolysosomal pathway.360 However, many of these small molecule

inhibitors of endosomal acidification, including Bafilomyin A1, are highly toxic because

endosomal trafficking is essential for the survival of cells,361 which limits the possibility for

downstream assays such as quantitative PCR or western blot.

A better option would be to express a mutant version of the aDT containing a point mutation in

the translocation domain that prevents the mechanism of translocation, and prove that the siRNA

delivery is no longer effective with the mutant toxin. The point mutation of a glutamic acid

residue to a lysine residue at amino acid position 349 (E349K) has been shown to strongly inhibit

cytotoxicity and translocation compared to native DT.311 A crystallographic model of DT shows

that Glu349 sits in a short loop connecting two long alpha-helixes of the translocation domain,

and the E349K mutation introduces a cationic amino acid at this site that interferes with the

mechanism of translocation.311 This method has been used to explore mechanisms of DT-

mediated delivery in the past; for example, Shaw et al. used the E349K mutant to demonstrate

that the delivery of T-cell antigens fused to DT was no longer effective with the mutant DT. This

strategy would require expressing the DT with the same structure as described in Chapter 2,

where there is an available cysteine for modification and pendant SUMO and His tags, and also

containing the E349K point mutation.

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6.1.2 IC50 shifts of modified DT

Another method to probe the delivery efficiency is to measure the shift in IC50 following

modification, if the native toxic domain is conserved during the modification process (ie. DT

instead of aDT).210 A higher IC50 would indicate a less efficient delivery, most likely from a

reduced capacity for either receptor binding or translocation. If the IC50 was conserved, it would

indicate that the cargo (ie. siRNA) is not interfering with the DT trafficking mechanism.

However, we were not able to use this strategy because a significant amount of unlabeled DT

(without siRNA) was present after synthesis and purification, and this unmodified DT would

interfere with any IC50 assays because it would retain the native IC50. In order to use this strategy

a labeling efficiency of at least 90% would be ideal.

We could have tried modifying the siRNA with the crosslinker first, instead of modifying the DT

with the crosslinker first to see if this different process of labeling could improve the reaction

efficiency and reduce the amount of unlabeled DT. The labeling of the siRNA with the

crosslinker, forming siRNA-maleimide, might be more efficient than labeling the DT with the

crosslinker, which formed DT-DBCO. However, even if this led to a more efficient conjugation

it is unlikely that we would reach >90% labeling of the DT with this method.

Another way to purify the DT-siRNA conjugate would be incorporate a tag for purification on

the distal end of the siRNA, for example a small peptide such as a Strep-tag that can be

recognized and purified using a streptavidin column.362 In this way, we could first use the Nickel

column to purify the DT from the siRNA, and then use a streptavidin column to purify the DT-

siRNA from the unlabeled DT. This would allow for downstream assays including

measurements of IC50 with the labeled DT, but could lead to a low recovery of the DT-siRNA

conjugate.

6.1.3 Split-reporter assays

Another way to localize the aDT fragment once it reaches the cytoplasm and confirm effective

delivery would be a split-reporter assay, where an enzyme or fluorescent reporter is split into two

parts. One part is included in the conjugate to be delivered and the other part is expressed in the

cytoplasm by the target cell population. Once the two parts rejoin, which requires successful

delivery of the conjugate into the cytoplasm, a signal such as fluorescence or luminescence can

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be detected. This has been used for reporters such as GFP, which fluoresces once it recombines

in the cytoplasm,363 or for luciferase, which can turn over its substrate following recombination

to give a bright luminescent signal.364 The first step would be to express a fusion toxin with the

split fragment and confirm effective delivery; the fusion protein could then be modified with a

cargo such as siRNA. This would be a very useful strategy if testing different variations of an

aDT-siRNA conjugate, since different levels of fluorescence/luminescence could be used to

compare efficacy of delivery.

6.2 Optimization of specificity, potency, and endosomal escape

Although the work in this thesis involved testing two distinct delivery vehicles, an antibody and

a bacterial toxin, there are many similarities between the two: they are both proteins that bind to

extracellular receptors, induce endocytosis, and can deliver oligonucleotides into the cytoplasm.

There are several ways that we could combine concepts learned using these two different

delivery vehicles to synthesize a targeted delivery vehicle that would escape the endosome and

deliver a highly potent oligonucleotide. In this section, four main ideas are proposed: re-targeting

the receptor binding domain of the aDT to target more specific cell populations; stabilization of

the siRNA to yield stable oligonucleotides that are still very potent; exploring nucleic acid

analogues (morpholinos or peptide nucleic acids) that could have the same action as

oligonucleotides but with different backbone character and better stability; and finally using

phenotypic assays as a first-line selection tool for the optimal oligonucleotide.

6.2.1 Re-targeting the receptor binding domain

As previously discussed, the native cell surface receptor of DT, HBEGF, is a promiscuous

marker and not specific to one cell population. To combine the specific targeting capacity of an

antibody with the trafficking and/or toxic properties of AB toxins, it is possible to replace the

receptor binding domain of the toxin with an antibody mimic called a single-chain variable

fragment (scFv). These scFvs can be engineered to bind to desired cell surface receptors in a

similar way to engineered antibodies.365 For example, this strategy has been used with DT

variants for the in vivo targeting of T cell lymphoma cells366 and even in the clinic to target B-

cell malignancies.367 A version of pseudomonas exotoxin A has been expressed with a CD133

scFv to specifically target CSCs in head and neck cancer;368 a similar strategy could be used to

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fuse a CD133 or CD44 scFv to DT to more specifically target the GSCs. However, transcytosis

of the DT across the blood brain barrier may be dependent on targeting HBEGF,217 so this trade-

off would have to be considered for future in vivo applications. However, a receptor mutant

could be propelled across the blood brain barrier by recent methods such as focused

ultrasound,369 or this re-targeting strategy could be used to target cancers in tissues other than the

brain.

6.2.2 Stabilization of the siRNA

As described in Chapter 3, the siRNAs that were tested against the gene targets eIF-3b and

ITGB1 were extremely potent for knockdown, reducing the mRNA expression by 80-90%.

However, these siRNAs were not very stable: we showed that almost all of the siRNA is

degraded by 24h when exposed to serum nucleases. These siRNAs were Dicer-substrate siRNAs,

meaning they were 27-mer RNA duplexes that must be recognized by an enzyme called Dicer to

be effective in the cells. Different modification patterns of these siRNAs should be explored to

determine structure-functional relationships between modifications and siRNA properties,

thereby optimizing the stability of the siRNAs while maintaining or increasing their potency.

A significant amount of work has already been done to optimize modification patterns for Dicer-

substrate siRNAs. Chan et al. used siRNAs with 2’-fluoro-modifications and observed similar

potency to unmodified siRNAs.370 Allerson et al. demonstrated that a combination of 2’-fluoro

and 2’-methoxy modifications to the siRNA was more effective than unmodified siRNA.325

Collingwood et al. showed that siRNAs with 2’-methoxy-based modifications were better at

evading immune activation than 2’-fluoro modifications and siRNAs containing 2’-methoxy

modifications were generally more potent than siRNAs containing 2’-fluoro modifications,

although these effects appeared to be target- and sequence-specific.371 Importantly, Patisiran, the

first FDA-approved siRNA therapeutic, incorporates siRNA with some combination of

unmodified and 2’-methoxy modified ribonucleosides.326 Thus, the most potent modification

pattern probably incorporates primarily 2’-methoxy modifications, although there is a whole

library of additional modifications that could be explored,89 and these may have to be re-

optimized for each target/sequence tested.

