An Adenoviral Vector Can Transfer lac2 Expression into Schwann Cells in Culture

and in Sciatic Nerve I

Michael E. Shy, MD," Mari Tani, MD,? Yi-jun Shi, PhD,? Shelley A. Whyatt, BA,? Taibi Chbihi, BS," Steven S. Scherer, MD, PhD,? and John Kamholz, MD, PhD"

Although a number of genetic defects in the PO, peripheral myelin protein-22, and connexin-32 genes recently were shown to cause the demyelinating forms of Charcot-Marie-Tooth disease, there is yet no effective treatment for these patients. Recent studies showed that replication defective adenoviral vectors can efficiently introduce genes into muscle, brain, lung, and other tissues, suggesting that this vector system may be useful for the treatment of a number of genetic diseases. In this work, we demonstrated that a replication deficient adenovirus expressing the Escherichia coli P-galactosidase gene (AdCMVLacZ) can introduce genes into Schwann cells, in culture as well as in sciatic nerve. Schwann cells cultured at a multiplicity of infection of 250: 1 did not demonstrate cytopathic effects. Following injection of AdCMVLacZ into sciatic nerve of rats, lacZ-expressing, myelinating Schwann cells could be detected for at least 45 days. These data suggest that in the future, these vectors may be useful both in perturbing Schwann cell gene expression and in designing therapies for the treatment of Charcot-Marie-Tooth disease.

Shy ME, Tani M, Shi Y , Whyatt SA, Chbihi T , Scherer SS, Kamholz J. An adenoviral vector can transfer lacZ expression into Schwann cells in culture and in sciatic nerve. Ann Neurol 1995;38:429-436

Charcot-Marie-Tooth disease type 1 (CMTI) is the most common inherited peripheral neuropathy in hu- mans, with a prevalence rate of 1 in 2,500 El). CMTl is usually inherited as an autosomal dominant disorder, and is associated with peripheral nervous system (PNS) demyelination as demonstrated by nerve conduction velocities and nerve biopsy 12). The average age at clinical onset of CMTl is 12 + 7 years 131, and the clinical course is slowly progressive. Patients may re- quire foot care or bracing to ambulate normally 121, and sometimes become unable to walk 12, 3). Among the nerves most severely affected in CMT1, the pero- neal nerves are often involved earliest, hence the alter- native name for this disease, peroneal muscular atrophy t41.

In a series of elegant transplantation experiments, Aguayo and coworkers [S] demonstrated that the de- fect leading to demyelination in CMT1 is probably caused by abnormalities intrinsic to the Schwann cell, the myelin-producing cell of the PNS. The molecular nature of the Schwann cell defect in CMTl was eluci- dated recently. The majority of cases, designated CMTlA, have been shown to be associated with a du- plication in the pll-p12 region of human chromo- some 17 16-81, which contains the peripheral myelin

protein-22 (PMP-22) gene. PMP-22 encodes one of the major PNS myelin proteins [9-12) and overex- pression of PMP-22 has been postulated to be the cause of CMTlA [9-12). In addition, point mutations in the PMP-22 gene have also been shown to produce an inherited demyelinating peripheral neuropathy in Trembler mice, as well as in rare patients with CMTlA without the chromosome 17 duplication [ 13- 17). A less common form of CMT1, CMTlB, recently was shown to be caused by mutations in the PO gene, which encodes another major PNS myelin structural protein [17). Finally, an X-linked form of demyelinating CMT, CMT X, is caused by point mutations in the con- nexin-32 gene, which is also expressed by myelinating Schwann cells, but incorporated into the incisures and paranodes and not compact myelin [18]. Mutations in the PMP-22, PO, and connexin-32 genes thus account for most of the cases of demyelinating CMT C19).

Although the genetic defects that cause CMTl have been elucidated, these discoveries have not yet led to new treatments, which will likely require the devel- opment of methods to repair genetically abnormal Schwann cells. One way that this might be done is to use replication defective adenoviruses. Adenoviral vectors are currently being used to introduce the cystic

From the *Department ofNcurology and Center ofMolecular Medi- cine and Genetics, Wayne State University School of Medicine, De- troit, MI, and the iDepartment of Neurology, University of Pennsyl- vania School of Medicine, Philadelphia, PA.

Received Jan 12, 1995, and in revised form Jun 15. Accepted for publication Jun 15, 1995.

Address correspondence to Dr Kamholz, Department of Neurology, Wayne State University School of Medicine, 6E University Health Center, 4201 St. Antoine, Detroit, MI 48201.

