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An adenoviral vector can transfer lacZ expression into schwann cells in culture and in sciatic nerve.

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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 humans, 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 require 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 peroneal nerves are often involved earliest, hence the alternative name for this disease, peroneal muscular atrophy
t41.
In a series of elegant transplantation experiments,
Aguayo and coworkers [S] demonstrated that the defect 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 elucidated recently. The majority of cases, designated
CMTlA, have been shown to be associated with a duplication in the pll-p12 region of human chromosome 17 16-81, which contains the peripheral myelin
From the *Department ofNcurology and Center ofMolecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI, and the iDepartment of Neurology, University of Pennsylvania School of Medicine, Philadelphia, PA.
protein-22 (PMP-22) gene. PMP-22 encodes one of
the major PNS myelin proteins [9-12) and overexpression 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 connexin-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 development 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
Received Jan 12, 1995, and in revised form Jun 15. Accepted for
publication Jun 15, 1995.
Address correspondence to D r 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
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 vectors can be used to express foreign genes in adult
Schwann cells, suggesting that they may be useful vectors for the gene therapy of CMT1.
fibrosis transmembrane conductance receptor into
bronchial epithelial cells of individuals with cystic fibrosis [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
[29-3 11.
Materials and Methods
Construction and Preparation of AdCMVLucZ
In this study we utilized an adenoviral vector expressing the lacZ gene under control of the cytomegalovirus (CMV) Promoter
described in
[32}) to demonstrate that a replication defective adenoviral vector can be used to transduce genes into
Schwann cells, both in vitro, and in vivo. At the appropriate titers, replication defective adenoviral vec-
The Eschericbia coli P-galactosidase (IacZ) gene under control
of the CMV promoter was inserted into a plasmid designed
to facilitate recombination into a replication defective adenovirus [ 3 3 ] (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 approximately 2 kb of adenoviral D N A (9-16 mu). The E l a
region of the adenoviral D N A , required for viral replication,
Fig 1 , Schematic representation of the construction of the recombinan t adenouirus, AdC M VLacZ.
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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. Recombinant virus was propagated in the Ela-transformed human
embryonic cell line, 293 [34]. The recombinant virus was
purified through two rounds of plaque purification, propagated 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-dayold Sprague Dawley rats [35]. The cells were expanded on
poly-L-lysine-coated, 100-mm tissue culture plates in Dulbecco’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 passages 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 (approximately 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 . 5 q glutaraldehyde, stained with X-gal, mounted,
and examined under a Zeiss inverted microscope for morphological 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% glutaraldehyde 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, dehydrated in a graded series of ethanol, infiltrated briefly with
propylene oxide, and embedded in expoxy resin. Thick sections ( 1 pm) were counterstained with pam-phenylaminediamine; thin sections were stained with lead citrate.
Results
Schwann cells infected with varying concentrations of
recombinant adenovirus expressing t h e E . coli lacZ
gene were analyzed for P-galactosidase expression 24
hours after infection. Schwann cell cultures, approximately 200,000 cells p e r 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 2 5 0 : 1
(Fig 2A, panel 1). T h e s e Schwann cells remained
tightly compacted in rows and whorls, and were morphologically indistinguishable from control Schwann
cells not infected with adenovirus. Schwann cells infected 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 t h e surviving Schwann cells stained
blue with X-gal (Fig 2 A , panel 2). These data thus
demonstrate that AdCMVLacZ can efficiently infect
cultured Schwann cells. A t lower titers, infected cells
express t h e lacZ gene, and appear morphologically
normal. Higher viral titers, however, clearly have a deleterious 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
t h e percentage of lacZ-expressing cells during this time
period. Finally lacZ-expressing Schwann cells appeared
morphologically normal and were able to ensheath axo 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 saline [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 microscope within 10 days of injection that extended
from 4 to 7.8 m m along the nerve fiber. No control 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 analysis of sciatic nerve 4 days after injection with
AdCMVLacZ, shown in Figure 2 B (panel l), demonstrated 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 2 B , panel 2). Although this type of analysis 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 adenoviral-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
C
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, including demyelination, axonal degeneration, myelin
debris, macrophages, and edema (Fig 2C, panel 1). Surrounding these damaged areas, there were zones of
partial damage, with a mixture of degenerating and intact, 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 injection, 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, viralinfected 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 identified 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 demonstrate that postmitotic, myelinating Schwann cells can
F i g 2. X-gal staining Schwann cells infected with
1AdCMVLacZ
in vitro and in vivo. (A) Cultured rat Schwann
of
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 Jection 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 membranes. 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.
express lac2 after injection of the AdCMVLacZ adenovirus 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 transplanted 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 myelinate regenerating axons. In order for the transplanted Schwann cells to contact axons and to myelinate 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 adenovirus to transduce genes into Schwann cells in the sciatic nerve obviated the difficulties described above.
The Schwann cells need not be cultured prior to infection, 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 myelinating 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 following infection with high-titer virus {31, 40). In our experiments there were areas of tissue injury near the
site of adenoviral injection. Since only a small proportion 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
Fig 3. Electron microscopy of X-ga/-stained myelinating
Schwann cells. Transversesections of an adult rat sciatic nerve
4 days after injection with adenov,;rus were analyzed as described 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 D N A can be expressed by infected cells.
Acsadi and colleagues [23] demonstrated that adenoviral genes can be expressed for several months in muscle, 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 dramatically with time after injection. The reason for this
decrease is not known, but is likely to involve immunemediated removal of viral-infected cells [44]. Since repeated administrations of adenoviral vectors, as a rule,
have not been successful, perhaps because of a host
immune response to the adenovirus [45}, we are currently 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 vectors, which are known to be less immunogenic. In addition, 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 location of Schwann cells to be repaired to produce a clinically 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 degeneration and denervation of muscle rather than demyelination per se, and surviving axons exhibit reduced numbers of neurofilaments and reduced axonal diameters
[46, 471. The axonal defect in CMTl is probably a
consequence of altered Schwann cell-axon interactions, which in turn are produced by the primary
Schwann cell defect. Data from Trembler mice, an animal model of CMTl, confirm this notion. Trembler
mice have been shown to have distal axonal degeneration and muscle denervation 1481, and more recent
studies demonstrated that demyelination can also lead
to local alterations in axon caliber, neurofilament phosphorylation, and axonal transport E49-5 11. In addition,
these axonal changes may be proportional to the severity of demyelination [52]. Recently, changes in neurofilament 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 axonal changes and thus prevent denervation of muscle
along with the subsequent clinical disability.
The fourth and most important issue to resolve before 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 overexpression of PMP-22, the role of gene therapy will
be to reduce expression of this gene, perhaps by delivery 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 delivery system for the peripheral nerve and further knowledge 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 National 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 preparing Figure 2, and Agnes Jani for her help in reviewing the manuscript.
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436 Annals of Neurology Vol 38 No 3 September 1995
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expressions, sciatic, can, adenoviral, schwann, nerve, transfer, culture, lacz, vectors, cells
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