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6.2.3 Exploring RNA analogues: morpholinos and peptide nucleic acids

A major concern when conjugating oligonucleotides to the aDT was that the negative nature of

the phosphodiester linkages within the RNA or DNA would interfere with the translocation of

the cargo. While the aDT was able to deliver siRNA, a backbone with a more neutral charge

might be more easily shuttled through the translocation pore. There are several options for

neutrally charged backbones with the same base-pair capacity as nucleic acids, including

morpholinos, where the 5-membered ring of RNA or DNA is replaced with a 6-membered

morpholino ring and the phosphodiester linkages are replaced by phosphorodiamidate

linkages;372 or peptide nucleic acids, where the DNA/RNA backbone is completely replaced by a

peptide backbone, but with the same pendant bases as a nucleic acid.373 The structures of these

analogues are shown in Figure 6.1. These structures have key advantages aside from their neutral

backbone: they are highly stable to nuclease degradation because they are not recognized in the

same way as DNA/RNA;374-375 and they can still be potent for gene knockdown in a sequence-

specific manner with a mechanism similar to AONs.374, 376 Conjugation of the morpholinos or

peptide nucleic acids would be done in a similar manner as the siRNA conjugation presented in

this thesis, and it could be an exciting way forward to achieve gene knockdown with a much

more stable biomolecule.

Figure 6.1. Structures of nucleic acid analogues including morpholinos and peptide nucleic

acids.

O Base

OPO OO

O Base

O

O

5’

OH

OH

3’

RNA

O

N

O Base

OPO OO

O Base

O

O

5’

3’

DNA

O

5’

PO NO

O

NO

3’

Base

Base

Morpholino

N

NH

N

O

NHO

N

N-terminus (5’)

C-terminus (3’)

O

Base

O

Base

O

Base

Peptide Nucleic Acid

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6.2.4 Phenotypic assays as a first-line screening tool for oligonucleotide

sequences

As discussed in section 4.1, there was a negative correlation between stability and potency for

the oligonucleotides tested in this work. This means that either the stability or potency was a

constant limitation. For most of this work, we used potency as a screening tool to pick the best

oligonucleotide; however, stability and duration of silencing also play significant roles in

achieving the desired effect when delivered to cells.

Screening antisense therapeutics on the basis of a phenotypic effect, such as cell death or

invasion as were used in this work, or any other desired phenotypes, would be a more rational

selection strategy wherever possible. For example, several modification patterns of the ITGB1

siRNA could be tested in the 3D hydrogel platform and the most efficient oligonucleotide would

give the strongest reduction in invasion. Once the best phenotypic outcome is observed, it would

be possible to work backwards and determine the best combination of stability and potency in

order to achieve a desired effect. In addition, screening in this way would provide greater

confidence in downstream phenotypic assays when delivering the oligonucleotides using novel

platform technologies.

6.3 Simultaneous delivery of siRNA and other therapeutics

An extension of this work would be to use the aDT delivery platform developed in this thesis and

combine it with the delivery of other therapeutics. Most of the literature concerning AB toxins

for delivery has focused on delivering protein- or peptide-based therapeutics, so these types of

cargo could be combined with the siRNA delivery for therapy of many diseases, not only cancer.

Small-molecule drugs, such as chemotherapeutics, could also be combined with the siRNA

delivery to increase the therapeutic effect. This section will suggest several combination

therapies that draw on both the ideas developed in this thesis as well as previous work that has

been done to explore aDT as a delivery vehicle.

6.3.1 Combination with native toxin domains

Fusion toxins combining the toxic active domain of DT with re-directed receptor binding regions

have already been explored for the treatment of GBM. For example, Weaver et al. fused

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transferrin, which is highly expressed in GBM, to the translocation and toxin domains of DT for

the treatment of malignant brain tumors and observed tumor response without significant off-

target toxicity in at least a third of patients in a phase I/II clinical trial.377 The work with DT

presented in this thesis was with an attenuated DT, where the active domain was mutated to

eliminate its cytotoxicity; this was important so that we could isolate the effects of siRNA

delivery. However, a future strategy could involve keeping the native toxic domain while also

conjugating an oligonucleotide for gene knockdown; with careful selection of the gene target,

this could result in synergistic effects between the toxin and the siRNA/AON for improved

toxicity towards cancer cells.

6.3.2 Combination with therapeutic proteins

Another delivery strategy that has been heavily pursued is the delivery of therapeutic proteins

that are depleted in diseased cells using attenuated toxins. An attenuated toxin variant could be

engineered that could bind to a desired receptor and maintain its translocation capacity, but it

would also contain a cysteine group for siRNA conjugation and also be expressed as a fusion

protein with a therapeutic protein domain of interest. For example, in cancer treatment, the toxin

could be used to carry both a therapeutic siRNA as well as a protein that would act as a tumor

suppressor such as caveolin-1 which represses apoptosis inhibitors.378

This combination delivery of therapeutic proteins and siRNA could also be a sophisticated

approach for complex diseases other than cancer, such as Alzheimer’s disease (AD). There are

many mis-regulated genes at work in AD, including downregulation of the MAPK/ERK pathway

and upregulation of clathrin proteins, which lead to increased internalization of amyloid

precursor proteins (APPs) and increased intracellular levels of amyloid beta plaques.379 Recent

studies have suggested that targeted RNA interference may be able to limit the progression of

AD, such as the study by Rodriguez-Lebron et al. which showed that short-hairpin RNA against

APP could reduce AD phenotypes in a mouse model.380 At the proteome level, brain-derived

neurotrophic factor (BDNF) plays an important role in synaptic plasticity and memory formation

and abnormalities in its expression have been reported in AD patients.381 Shin et al. delivered

Neuropep-1, a peptide that increases intracellular levels of BDNF, to a mouse model of AD and

observed increased BDNF expression and dramatically reduced amyloid beta plaque formation, a

promising sign of an effective AD treatment strategy.382 aDT could be engineered to carry

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siRNA against APP in combination with Neuropep-1 or BDNF for a dual-factor treatment

strategy against Alzheimer’s disease. Of course, this is just one example of a dual delivery

strategy using the aDT platform; it could be used for virtually any disease where it is important

to downregulate expression of some genes and also introduce therapeutic proteins into cells.

6.3.3 Combination with chemotherapeutic drugs

aDT could be used to deliver other chemotherapeutic drugs alone or in combination with siRNA.

There is abundant literature where antibodies are used as carriers for small-molecule

chemotherapeutics; in a similar manner, aDT could be conjugated to chemotherapeutic drugs and

siRNA for co-delivery. This could result in synergistic effects if the siRNA target was chosen to

modulate the response of the cells to the chemotherapeutic drug. Chemotherapeutic resistance is

a significant challenge in cancer treatment and often arises from underlying genetic mutations.383

Triple-negative breast cancer often acquires chemotherapeutic resistance through over-

expression of the multidrug resistance protein 1 (MDR1), and siRNA-mediated knockdown of

MDR1 can lead to enhanced susceptibility of the tumor cells to common chemotherapeutics such

as doxorubicin; aDT could simultaneously deliver MDR1 siRNA and doxorubicin to cancer

cells, overcoming their chemotherapeutic resistance while simultaneously delivering a potent

chemotherapy drug. In GBM, resistance of DNA damage by TMZ is mediated by poly(ADP-

ribose) polymerase-1 and -2 (PARP-1 and -2), and inhibition of PARP can increase the

sensitivity of GBM cells to TMZ.384 Although TMZ is not the most potent chemotherapeutic,

aDT could be used to deliver PARP siRNA and TMZ synergistically and this would align with

the current standard of care of TMZ chemotherapy.