Copyright 0 1095 by the American Neurological Association 429

fibrosis transmembrane conductance receptor into bronchial epithelial cells of individuals with cystic fi- brosis [20, 211 and the dystrophin gene into muscle of patients with Duchenne muscular dystrophy {22-26]. These vectors can transiently express foreign proteins (reviewed in {27}) , but un1ik:e retroviral vectors, they can also infect nondividing cells. This is important in the PNS, since the Schwann cells that ensheath axons are no longer dividing [28}. .Additionally, the majority of the viral DNA remains episomal in the nucleus, avoding possible disruption of the host genome

tors do not significantly alter normal Schwann cell morphology, and terminally differentiated, myelinating Schwann cells in adult sciatic nerve can be transduced to express the lacZ gene. In addition, IacZ expression can be detected for at least 45 days after injection of the virus into the nerve. These data thus demonstrate that replication defective recombinant adenoviral vec- tors can be used to express foreign genes in adult Schwann cells, suggesting that they may be useful vec- tors for the gene therapy of CMT1.

Materials and Methods Construction and Preparation of AdCMVLucZ The Eschericbia coli P-galactosidase (IacZ) gene under control

to facilitate recombination into a replication defective adeno- virus [33] (Fig 1). The 5' end of the lacZ gene was flanked by adenoviral D N A (0-1 map unit [mu)) required for viral replication. The 3' end of the lacZ gene was flanked by ap- proximately 2 kb of adenoviral DNA (9-16 mu). The E l a region of the adenoviral DNA, required for viral replication,

[29-3 11. In this study we utilized an adenoviral vector ex-

pressing the lacZ gene under control of the cytomega-

[32}) to demonstrate that a replication defective adeno- viral vector can be used to transduce genes into Schwann cells, both in vitro, and in vivo. At the ap- propriate titers, replication defective adenoviral vec-

lovirus (CMV) Promoter described in of the CMV promoter was inserted into a plasmid designed

Fig 1 , Schematic representation of the construction of the recom- binan t adenouirus , AdC M VLacZ.

xbd14769( 'Nod145731

Nhel Digea I xh119701

is

I /- 100 m u Ckd DigencdAd

Co-Trnnsfeciwn irvo 293 cellt followed by ituracellvlnr homologous recombination. I

xh119701

Nodl45?31 M I 4 7 6 9 1 I ,

I n / / 4

0 1 9 100 mu Larz. Recom5uuDu Ade~wvinu

430 Annals of Neurology '(01 38 No 3 September 1995

was deleted from the adenoviral sequences of this plasmid, as was most of the E3 gene, providing space for foreign gene insertion. The plasmid, including the ampicillin resistance gene and bacterial origin of replication, was linearized and cotransfected with wild-type adenovirus D N A whose 5‘ end (0-2 mu) had been deleted by digestion with Cla I. Recombi- nant virus was propagated in the Ela-transformed human embryonic cell line, 293 [34]. The recombinant virus was purified through two rounds of plaque purification, propa- gated in 293 cells, and purified on a discontinuous cesium chloride gradient. The viral band was collected and desalted over a Sephadex column. Viral particles were estimated by measuring the optical density at 260 nm and the viral titer determined by direct plaque assay.

Schwann Cell cultures Schwann cells were isolated from the sciatic nerves of 3-day- old Sprague Dawley rats [35]. The cells were expanded on poly-L-lysine-coated, 100-mm tissue culture plates in Dul- becco’s modified Eagle medium (DMEM) supplemented with 10% heat inactivated fetal calf serum (FCS), 2 pM forskolin (Fsk), and pituitary extract [36]. The cells were re-fed every 3 to 4 days and subcultured every 7 days. Schwann cells infected with AdCMVLacZ had undergone less than five pas- sages in culture.

Infection of Schwann Cell Cultures Rat Schwann cell cultures were plated in 24 well plates at 50,000 cells per well and allowed to grow to confluence (ap- proximately 200,000 cells/well) over the next week. The cells were then infected with AdCMVLacZ at titers ranging from 10‘ to lo’ plaque-forming unit (pfu)/ml of media, 0.5 ml/well. Twenty-four hours after infection the cells were fixed in 0 .5q glutaraldehyde, stained with X-gal, mounted, and examined under a Zeiss inverted microscope for mor- phological appearance and the presence of‘ blue color.