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Abbreviations

AD Alzheimer's disease

ADC Antibody-drug conjugate

aDT Attenuated diphtheria toxin

Akt Protein kinase B

ALDH1A3 Aldehyde dehydrogenase 1A3

AON Antisense oligonucleotide

ApoE Apolipoprotein E

APP Amyloid precursor proteins

AT Anthrax Toxin

BBB Blood-brain-barrier

BCL-2 B-cell lymphoma 2

BDNF Brain-derived neurotrophic factor

ccRCC Clear cell renal cell carcinoma

CD133 Cluster of differentiation 133

CD15/SSEA1 Cluster of differentiation 15/stage-specific embryonic antigen 1

CD171/L1CAM Cluster of differentiation 171/L1 cell adhesion molecule







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CPP Cell penetrating peptide

CSC Cancer stem cell

DNA Deoxyribonucleic acid

DRR Downregulated in renal cell carcinoma

DT Diphtheria toxin

EGFR Epidermal growth factor receptor

eIF-3b Eukaryotic translation initiation factor 3 subunit B

EphA2 Ephrin receptor A2

EphA3 Ephrin receptor A3

FAK Focal adhesion kinase

FAM107A Family with sequence homology 107A

FANA 2ʹ-deoxy-2ʹ-fluoro-beta-D-arabinonucleic acid

FDA Food and drug administration

GalNAc N-acetylgalactosamine

GBM Glioblastoma

GO Gemtuzumab ozogamicin

GSC Glioblastoma stem cell

HBEGF Heparin-binding epidermal growth factor receptor

HER2 Human epidermal growth factor receptor 2

HIR Human insulin receptor

HIV Human immunodeficiency virus

HRP Horseradish Peroxidase

IDH Isocitrate dehydrogenase

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IgG Immunoglobulin G

ITGB1 Integrin beta 1

JAK Janus kinase

KRAS Kirsten rat sarcoma viral oncogene homolog

mAb Monoclonal antibody

MALDI-TOF Matrix-Assisted Laser Desorption/Ionization-Time Of Flight

MAPK Mitogen-activated protein kinase

MDR1 Multi-drug resistance protein 1

MFI Median fluorescent intensity

MGMT O6-methylguanine-DNA methyl-transferase

MMAF Monomethyl auristatin F

MMP Matrix metalloprotease

mRNA Messenger RNA

mTOR Mammalian target of rapamycin

MXD3 MAX dimerization protein 3

NADPH Nicotinamide adenine dinucleotide phosphate hydrogen

NHS N-hydroxysuccinimide

NLS Nuclear localization signal

PAGE Polyacrylamide gel electrophoresis

PARP Poly(ADP-ribose) polymerase

PCL Poly(caprolactone)

PCR Polymerase chain reaction

PDI Protein disulfide isomerase

PE Pseudomonas exotoxin A

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PEG Poly(ethylene glycol)

PEI Polyethyleneimine

PFS Progression-free survival

PI3K Phosphoinositide 3-kinase

PLGA Poly(lactic-co-glycolic) acid

PLO Poly(L-ornithine)

PNP Human purine nucleoside phosphorylase

PO-DNA Phosphodiester backbone DNA

PS-DNA Phosphorothioate backbone DNA

RFP Red fluorescent protein

RISC RNA-induced silencing complex

RNA Ribonucleic acid

RNAseH Ribonuclease H

siRNA Small interfering ribonucleic acid

STAT Signal transducer and activator of transcription proteins

TAT Transactivator of transcription

TMZ Temozolomide

TP10 Transportan 10

TRPC6 Transient receptor potential channel 6

VEGFR1 Vascular endothelial growth factor receptor-1

Wnt Wingless-related integration site

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Appendix A: Supporting information for “Antibody-antisense

oligonucleotide conjugate downregulates a key gene in

glioblastoma stem cells”

A.1 Melting Point Determination

Equimolar amounts (1.5 nmol) of complementary sequences were combined and dried. To the

resulting pellet was added 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl

and 0.1 mM EDTA (1 mL). They were then transferred into cuvettes in a Varian UV

spectrophotometer. The samples were heated to 90 °C and then cooled to 5 °C. The change in

absorbance at 260 nm was then monitored upon heating from 5 to 90 °C at a rate of 0.5 oC/min.

The dissociation temperatures were calculated as the midpoint of the transition (T1/2) using the

first derivative of the melting curve.

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Figure A.1. Melting point determination between the DRR sense strand and the Gapmer and

Gapmer MeC Strands. (A) Melting curve of 28E1-15E1. (B) Melting curve of 28E1-

15E1(5MeC). (C) Table with duplex IDs, descriptions, sequences, and calculated Tm.

Figure A.2. Internalization of CD44 and EphA2 mAbs at 15, 45, and 90 mins determined via

flow cytometry. No significant differences between time points were observed. 45 mins was

chosen as the comparison point between CD44 mAb, EphA2 mAb, and CTL as there is a trend

towards the most internalization at 45 min for the CD44 mAb (Figure 3). Data was analyzed

using two-way-ANOVA followed by Sidak’s post-hoc test. Data is shown as mean+SD, n=3.

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Figure A.3. (A) MALDI-TOF mass spectrometry analysis of CD44 mAb (Mobt = 150.2 kDa vs

Mth = 150.0 kDa). (B) MALDI-TOF mass spectrometry analysis of CD44 mAb-DRR sense

strand conjugate. For CD44-DRR sense strand, two shifted peaks were obtained: the first

represents the azide-modified CD44 (Mobt = 154 kDa vs. Mth = 150.8 kDa) and the second for the

CD44-DRR sense strand (Mobt = 160.0 kDa vs. Mth = 160.2 kDa).

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Figure A.5. Comparison of uptake at 75 nM vs. 150 nM for EphA2 mAb-dsDRR and CD44

mAb-dsDRR. DsDRR only is shown as a negative control. A significant increase in uptake was

observed for both EphA2 mAb-dsDRR and CD44 mAb-dsDRR at 150 nM. Cell nucleus

(Hoechst, blue); AON (Cy3, green); lysosome (Dextran647, red). Representative z-stack images

shown. All scale bars are 50 µM.

Table A.1. Mass analysis of the oligonucleotide strands used in this study. High-resolution mass

spectrometry (HRMS) acquisition was performed in negative ion mode using electrospray

ionization (ESI).

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Oligo ID Expected Mass Obtained Mass

Gapmer 6476.9 6478.3

Gapmer MeC 6490.9 6492.2

Altmer 6476.9 6478.3

Altmer MeC 6490.9 6492.2

Scrambled 6431.1 6430.5

Scrambled (Sense) 8689.4 8690.3

DRR (Sense) 8870.5 8869.8

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Appendix B: Supporting information for “Attenuated diphtheria

toxin mediates siRNA delivery”

Supplemental Methods

Preparation of aDT-siRNA conjugate (incubation at 37 °C)

The synthesis was performed as described in the main methods, with the exception of the

incubation at -20 °C. Instead, the mixture was held at 6h or 24 h at 37 °C with between 1 and 10

equivalents of oligonucleotide to 1 equivalent of diphtheria toxin.

Nuclease stability assay

siRNA or aDT-siRNA was incubated with a solution of 10% FBS in 20 mM Tris, 150 mM NaCl,

1% glycerol, pH 7.5 for 1, 4, 8, or 24 h. The entire contents of the sample were analyzed using

acrylamide electrophoresis to separate nucleic acid material based on size. The remaining full

length siRNA was quantified by imageJ and the data was reported as a percentage of intact

siRNA.

IC50 of native diphtheria toxin

Cells were plated to reach 60-80% confluency on 96-well Primaria plates (Corning 353872).

Native diphtheria toxin was added at varying concentrations to cells in full media and the cells

were incubated with the treatment for 48 h. At 48 h, PrestoBlue Cell Viability reagent (Thermo

Fisher A13262) was added for a final concentration of 10% in media and the viability was

measured by reading the fluorescent signal in each well. The IC50 was estimated using an online

calculator (aatbio.com/tools/ic50-calculator).

Cell viability assays following transfection with eIF-3b siRNA

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siRNA and Lipofectamine RNAiMAX were incubated together with Opti-MEM for the desired

final concentration. The transfection complex was incubated at RT for at least five minutes then

added to 96-well plates coated with PLO/laminin. Cells were then added to the plates with the

transfection solution at a density of 8,000 cells per well. Fresh media was added to each well 24h

following treatment. At 72 hours, cell viability was measured via PrestoBlue viability assay and

quantified as a percentage of untreated controls.

Figure S1. Incubation at 37 °C leads to inefficient aDT-siRNA conjugation. A) Coomassie-stained SDS-PAGE demonstrating conjugation of DT with different equivalents of oligonucleotides conjugated for 6 or 24h at 37 °C. B) Quantification of % conjugation of aDT to

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siRNA at different equivalents and different incubation times. Green star represents approximate conjugation efficiency using the freeze-thaw method presented in the main methods (~50-60%).

Figure S2. aDT conjugation does not change siRNA susceptibility to nuclease degradation. A) Coomassie-stained SDS PAGE gel shows that siRNAs are degraded in serum-containing solutions over a period of 24 hours. The lanes show the 0, 1, 4, and 8 h timepoints (left-right). No band was detected at 24 h. B) Quantification of remaining intact siRNA at 1, 4, and 8 hours demonstrates no significant difference between the degradation of siRNA and aDT-siRNA. Data is shown as n=3, mean±SD, and was analyzed using 2-way-ANOVA.

Figure S3. Native diphtheria toxin mechanism is conserved in GSCs. The IC50 of native diphtheria toxin in HB-EGF was determined; data is shown as cell viability normalized to an untreated control, n=3, mean+SD.

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Figure S4. Transfected eIF-3b siRNA reduces cell viability at 72h in GSCs. Cells were transfected with Lipofectamine RNAiMAX and cell viability was measured at 72 h with the siRNA for eIF-3b (eIF-3b +CTL, positive control) vs. the non-targeting siRNA control (NT CTL). Data are reported as n=3, mean+SD and was analyzed using an unpaired t-test (**p<0.01).

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Appendix C: Effect of sugar 2’,4’-modifications on gene silencing

activity of small interfering RNA duplexes

This Appendix was published in Nucleic Acid Therapeutics:

Malek-Adamian, E.; Fakhoury, J.; Arnold, A. E.; Martínez-Montero, S.; Shoichet, M. S.; Damha,

M. J. Effect of Sugar 2’,4’-Modifications on Gene Silencing Activity of Small Interfering RNA

Duplexes. Nucleic Acid Ther 2019.

E.M.-A. synthesized and characterized all oligonucleotide materials and wrote the manuscript.

A.E.A. and J.F. performed cell experiments and edited the manuscript. S.M.-M. conceptualized

fundamental work on the project and provided feedback on the manuscript. M.J.D. and M.S.S.

secured funding, supervised and guided the research, and participated in writing the manuscript.

ABSTRACT

In this study, we explore the effect of a library of 2’-, 4’-, and 2’,4’-modified uridine

nucleosides and their impact on silencing firefly luciferase and on Downregulated in Renal Cell

Carcinoma (DRR) gene targets. The modifications studied were 2’-F-ribose, 2’-F-arabinose, 2’-

OMe-ribose, 2’-F,4’-OMe-ribose, 2’-F,4’-OMe-arabinose, and 2’-OMe,4’-F-ribose. We found

that 2’,4’-modifications are well tolerated within A-form RNA duplexes, leading to virtually no

change in melting temperature as assessed by UV thermal melting. The impact of the dual

(2’,4’) modification was assessed by comparing gene silencing ability to 2’- or 4’- (singly)

modified siRNA counterparts. siRNAs with (2’,4’)-modified overhangs generally outperformed

the native siRNA as well as siRNAs with a 2’- or 4’-modified overhang, suggesting that 2’,4’-

modified nucleotides interact favorably with Argonaute protein’s PAZ domain. Among the most

active siRNAs were those with 2’-F,4’-OMe-ribose or 2’-F,4’-OMe-arabinose at the overhangs.

When modifications were placed at both overhangs and internal positions, a duplex with the 2’-F

(internal) and 2’-F,4’-OMe (overhang) combination was found to be the most potent, followed

by the duplex with 2’-OMe (internal) and 2’,4’-diOMe (overhang) modifications. Given the

nuclease resistance exhibited by 2’,4’-modified siRNAs, particularly when the modification is

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placed at or near the overhangs, these findings may allow the creation of superior siRNAs for

therapy.

INTRODUCTION

Exploiting the therapeutic potential of RNAi has been fueled with the recent approval of

Patisiran, the first siRNA-based drug approved for the treatment of hereditary transthyretin

amyloidosis.326 This siRNA is comprised of unmodified and 2’-OMe pyrimidine nucleotides

that require encapsulation in a lipid-based nanoparticle for efficient delivery to the target organ

[326,2]. The pursuit of novel chemical modifications that improve the biochemical (nuclease

resistance, potency) and biophysical properties of oligonucleotides continues to be an important

endeavour towards the development of more effective siRNA drugs that do not require a lipid-

complex formulation. In this regard, although the 2’-F modification is widely introduced into

siRNAs, it provides minimal stabilization towards nuclease degradation. Modifications that

retain some of the properties of 2’-F nucleotides while increasing nuclease resistance are desired.

In a collaborative study, we reported recently on the synthesis, conformational analysis and gene

silencing activity of 2’-F,4’-OMe modified siRNAs in cultured cells, and showed that they

promote efficient gene silencing [7]. More recently, our laboratory reported on the synthesis and

hybridization properties of duplexes containing a variety of other 2’,4’-modified uridine

nucleosides, e.g., 2’,4’-di-OMe, 2’-OMe,4’-F, and ara-2’-F, 4’-OMe uridines[3]. As previously

found with 2’-F, 4’-OMe uridine, this new library of uridines exhibited primarily a C3’-endo

(“North”) sugar conformation[4] and their incorporation into siRNAs led to virtually no change

in duplex melting temperature [3,5]. This property, together with the observation that C2’/C4’

modifications impart much desirable resistance towards endo/exo nuclease degradation [6-8],

prompted us to evaluate whether these analogues are compatible with RISC and able to regulate

gene expression via the RNAi pathway. Herein we report studies which assess the gene

silencing efficiency of C2’/C4’ modified siRNA duplexes (Figure C.1). Modifications were first

introduced at either internal (positions 6, 13 and 14 of the guide strand) or terminal positions

(positions 20 and 21 of the guide strand); the most potent modifications were then combined to

produce siRNAs with modifications at both overhangs and internal positions. Of the various

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modifications tested, duplexes with a C2’-F/OMe and C4’-OMe modified ribose sugar at the 3’-

overhangs were among the most potent.

Figure C.1. A) Sequence of siRNA targeting luciferase mRNA. Modified nucleotides were

introduced at positions 6, 13, and 14 and 3’-overhang of the antisense (guide) strand; B) 2’,4’-

modified nucleosides in this study.

MATERIALS AND METHODS

Oligonucleotide Synthesis. All 2’,4’-modified phosphoramidites and oligonucleotides were

synthesized as previously reported [3]. The 4’-OMe-dT phosphoramidite was synthesized from a

C4’-C5’ alkene dT precursor according to Liboska et al. [9] (Section S3, Supplementary

Information). Oligonucleotides were purified by ion exchange HPLC (1 M lithium perchlorate

buffer) or by PAGE (22% acrylamide), and desalted as previously described [7]. All

oligonucleotides were characterized by ESI or QTOF mass spectrometry (Table S1,

Supplementary Information).

Thermal Denaturation Studies. Complementary sequences were combined in equimolar

amounts (1.5 nmol), dried, and diluted in a buffer containing 10 mM sodium phosphate (pH 7.2)

with 100 mM NaCl and 0.1 mM EDTA (1 mL). Each solution was then transferred into cuvettes

in a Varian UV spectrophotometer. Each sample was heated to 93 °C and the cooled to 15 °C at

a rate of 0.5 oC/min. The change in absorbance at 260 nm was then monitored upon heating from

15 to 93 °C. The melting temperatures were calculated from the first derivative of the melting

curves (local maxima at dy/dt=0).

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Luciferase Assays. HeLa cells stably expressing Luciferase were counted and seeded at a

density of 7500 cells/well in a 96-well plate. Cells were then left to recover for 24 hours at 37 °C

with 5% CO2. Subsequently, cells were washed once with serum-free DMEM media and then 80

µl of serum-free DMEM media was added. siRNA and control nucleic acid preparations were

prepared by heating at 93 oC in a heat block in 1x siRNA buffer (instructions for preparation by

Dharmacon) and were allowed to anneal slowly followed by overnight cooling at 5 oC in a

fridge. siRNA duplexes were then diluted up to 20 µl with serum-free media and transfection

reagent (Oligofectamine, Invitrogen) and added to the appropriate well (for a total of 100 uL) at

increasing concentrations (0.16, 0.8, 4, 20, and 100 nM). Cells were incubated overnight (for a

total of 24 hours post-siRNA addition). Then 50 µL of ONE-Glo luciferin reagent (Promega,

USA) was added to each well and luminescence was measured and normalized to protein levels

using a Biotek Synergy HT plate reader. Data was acquired with the Gen5 software suite and

data was manipulated and plotted using Graphpad Prism software suite.