Injection and Morphological Analysis of Sciatic Nerves Two-week-old Sprague Dawley rats were anesthetized with an intraperitoneal injection of 3% chloral hydrate. The sciatic nerves were exposed and injected with 1 pl of AdCMVLacZ viraI suspension at titers ranging from 109 to 10’’ pfu/mI. The animals were killed 4 to 45 days after injection. The sciatic nerves were then removed, fixed in 0.5% glutaralde- hyde for 3 hours, stained with X-gal overnight, and postfixed in 3.6% glutaraldehyde. Nerves to be teased were immersed in glycerol. Nerves to be sectioned were osmicated, dehy- drated in a graded series of ethanol, infiltrated briefly with propylene oxide, and embedded in expoxy resin. Thick sec- tions ( 1 pm) were counterstained with pam-phenylaminedia- mine; thin sections were stained with lead citrate.

Results Schwann cells infected with varying concentrations of recombinant adenovirus expressing the E . coli lacZ gene were analyzed for P-galactosidase expression 24 hours after infection. Schwann cell cultures, approxi- mately 200,000 cells per well, were infected with AdCMVLacZ at titers between 5 x 10’ and 5 x 10’

pfu (multiplicity of infection [moil between 2.5 and 2,500: 1). As the moi increased, there were increasing numbers of infected cells, so that nearly all the cells were blue following X-gal staining at a n moi of 250: 1 (Fig 2A, panel 1). These Schwann cells remained tightly compacted in rows and whorls, and were mor- phologically indistinguishable f rom control Schwann cells not infected with adenovirus. Schwann cells in- fected with 5 x lo8 pfu (moi of 2,500:1), however, demonstrated significant cell loss. Surviving cells n o longer formed whorls and there were fewer cell-to-cell contacts, although the surviving Schwann cells stained blue with X-gal (Fig 2A, panel 2). These data thus demonstrate that AdCMVLacZ can efficiently infect cultured Schwann cells. A t lower titers, infected cells express the lacZ gene, and appear morphologically normal. Higher viral titers, however, clearly have a del- eterious effect o n Schwann cells.

We subsequently demonstrated that Schwann cells maintain lacZ expression for at least 2 weeks in culture in defined media. I n addition there was n o change in the percentage of lacZ-expressing cells during this time period. Finally lacZ-expressing Schwann cells appeared morphologically normal and were able to ensheath ax- o n s (data not shown).

To infect Schwann cells in vivo, 1 billion pfu of AdCMVLacZ (10” pfu/ml; 1 Fl/nerve) were injected into adult rat sciatic nerves, and analyzed at various time points after injection for lacZ expression by X-gal staining. Noninjected nerves and nerves injected with viral vehicle (10q glycerol in phosphate-buffered sa- line [PBS]) served as controls. Twenty-nine rats were injected, and analyzed at 4 days (n = 8), 10 days (n = 6 ) , 2 1 days (n = 14), and 45 days (n = 1) after injection. All adenoviral-injected nerves demonstrated blue staining when examined under the dissecting mi- croscope within 10 days of injection that extended from 4 to 7.8 m m along the nerve fiber. No con- trol nerves appeared blue. By 3 weeks after injection, however, it was difficult to detect blue areas in either adenoviral-injected or control nerves. Teased fiber anal- ysis of sciatic nerve 4 days after injection with AdCMVLacZ, shown in Figure 2 B (panel l), demon- strated many elongated cells with blue cytoplasmic staining which were not seen in control nerves. By 3 weeks after injection many fewer elongated blue cells could be detected by teased fiber analysis, and by 45 days after injection we could only find an occasional blue cell (Fig 2B, panel 2). Although this type of analy- sis cannot be used to precisely quantitate the extent o f adenoviral infection in injected nerve, w e estimate that there were approximately 10% the number of adeno- viral-infected cells identified at 3 weeks after injection than at 4 days after injection. T h e elongated shape of these cells and their nuclei strongly suggest that they are myelinating Schwann cells.

Shy et al: Adenoviral Vector Expresses in Schwann Cells 431

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432 Annals of Neurology Vol 38 No 3 September 1995

To better evaluate the histological changes in nerves following AdCMVLacZ injection, we also examined transverse sections of nerves that had been embedded in epoxy resin by light microscopy. At 4 days after infection there were focal areas of tissue damage, in- cluding demyelination, axonal degeneration, myelin debris, macrophages, and edema (Fig 2C, panel 1). Sur- rounding these damaged areas, there were zones of partial damage, with a mixture of degenerating and in- tact, myelinating Schwann cells (Fig 2C, panel 2). The appearance of the regions surrounding the damaged areas was relatively normal. Control nerves injected with vehicle alone (10% glycerol in PBS) exhibited similar, but less pronounced changes at the site of in- jection, as has been reported in other studies 137, 38). Both areas, however, contained Schwann cells with perinuclear cuffs of blue-staining cytoplasm, suggesting they had been infected by the adenovirus. An example of a viral-infected, demyelinating Schwann cell is shown in panel 1 of Figure 2C, while an intact, viral- infected myelinating Schwann cell is shown in panel 2. In addition, occasional globular cells containing blue cytoplasmic inclusions were also seen. These cells are macrophages, which are known to have endogenous P-galactosidase activity 1391.