DsRed DRR Cell Transfection. This protocol was adapted from Anzahaee et al. [10]. Briefly,

DsredDRR cells were seeded in a 6-well or 24-well plate such that they would reach 60-80%

confluency at the time of transfection. siRNAs were complexed to Lipofectamine RNAiMax

(Thermo 13778075) for five minutes in Opti-MEM media (Thermo 31985062) according to the

manufacturer’s reagent protocol and added to the cells in DMEM media (Gibco 11995605) for a

final DMEM:Opti-MEM ratio of 1.5:0.5 and desired siRNA concentration. At 24 h, additional

serum-containing DMEM media was added. After a total of 72 hours of incubation at 37 oC, cells

were collected and lysed using 0.1% NP-40 (Fluka 74385). Protein expression was assessed via

western blot analysis of DRR with alpha-tubulin as a loading control.

MTS Assay. Cytotoxicity of tested nucleosides was assayed by measuring cell viability using the

Cell Titer Blue Assay (Promega). Briefly, HeLa cells were seeded at 5,000 cells/well in a 96-

well plate. The following day, samples were prepared at stock concentrations starting at 1 M in

autoclaved H2O, and were serially diluted 10-fold down to 1 µM. 10 µL of each stock solution

was then added per well at the desired concentration to be incubated with HeLa cells for 24

hours. Incubations were performed in triplicate for each concentration. After overnight

incubation, 20 µL of Cell-Titer Blue reagent was added per well and sample fluorescence was

measured using a Biotek Synergy Plate Reader (Excitation = 560 nm and Emission = 590 nm)

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after 1 hour following addition. Analysis was performed using Graphpad Prism software and

samples were normalized to the buffer control.

RESULTS AND DISCUSSION

siRNAs with internal 2ʹ/4ʹ-modifications in the guide strand

Melting temperature of internally modified duplexes were studied first using a model siRNA

duplex targeting firefly luciferase as previously studied [11]. This model siRNA was chosen due

to its modification pattern which allocates a modified nucleotide in the sensitive seed region

(position 6) and allows exploration of the impact of two centrally located and consecutive

modified nucleotides (positions 13 and 14). Each duplex was analyzed by UV thermal melting in

order to assess how these modifications affected duplex formation. In all cases, no distortions in

duplex formation were noted during the melting-annealing cycle (Section S1, Supplementary

Information). Relative to the native siRNA, Tm values of duplexes increased upon incorporation

of modified ribouridines and 4’-OMe-dT (+1-2 oC/nt), whereas a slight decrease (ca. -0.3-0.7 oC/nt) was observed for the arabinose modifications (Table 1; Figure S1-S3, Supplementary

Information). As previously observed, duplexes with 2’,4’ modifications were more stable than

the unmodified duplexes (+0.8-1.4 oC/nt), although they were not as stable as duplexes with 2’-

modifications at the same positons [3,7,11].

Table C.1. Melting Temperature of Luciferase siRNA Duplexes with Internal Modifications at Positions 6, 13, and 14 in the Guide Strand

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Duplexes were transfected into HeLa cells stably expressing firefly luciferase, and the

reduction of firefly luciferase protein levels was evaluated in a dose-response manner (Figure

C.2). The best activity was granted by the 2’-OMe-rU modification, performing similarly to rU.

As previously observed with 2’,4’-diF-rU [11], siRNAs with 2’-F,4’-OMe-rU and 2’-OMe,4’-

F-rU modifications also conferred excellent activity. By contrast, decreased activity was

observed with 2’,4’-diOMe-rU and 2’-F,4’-OMe-araU modifications, in line with observed

results when incorporating LNA at these positions [11]. The negative impact of 2’,4’-diOMe-rU

on gene silencing was not expected given the excellent activities of siRNAs with 2’-OMe-rU or

4’-OMe-dT at the same positions. This may reflect possible disruption of hAGO2-RNA

interactions caused by the bulkier 2’,4’-diOMe-rU residues [12-14].

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Figure C.2. Luciferase assay results of siRNA with internal 2’,4’ modifications at positions 6,

13 and 14 of the guide strand. A) Sequence design of the siRNA. Locations of modified

nucleotides are underlined. B) Activity of siRNA duplexes. The concentrations used (organized

left-right are 0.16 nM, 0.8, 4, 20, and 100 nM, respectively. C) Relative gene silencing activity

of duplexes at 4 nM concentration. 4’-OMe refers to the 4’-OMe-dT modification. SCRM CTL

is the scramble negative control.

siRNAs with 2ʹ/4ʹ-modifications at the 3′-overhang of the guide strand

Structural studies by Patel and co-workers revealed that the PAZ domain from human Argonaute

serves as a binding module and anchoring site for the 3′ end of guide RNA, requiring an essential

2-nt, single-stranded segment [15]. To study the potential interaction of our 2’,4’-modifications

with the PAZ domain, they were introduced at the 2-nt 3′-overhangs of several siRNA duplexes

(positions 20 and 21 of the guide strand). Their gene silencing activity was measured at a range

of concentratons from 0.16, 0.8, 4, 20, to 100 nM, and depicted in Figure C.3.

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Figure C.3. Luciferase assay of duplexes with indicated modifications at overhang positions. A)

Sequence of the siRNA; B) Activity of siRNAs modified at positions 20 and 21 of the guide

strand. The concentrations used are 0.16 nM, 0.8, 4, 20, and 100 nM (organized left to right),

respectively. C) Relative gene silencing activity of duplexes at 4 nM concentration. Sequence

of the native siRNA is shown in Figure 2 and contains a dGG 3’-overhang. 4’-OMe refers to the

4’-OMe-dT modification. SCRM CTL is the scramble negative control.

The slightly better activity of ara-2’-F vs 2’-H had previously been reported by Dowler et al., and

is fully consistent with the present results [16]. Regarding the 2’,4’-modified siRNAs, the

instructive findings were that overhangs with C4’-α-OMe substituted furanoses provided the

most active duplexes across the series. This suggests that the hydrophobic pocket of the PAZ

domain interacts favorably with C4’-O-methyl (and even C4’-fluorine) substituents. For

example, the following trends in activities were observed: 2’-F-4’-OMe > 2’-F; also, ara-2’-F-

4’-OMe > ara-2’-F; furthermore 2’,4’-diOMe > 2’-OMe; also ara-2’-F-4’-OMe > ara-2’-F.

Further contribution to the activity of these duplexes may arise from the enhanced nuclease

stability provided by the C4’-substituent which hinders hydrolysis of the vicinal 3’,5’-

phosphodiester linkage [7].

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siRNAs combining internal and overhang 2’/4’-modifications

Next, we sought to combine some of the best performing internal modifications (modifications at

positions 6, 13 and 14) with the best performing overhang modifications (modifications at

positions 20 and 21) with the aim of maximizing chemical modification without compromising

activity (Table 2). The sequence and melting temperatures of the duplexes prepared are provided

in Table 2, and their activity summarized in Figure C.4. For comparison, the Tm and gene

silencing activity of these duplexes were compared to siRNAs containing modifications at

overhangs or internal positions.

Table C.2. Melting Temperature of Luciferase siRNA Duplexes with a Modified Guide Strand.

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Figure C.4. Luciferase assay of duplexes modified at at positions 6, 13, 14, 20 and 21. A)

Sequence of the siRNA; B) Activity of modified duplexes. The concentrations used are 0.16 nM,

0.8, 4, 20, and 100 nM (left to right), respectively; C) Relative gene silencing activity of

duplexes at 4 nM concentration. Sequence of the native siRNA is shown in Figure 2 and

contains a dGG 3’-overhang. 4’-OMe refers to the 4’-OMe-dT modification. SCRM CTL is the

scramble negative control.