To demonstrate further that myelinating Schwann cells in sciatic nerve could be infected with adenovirus, we examined the adenoviral-in jected nerve by electron microscopy, one example of which is shown in Figure 3. As can be seen in this figure, Schwann cells that contain numerous rectangular crystals associated with the smooth endoplasmic reticulum, nuclear envelope, and lysosomes, similar in size, shape, and location to the known products of the X-gal reaction, were identi- fied in an injected nerve 137). In addition, these Schwann cells produced intact multilamellar myelin sheaths and were surrounded by a basal lamina. Taken together, the data presented in Figures 2 and 3 demon- strate that postmitotic, myelinating Schwann cells can

F i g 2. X-gal staining of Schwann cells infected with AdCMVLacZ in vitro and in vivo. (A) Cultured rat Schwann cells infected with AdCMVLdcZ at a multiplicity of infection (moil of 250:1 (panel 1) or 2,500:1, and stained with X-gal 24 hours later. (B) X-gal staining of teased fibers from sciatic nerve 4 days (panel 1) and 45 days (panel 2) after injection with AdCMVLacZ. (C) X-gal staining of sciatic nerve cross Jec- tion 4 days after adenoviral injection. Degenerating myelin sheaths (dark blue material in panels 1 and 2) can be seen at the site of injection. The arrows in panels 1 and 2 indicate Schwann cells that have X-gal clystals in thew nuclear mem- branes. The Schwann cell in panel 1 probably had a myelin sheath; the Schwann cell in panel 2 has an intact myelin sheath. Scale bars = 10 pm.

1

express lac2 after injection of the AdCMVLacZ ade- novirus into the adult rat sciatic nerve.

Discussion Transduction of genes into Schwann cells in vivo using a recombinant adenovirus has several advantages over that using a retrovirus. Most importantly, recombinant adenovirus can infect and transduce cells that are not dividing, which is an essential consideration for gene therapy of CMT1, since differentiated Schwann cells in vivo are postmitotic E28). In a previous study, we used a recombinant retrovirus to transduce the lacZ gene into cultured Schwann cells that were then trans- planted into the sciatic nerve C37). Using this ex vivo approach, we demonstrated that once transduced, Schwann cells could be transplanted into the sciatic nerve after retroviral infection and successfully my- elinate regenerating axons. In order for the trans- planted Schwann cells to contact axons and to my- elinate them, however, endogenous Schwann cells first had to be removed to give transplanted Schwann cells an opportunity to ensheath axons, which are already ensheathed by endogenous Schwann cell processes. The use of a replication deficient, recombinant adeno- virus to transduce genes into Schwann cells in the sci- atic nerve obviated the difficulties described above. The Schwann cells need not be cultured prior to infec- tion, but can be infected by direct injection of the virus into nerve. In addition, since the virus can infect postmitotic, myelinating cells, already in contact with axons, removal of the endogenous Schwann cells is not necessary to transduce exogenous genes into myelinat- ing Schwann cells.

Although adenoviral vectors can introduce genes into Schwann cells, there are several issues to resolve before they become useful tools for gene therapy of CMT1. The first issue to resolve is the cytotoxicity of the injected adenovirus. Previous studies suggested that endosmolytic properties of adenoviral structural proteins may induce dose-related cytotoxicity follow- ing infection with high-titer virus {31, 40). In our ex- periments there were areas of tissue injury near the site of adenoviral injection. Since only a small propor- tion of the injury can be attributed to the injection itself 137, 38) (M. E. Shy et al, unpublished results, 1995), most of the cytotoxicity is probably due to the injected adenovirus. This is particularly relevant, since in tissue culture many Schwann cells were damaged by an exposure to virus at a multiplicity of infection of 2,500: 1. Le Gal la Salle and coworkers {36}, however, did not detect any toxicity following injection of 30 to 50 x lo6 pfu of a similar recombinant adenovirus into various locations within the central nervous system (CNS), and a similar result was found by others in both the CNS C42, 431 and muscle E25). The amount of