UV experiments were performed in order to verify if the additional modifications affected the

melting temperature of the duplexes (Table 2, Figures S1-S3). Interestingly, the Tm of duplexes

with combined modifications at positions 6, 13, 14, 20 and 21 displayed similar or slightly higher

values compared to duplexes with internal modification at positions 6, 13, and 14 (DTm = + 0.3-

3.0 oC). It is also interesting to note that the modified rPy overhangs tested at positions 20 and

21, namely 2’-F-4’-OMe-rU, 2’,4’-diOMe-rU and 2’-F-4’-OMe-araU all provided significant

increases in Tm (+2.2 oC /nt) relative to the duplex with a native rPu (dGG) overhang. While the

origin for this difference cannot be reconciled with the data available, the larger stabilizing effect

of the modified rPy ‘dangling’ ends may be attributed to an overall more cross-stacking of the

modified residues which better conform to A-form structure of the siRNA duplex [17]. In other

words, the propensity of the unstacked dangling nucleotide to a stacked geometry should be

112

easier for a nucleotide pre-organized in the A-like C3’-endo conformation (such as 2’,4’-

modified rU) relative to a more flexible deoxynucleotide.

Regarding gene silencing activity, the combinations of modifications afforded siRNAs with

potencies similar to those seen for siRNAs with a solely overhang or a solely internally modified

guide strand. This implies that a desired level of activity can be achieved while maintaining a

high level of modification necessary for nuclease protection and immune system evasion.

Activity of the combination of best internal and overhang motifs against DRR gene

To validate the generality of our observations, we designed a new set of siRNAs targeting the

Downregulated in Renal (DRR) Cell Carcinoma gene as previously reported [18,19]. The DRR

protein contributes to the aggressive and invasive nature of glioblastoma stem cells by cross

linking microtubules and actin at focal adhesions and activating downstream pathways of Protein

Kinase B [18]. Knockdown of the DRR gene is a promising therapeutic strategy for decreasing

the invasion of brain cancer stem cells [19,20]. One duplex contained 2’-F-4’-OMe-rU residues

at the 2-nt 3’-overhang of the guide strand; the other contained three additional 2’-F -rU residues

at internal positions. Their sequences are shown in Table 3, along with that of the unmodified

siRNA control. DsedDRR cells were transfected with modified and control siRNA duplexes.

All siRNA duplexes targeting the DRR gene provided significant knockdown compared to the

scrambled control; the DRR positive CTRL duplex was the most active, followed by the siRNA

duplex with modified overhangs (Figure C.5). The least active was the most modified (112-2),

however it still exhibited excellent activity (IC50 ca. 4 nM) under the conditions tested.

Table 3. Sequences of Down-Regulated in Renal Cell Carcinoma (DRR) siRNAs.

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Figure C.5. DRR knockdown following transfection of siRNAs with overhang or combined

internal and overhang modifications in DsredDRR cells. A) Assay results grouped by

modification type. B) Relative gene silencing activity of duplexes at 4 nM concentration . C) Gel

image of the Western Blot analysis grouped by concentration. SCRM CTL is the scramble

negative control.

Metabolic activity of HeLa cells upon treatment with excess modified nucleosides

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A common concern with chemically modified nucleotides is that if they were to be excised by

the strand by endogenous nucleases, especially at vulnerable positions for cleavage such as at the

3’-end, they could potentially cause toxicity [21]. We addressed this concern by performing a

cell viability assay of each nucleoside in this tested library at concentrations up to 0.1 M with a

MTS assay in HeLa cells, and none other than 2’,4’-diOMe-rU was shown to reduce

significantly cell viability at this concentration (Figure C.6). It is important to note that 0.1 M

also far exceeds physiological conditions within cells, where dUTP in mammalian cells normally

hovers at ca. 0.5 mM [22]. Indeed, it was also recently shown that 2’-F nucleotide modifications

do not cause significant toxicity in cells [23]. As such, we expect that these chemically modified

nucleosides, if released during siRNA hydrolysis by nucleases and phosphatases, should not be

toxic to human cells.

Figure C.6. Cell viability assay results. A) Normalised cell viability at highest tested

concentration (0.1 M). B) Normalised cell viability at range of concentrations tested.

CONCLUSIONS

Rapid progress has been made toward RNAi-based therapy by rationally optimizing chemically

modified siRNAs for high specificity and potent gene silencing. In this study we analyzed the

effect of novel 2’,4’-modifications at different positions in the antisense strand of an siRNA

targeting the firefly luciferase and DRR genes. We found that 4’-OMe or 4’-F modifications are

effective at inducing gene knockdown, especially when placed at the 3’-overhang of the guide

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strand. We hypothesize that favourable interaction with the PAZ domain of hAGO2 and

enhanced nuclease stability provided by these modifications both contribute to the improved

activity of these modified siRNAs. This study expands the toolbox of desirable chemical

modifications for gene silencing applications and opens the avenue for further studies of these

chemical modifications against other gene targets.

ACKNOWLEDGEMENTS

We would like to thank Dr. Hanadi Sleiman for her generous contribution of resources to this

project. Financial support was provided by the Natural Sciences and Engineering Research

Council of Canada (Discovery grant to M.J.D.; CGSD to A.E.A), and the Canadian Institute for

Health Research (CHRP to M.J.D., M.S.S.).

Conflict of Interest Statement: No competing financial interests exist.

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20. Arnold AE, E Malek-Adamian, PU Le, A Meng, S Martínez-Montero, K Petrecca, MJ Damha and MS Shoichet. (2018). Antibody-antisense oligonucleotide conjugate downregulates a key gene in glioblastoma stem cells. Mol Ther Nucleic Acids 11: 518-527.

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Appendix D: Additional characterization of AONs and siRNAs

against DRR

Purpose

Many of the AONs and siRNAs used in during the course of this thesis were characterized in

terms of their stability and functional effects; not all of this data was published, but it is

important for the understanding of the stability and potency of the different AONs and siRNAs

against DRR used in this thesis.

Methods

Nuclease stability assays

AON samples (dsDRR and mAb-dsDRR) or DRR siRNAs were incubated with a solution of

10% FBS in PBS, pH 7.4 for up to 24h. At each timepoint, the entire contents of the sample were

analyzed using acrylamide electrophoresis to separate nucleic acid material based on size. The

remaining full length siRNA or AON was quantified by imageJ and the data was reported as a

percentage of intact AON or siRNA.

Invasion assay

Hydrogels were synthesized as previously described by Tam et al.7 Cells were plated on

hydrogels at a density of 3500 cells/hydrogel and allowed to adhere for 24h. Cells were then

treated with transfection complexes at a concentration of approximately 25 nM. 48h after

treatment fresh media was added to each well and cells were fixed with 4% PFA 4-5 days after

treatment. Cells were stained with Hoechst to identify cell nuclei and 15 µM beads were added to

each well to label the surface of the hydrogel. Hydrogels were imaged using confocal

microscopy and depth of invasion was analyzed using custom computational software.

Figures

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Figure D.1. mAb conjugation increases AON stability against nuclease degradation. A) Gel

separation of nucleic acid products using PAGE. Arrows indicate the progression of time;

degradation of the DRR AON, dsDRR (red box) was compared to the antibody conjugated

AON, mAb-dsDRR (blue box). B) The remaining amount of intact AON was quantified at

each time point (dsDRR, red line and mAb-dsDRR, blue line). Data was analyzed using

two-way-ANOVA followed by Tukey’s post hoc correction (***p<0.001). Data is shown as

mean±SD, n=3.

Table D.1. DRR siRNAs used in Appendix D (and Appendix C).U = 2'-F-rU; U = 2‘-F,4‘-

OMe-rU.

Strand Duplex Name Sequence

Sense --- 5'- r(GGA ACC AGC UCA UCA AGA AUU) -3' (S)

+DRR Unmodified DRR siRNA 5' r(UUC UUG AUG AGC UGG UUC CUU) 3' (AS)

112-1 Modified DRR siRNA 1 5' r(UUC UUG AUG AGC UGG UUC CUU) 3' (AS)

112-2 Modified DRR siRNA 2 5' r(UUC UUG AUG AGC UGG UUC CUU) 3' (AS)

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Figure D.2. Chemical modifications increase siRNA stability against nuclease degradation.

At each time point, the amount of intact siRNA was quantified. Data was analyzed using

two-way-ANOVA followed by Tukey’s post hoc correction (***p<0.001). Data is shown as

mean±SD, n=3.

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Figure D.3. In this work, oligonucleotide potency and stability are negatively correlated.

Data was pulled from Chapter 2, Appendix C, and Appendix D to compare the potency of

three differently modified DRR siRNAs and DRR AON at 20 nM and stability, ie. the

intact oligonucleotide at 8h of incubation in serum-containing buffer.

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Figure D.4. Invasion of glioblastoma cells was reduced following DRR transfection but cell

proliferation was significantly increased. A) The transfected DRR AON (dsDRR) was

compared to transfected scrambled AON and an untreated (No Treatment) group.

Representative images shown. B) The invasion depth and cell number were quantified

using image analysis and a computational software, and data was analyzed by one-way-

ANOVA followed by Tukey’s post hoc correction (**p<0.01). Data is shown as mean+SD,

n=3.

Results and Discussion

We wanted to determine the stability of our AON and siRNA material so we could compare it to

the potency we observed and also downstream functional effects. In Figure D.1, we determined

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that the AON was relatively stable (~50% remaining at 24h) and that conjugation to a protein,

such as an antibody, could further significantly increase its stability.

Several siRNAs against DRR were described in Appendix C; in Figure C.5, these are labelled

Positive control, 112-1, and 112-2; they are also known as Unmodified DRR siRNA, Modified

DRR siRNA 1, and Modified DRR 2, respectively. These siRNA strands are shown in Table D.1

and they contain different modification patterns. The stability of these siRNAs is shown in

Figure D.2, and the Modified DRR siRNA 1 is significantly more stable than the other strands at

8h.

In Figure D.3, data was pulled from Chapter 2, Appendix C, and Appendix D to correlate the

potency of these AONs and siRNAs at 25 nM and the stability at 8h of nuclease incubation. We

observed a negative correlation between stability in potency; that is, the more intact nucleic acid

material persisted, the less the oligonucleotides worked to reduce expression of DRR. This is

discussed further in the thesis discussion chapter.

Finally, in Figure D.4, we wanted to test the effect of transfected DRR antisense oligonucleotide

(dsDRR) on invasion of glioblastoma cells in a 3D hydrogel model. We saw that the invasion

was reduced, but it was not a statistically significant reduction in invasion. Interestingly, we also

observed that the proliferation of the cells was significantly increased following transfection of

the DRR antisense oligonucleotide in the 3D hydrogel model.

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Appendix E: In vivo biodistribution of a CD44 antibody conjugated

to a fluorescent dye

Purpose

In order to determine the best injection route for an antibody conjugate to reach glioblastoma

stem cells, we carried out a biodistribution study using a Cy5-labelled antibody (ADC(CD44-

Cy5)). The control was a non-specific IgG (ADC(CTL-Cy5)). We injected the antibodies in five

different ways, including intravenous, intraventricular, and intratumoral injections into mice with

pre-established tumors from GSC injections, as well as co-injection with GSCs into naïve mice.

Methods

Conjugation of CD44 Ab to Cy5 Fluorophore

CD44 Ab or IgG control was reacted with Sulfo-Cy5-NHS Ester (ab146459) according to the

manufacturer’s protocols. Briefly, 10 eq. of the Cy5-NHS Ester was mixed with the antibody for

30 min at RT. The excess Cy5 was then purified by dialysis and successful labeling of the

antibody was confirmed by absorbance at 650 nM for the Cy5 fluorophore.

In Vivo Injections of CD44-Cy5 Conjugate

ADC(CD44-Cy5) or ADC(CTL-Cy5) were dialyzed into PBS and sterile filtered prior to

injection. All animal procedures were approved by the Institution’s Animal Care Committee and

performed according to the guidelines of the Canadian Council of Animal Care. Female NSG

mice (Charles River, Canada) were anesthetized at 6 weeks of age using intraperitoneal injection

containing Ketamine, Xylazine and Acepromazine. For all injections, the mice were placed on a

stereotaxic apparatus and a midline scalp incision was made. A burr-hole (3–5 mm) was created

2.2 mm lateral to the bregma using a high-powered drill. Tumor cells were GFP-labeled GSCs

provided by Dr. Kevin Petrecca, McGill University. The conjugate was injected at 15 ug/mouse

for all groups, except for the IV injection high dose, which was injected at 100 ug/mouse. IV

injections, intraventricular injections, and intratumoral injections were carried out in mice that

had pre-established tumors by stereotactically injecting GSCs 2 weeks before the start of the

study. IV injections were via tail vein; intraventricular injections were into the left ventricle; and

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intratumoral injections were carried out by stereotactically injecting at the same location as the

tumor. Co-injections with the antibody conjugates and tumor cells were carried out in naïve mice

using stereotactic injections. 48h following antibody injection, mice were sacrificed and tumors

were sectioned. GFP staining was used to locate the injected GSCs and the fluorescence of the

Cy5 fluorophore indicated the position of the antibody.

Figures

Figure E.1. Synthesis of Antibody-Cy5 conjugate. A) Antibodies were reacted with NHS-

Cy5 to form antibody-Cy5 conjugates. B) Absorbance peaks at 280 nm (antibody) and 650

nm (Cy5) confirmed successful conjugation.

Table E.1. Study Design. In total, there were 5 injection modalities tested: Intravenous (IV)

high (100 ug/mouse), IV low (15 ug/mouse), intraventricular (15 ug/mouse), intratumoral

(15 ug/mouse), and co-injection into naïve mice (15 ug/mouse). Within each of these, the

ADC(CD44-Cy5) and the non-targeted ADC(CTL-Cy5) were compared for a total of 10

experimental groups.

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Figure E.2. No evidence of antibody-Cy5 uptake into GSCs was observed in intravenous or

intraventricular injections. A) Intravenous injections. Only the high intravenous injection

group (100 ug/mouse) is shown; even at the high dose, no Cy5 signal (red) was found

overlapping with the GFP signal of the GSCs (green). Representative images shown for

Tumor EstablishedOPK164TumorinNODscidmice Noestablishedtumor

InjectionRoute Intravenous Ventricle Tumor Co-injectionwithtumorcells

Groups ADC(CD44-Cy5)15ug/mouse

ADC(CD44-Cy5)15ug/mouse



ADC(CTL-Cy5)15ug/mouse





X X X


X X X

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ADC(CTL-Cy5) and ADC (CD44-Cy5). B) Intraventricular injections. No evidence of Cy5

signal (red) was found to be colocalized with the GFP (green) following injection into the

left ventricle.

Figure E.3. Intratumoral injections. Some colocalization with the antibody (red) and the

GSCs (green) was observed following direct intratumoral injection, and potentially a

higher amount of overlap between the ADC(CD44-Cy5), as indicated with yellow arrows,

compared to the ADC(CTL-Cy5). Representative images shown.

Figure E.4 Co-injections. Following co-injection of antibody conjugates (red) and GSCs

(green) into naïve mice, a significant amount of colocalization (yellow) was observed

between the ADC(CD44-Cy5) and the GSCs. Little to no overlap was observed between the

ADC(CTL-Cy5) and the GSCs. Representative images shown.

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Results and Discussion

In order to track the biodistribution of an antibody in vivo, we modified the antibody with a

fluorophore, Cy5 (Figure E.1). We then designed a study that would compare multiple injection

routes and doses to find the best overlap between the delivered fluorescent antibody and the

GSCs in vivo (Table E.1). No uptake into the GSCs was observed when the antibody conjugates

were injected into the tail vein (Figure E.2A) or into the ventricle (Figure E.2B), indicating that

these delivery methods would not have a high chance of success for delivering an antibody-

conjugate to cells within the brain tissue.