Shy et al: Adenoviral Vector Expresses in Schwann Cells 433

F i g 3. Electron microscopy of X-ga/-stained myelinating Schwann cells. Transverse sections of an adult rat sciatic nerve 4 days after injection with adenov,;rus were analyzed as de- scribed in Materials and Methods. The myelinating Schwann cell in (A) has X-gal crystah within the nuclear membrane and smooth endoplasmic reticulum (arrowheads); some of these are shown at higher magnzjication (B). Scale bars = 1 pm (A) and 0.1 p i (B) .

adenovirus we injected is thus likely to have had a deleterious effect on the Schwann cells at the site of injection, and the ideal amount of virus to inject, as well as alternative delivery systems, will have to be determined in future studies.

A second issue to resolve is the length of time that adenoviral DNA can be expressed by infected cells. Acsadi and colleagues [23] demonstrated that adenovi- ral genes can be expressed for several months in mus- cle, and several groups [41, 42) found similar results in the CNS. From our preliminary studies we know that AdCMVIacZ is expressed in some sciatic nerve Schwann cells for at least 45 days following infection, although the number of infected cells decreases dra- matically with time after injection. The reason for this decrease is not known, but is likely to involve immune- mediated removal of viral-infected cells [44]. Since re- peated administrations of adenoviral vectors, as a rule, have not been successful, perhaps because of a host immune response to the adenovirus [45}, we are cur- rently attempting to develop strategies to increase the duration and number of adenoviral-infected cells after a single injection of virus. ‘Chis strategy includes in-

jecting younger and/or immunosuppressed animals, as well as using the second generation of adenoviral vec- tors, which are known to be less immunogenic. In addi- tion, a new generation of viral vectors, such as the adeno-associated viruses, may be required to introduce genes indefinitely into nondividing cells.

A third issue to be resolved is the number and loca- tion of Schwann cells to be repaired to produce a clini- cally observable effect. This is important, since it will be technically difficult to repair most of the Schwann cells along the length of a peripheral nerve or its branches. The major clinical disability in patients with CMTl, however, is caused by distal axonal degenera- tion and denervation of muscle rather than demyelin- ation per se, and surviving axons exhibit reduced num- bers of neurofilaments and reduced axonal diameters [46, 471. The axonal defect in CMTl is probably a consequence of altered Schwann cell-axon interac- tions, which in turn are produced by the primary Schwann cell defect. Data from Trembler mice, an ani- mal model of CMTl, confirm this notion. Trembler mice have been shown to have distal axonal degenera- tion and muscle denervation 1481, and more recent studies demonstrated that demyelination can also lead to local alterations in axon caliber, neurofilament phos- phorylation, and axonal transport E49-5 11. In addition, these axonal changes may be proportional to the sever- ity of demyelination [52]. Recently, changes in neu- rofilament phosphorylation also were found in CMTl [19], suggesting that similar mechanisms occur in this

434 Annals of Neurology 1/01 38 No 3 September 1995

disease. Thus minor improvement in Schwann cell function for a small number of cells in the distal nerve, early in the course of CMTl, might prevent these axo- nal changes and thus prevent denervation of muscle along with the subsequent clinical disability.

The fourth and most important issue to resolve be- fore gene therapy of CMTl can become a reality is the nature of the gene product to be delivered to the defective Schwann cells. CMTl A is probably caused, at least in the majority of patients, by overexpression of PMP-22 {b], although some cases of CMTlA are associated with point mutations in PMP-22 {lb, 17). In contrast, CMTlB is caused by a point mutation in the major myelin protein PO { 171. Precisely how these genetic defects lead to dysmyelination, however, has not yet been determined. If CMTlA is caused by over- expression of PMP-22, the role of gene therapy will be to reduce expression of this gene, perhaps by deliv- ery of a recombinant adenovirus expressing PMP-22 antisense messenger RNA. For patients with CMTlB, however, as well as those with CMTlA without a gene duplication, a different strategy must be developed. Successful gene therapy of CMTl thus depends on both the development of a sophisticated genetic deliv- ery system for the peripheral nerve and further knowl- edge of the molecular pathogenesis of demyelination in these diseases.

This work was supported by grants from the Muscular Dystrophy Association (to M. E. S. and J. K.) and by NS08075 from the Na- tional Institutes of Health (to S. S. S.).

The authors gratefully acknowledge Drs James Wilson and Steven Eck for their help with the adenoviral vector, Ms Susan Shumas for her expert technical assistance, Rosemary Shy for her help in prepar- ing Figure 2, and Agnes Jani for her help in reviewing the manu- script.

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