With a future in vivo study in mind, we also explored injection directly into pre-established

tumors or co-injection that involved pre-mixing of the antibody and the GSCs immediately

followed by injection of the cocktail into naïve mice. When we injected the antibody-Cy5

conjugates into pre-established tumors, we observed some overlap between the antibody and the

GSCs (Figure E.3) and perhaps more areas of overlap with the targeted CD44 antibody

compared to the non-targeted CTL antibody, suggesting that this could be a possible route for

delivering antibody conjugates in vivo. Finally, we also co-injected GSCs and the antibody

conjugates, and observed a significant amount of overlap between the targeted CD44 antibody

and the GSCs but not for the non-targeting CTL antibody (Figure E.4). Although this would not

be practical as a treatment strategy, it could be a good way to carry out a proof-of-concept in

vivo study and ensure plenty of uptake of the antibody conjugate into the GSCs. This study had

quite a few limitations: for most groups we only tested one dose of the antibody conjugates; we

only analyzed images from one mouse for each injection strategy; and we did not quantify the

extent of colocalization between the antibody and the GSCs. Nevertheless, this data qualitatively

suggests that the best injection route for achieving colocalization between antibodies and tumor

cells is co-injection of tumor cells and the antibody treatment.

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Appendix F: Polymeric nanomicelles for targeted therapeutic

delivery to glioblastoma

Rationale

Glioblastoma multiformes (GBMs), a type of brain cancer, is one of the deadliest cancers and

represents a significant clinical challenge. It is hypothesized that GBM cancer stem cells (GSCs)

are the major cell type responsible for glioblastoma invasion, resistance, and recurrence.21 Small

interfering ribonucleic acids (siRNAs) are potent therapeutics that can be engineered to target

specific proteins responsible for cancer cell proliferation and invasion.81 For this work, DRR

(downregulated in renal cell carcinoma), a gene associated with the GSC invasion, resistance,

and stemness, was chosen as the genetic target in a series of primary brain cancer cell lines.64, 66

Polymeric nanomicelles are promising vehicles for the delivery of genetic therapeutics to

cancerous tissue, stabilizing the nucleic acids with respect to cleavage and enabling targeted

delivery strategies. We have synthesized poly(D,L-lactide-co-2-methyl-2-carboxytrimethylene

carbonate)-graft-poly(ethylene glycol)-azide (P(LA-co-TMCC)-g-PEG-N3), a polymeric

nanomicellular platform with azide reactive sites for ligand conjugation. The azide functional

groups at the terminal ends of the poly(ethylene glycol) polymers are capable of undergoing

Huisgen [3+1] click-type conjugations. This delivery platform is being investigated for the

simultaneous conjugation of both targeting antibodies and oligonucleotides specific to GSCs.

This appendix describes the synthesis and application of polymeric nanomicelles functionalized

with anti-EphA2 Fabs and anti-DRR oligonucleotides.

Methods

P(LA-co-TMCC)-g-PEG-N3 Synthesis

P(LA-co-TMCC)-g-PEG-N3 micelles were synthesized and characterized according to

previously reported methods.385-386 Briefly, The P(LA-co-TMCC) backbone is synthesized by a

ring-opening polymerization of the monomers D,L-lactide and 5-methyl-5-benzyloxycarbonyl-

1,3- trimethylene carbonate (TMCC-Bn) initiated by 1-pyrenebutanol and catalyzed by an

organic thiourea compound. The TMCC-Bn is deprotected by palladium-catalyzed

hydrogenolysis. A graft-to approach is used bifunctional azide-poly(ethylene glycol)-amine

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(NH2−PEG-N3) onto the P(LA-co-TMCC) backbone using diisopropylcarbodiimide (DIC) and

hydroxybenzotriazole (HOBt) coupling chemistry in DMF. The resulting polymer is 90 mol %

LA and 10 mol % TMCC, with an average of 2-3 PEG chains per backbone. Finally, micelles are

formed by a dialysis self-assembly procedure against phosphate buffered saline (1×, PBS).

P(LA-co-TMCC)-g-PEG-N3 Functionalization

P(LA-co-TMCC)-g-PEG-N3 micelles were mixed with DBCO-modified anti-DRR

oligonucleotides and DBCO-modified streptavidin at a 1:1:1 ratio for 1h at RT. The micelles

were then mixed with biotinylated Fab at a 1:1 ratio for 1h at RT. Conjugates were analyzed by

PAGE.

Cellular uptake

Functionalized nanomicelles were added to DsredDRR GBM cells or to GSCs at a desired

concentration (50-150 nM) and incubated for 3h at 37 °C. Cells were then fixed with 4% PFA,

counterstained with Hoechst, images were captured on an Olympus FV1000 confocal

microscope.

Gene knockdown

Functionalized nanomicelles were added to DsredDRR GBM cells or to GSCs at a desired

concentration (50-100 nM) and incubated for 72h at 37 °C in a 6-well format. Cells were then

lysed and protein expression was analyzed by western blot.

Figures and Results

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Figure E.1. Oligonucleotides can be conjugated to azide-functionalized nanomicelles. A)

Synthetic scheme showing the reaction between azide-functionalized nanomicelles and

DBCO-modified oligonucleotides. B) Micelle-oligonucleotide conjugates were analyzed by

PAGE, with approximately 80% conjugation efficiency of the oligonucleotides.

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Figure E.2. Anti-EphA2 Fabs can be conjugated to azide-functionalized nanomicelles. A)

Synthetic scheme showing reaction between azide-functionalized nanomicelles and DBCO-

functionalized streptavidin, followed by the addition of biotinylated anti-EphA2 Fab. B)

Modification of micelles with streptavidin was confirmed by PAGE. Biotin-Alexa Fluor 488

(Biotin-488) was then added to streptavidin-micelles to confirm the binding activity of the

streptavidin. C) Successful biotinylation of the Fab was confirmed by monitoring the

mobility shift of the Fab when mixed with streptavidin (SA). D) Binding of Fab-biotin to

streptavidin-micelles (micelle-strep) was confirmed by PAGE.

Figure E.3. Gene knockdown following treatment with DRR oligonucleotides or micelles

conjugated to DRR oligonucleotides (anti-DRR micelles). A) Quantification of DRR

expression following treatment with micelles conjugated to scrambled oligonucleotides

(scrambled micelles) compared to anti-DRR micelles with negative controls of Fab only

(Free Fab) or oligonucleotide only (Free AON) and positive control of lipofectamine-

transfected oligonucleotide (Lipo+15.E1). No knockdown was observed with any of the

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micelle-AON treatment conditions. Data is shown as n=3, mean+SD. B) Representative

western blot is shown.

Figure E.4. Uptake of micelle-oligonucleotide (micelle-AON) into GBM cells (DsredDRR)

and GBM stem cells (OPK126) compared to free AON control and no treatment controls.

No uptake was observed of micelles into glioblastoma stem cells (OPK126). A significant

amount of background uptake of free AON was observed in DsredDRR cells, and did not

appear to increase with the micelle formulation.

Discussion

Conjugation of the azide-functionalized nanomicelles to oligonucleotides and anti-EphA2 Fabs

was successful, with approximately 80% conjugation of the oligonucleotides (Figure E.1) and

nearly 100% conjugation of the anti-EphA2 Fabs (Figure E.2). Unfortunately, although the

positive control transfected by lipofectamine effectively knocked down DRR expression, no

DRR knockdown was observed in any of the anti-DRR-micelle treatment conditions (Figure

E.3). Finally, we examined uptake of the micelle formulations into GBM (DsredDRR) and GSC

(OPK126) cells (Figure E.4); although significant background uptake of the free AON was

observed in the DsredDRR cells, this did not translate to DRR knockdown as described in Figure

E.3. No uptake of the AON was observed with any treatment conditions for the GSCs (OPK126).

133

Following these results, we decided to pursue a direct conjugate between targeting antibodies

and antisense oligonucleotides instead of continuing with the functionalized nanomicelle system

due to a lack of observed efficacy. One hypothesis for the challenges faced with this approach is

the inefficient PEG conjugation achieved when using the graft-to approach for conjugating the

PEG chains to the backbone PLA-co-TMCC polymer; however, this hypothesis was not robustly

explored.

134

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