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Journal of Virology, August 1999, p. 6841-6851, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Neural Stem Cells as Engraftable Packaging Lines
Can Mediate Gene Delivery to Microglia: Evidence from Studying
Retroviral env-Related Neurodegeneration
William P.
Lynch,1,2,*
Arlene H.
Sharpe,3 and
Evan Y.
Snyder2,*
Department of Microbiology/Immunology, Northeastern Ohio
Universities College of Medicine, Rootstown, Ohio
44272,1 and Departments of Neurology
and Pediatrics, Children's Hospital and Harvard Medical
School,2 and Departments of
Pathology, Brigham and Women's Hospital and Harvard Medical
School,3 Boston, Massachusetts 02115
Received 6 January 1999/Accepted 12 April 1999
 |
ABSTRACT |
The induction of spongiform myeloencephalopathy by murine leukemia
viruses is mediated primarily by infection of central nervous system
(CNS) microglia. In this regard, we have previously shown that
CasBrE-induced disease requires late, rather than early, virus
replication events in microglial cells (W. P. Lynch et al., J. Virol. 70:8896-8907, 1996). Furthermore, neurodegeneration requires the presence of unique sequences within the viral
env gene. Thus, the neurodegeneration-inducing events could
result from microglial expression of retroviral envelope protein alone or from the interaction of envelope protein with other viral structural proteins in the virus assembly and maturation process. To distinguish between these possible mechanisms of disease induction, we engineered the engraftable neural stem cell line C17-2 into packaging/producer cells in order to deliver the neurovirulent CasBrE env gene
to endogenous CNS cells. This strategy resulted in significant CasBrE env expression within CNS microglia without the appearance
of replication competent virus. CasBrE envelope expression within microglia was accompanied by increased expression of activation markers
F4/80 and Mac-1 (CD11b) but failed to induce spongiform neurodegenerative changes. These results suggest that envelope expression alone within microglia is not sufficient to induce neurodegeneration. Rather, microglia-mediated disease appears to
require neurovirulent Env protein interaction with other viral proteins
during assembly or maturation. More broadly, the results presented here
prove the efficacy of a novel method by which neural stem cell biology
may be harnessed for genetically manipulating the CNS, not only for
studying neurodegeneration but also as a paradigm for the disseminated
distribution of retroviral vector-transduced genes.
| |
INTRODUCTION |
The simplest and best-defined model
for analyzing the details of retroviral neuropathogenesis resides in a
group of murine C-type leukemia viruses (MuLVs) that cause spongiform
neurodegeneration of motor system neurons from the neocortex through
the spinal cord (reviewed recently in reference 43).
The prototypic virus of this class is the ecotropic host range virus
referred to as CasBrE (1, 15). Genetic recombination
analyses indicate that the principal determinants of MuLV
neurovirulence map to the env gene (11, 41, 42,
63), which encodes the surface glycoprotein responsible for
binding and entry of retrovirus into cells. It has been widely proposed
that the gene product of env (the envelope protein) of
neurovirulent retroviruses may be directly toxic to the central nervous
system (CNS) (21, 22, 43, 62). However, indiscriminate
overexpression of env alone in the brains of susceptible mice is not sufficient to precipitate acute clinical or histopathologic disease (32). Induction of neurodegeneration requires late
retroviral replication events within host microglia, in particular
those events associated with envelope synthesis (32).
Hence, the study of retroviral pathogenesis has focused on the
infection of microglia. In vivo, microglial infection by CasBrE appears to result in the generation of unique Env proteins (10, 28, 30, 32). It remains unresolved, however, whether the microglial Env proteins themselves are directly neurotoxic, or whether
the synthesis and assembly of Env protein corrupts microglial function
and compromises neuronal survival from a loss of microglial support. To
investigate how retroviral interactions within microglia lead to
disrupted CNS function, we sought ways to genetically manipulate the
microglial compartment. Prior transgenic approaches to achieve either
global or cell-type-specific CNS CasBrE env expression have
been unsuccessful in approximating the expression associated with CNS
viral infection (22, 31a, 62). Furthermore, attempts to
genetically manipulate the microglial compartment by using bone marrow
chimeras have been hampered because of a very slow turnover of
parenchymal microglial cells (20, 24, 26). Virus-based
vectors offer a potential alternative for manipulating the microglial
compartment since they have been demonstrated to be effective vehicles
for the in vivo transfer of exogenous genes directly to endogenous
cells in the CNS (58). However, delivering genes of interest
to microglia throughout the brain is a challenge to viral vectorology
(49, 58). In fact, the relatively anatomically restricted
effectiveness of retrovirus- and, indeed, many virus-based vector
systems has been one of the obstacles to their broader use for
therapeutic gene transfer to the CNS. We recognized that surmounting
this limitation to answer our particular research question might
provide a method for improving the efficacy of viral vector-mediated
gene transfer for much broader applications.
Neural stem cells (NSCs) are immature, uncommitted cells that exist in
the developing and adult nervous system and are responsible for giving
rise to the vast array of more specialized neural cells of the CNS
(reviewed in references 33, 36, 39, 49, 57, 59, and
60). They are operationally defined by their ability to self-renew and to differentiate into cells of most (if not all)
neuronal and neuroglial lineages and to populate developing or
degenerating CNS regions. We previously demonstrated that migratory NSCs are well suited for gene therapy of broad regions of CNS because
they are easily expanded and genetically manipulated in culture and,
following transplantation into germinal zones, are integrated in a
cytoarchitecturally appropriate manner throughout the brain, where they
express the foreign genes. We have shown them to be capable of
delivering therapeutic gene products in a widely disseminated manner,
cross-correcting host neurons and glia by creating virtually chimeric
regions of the brain (25, 51, 54). Their facility to
distribute themselves extensively and disseminate foreign gene products
prompted us to use these NSCs in a somewhat unconventional manner. We
explored the possibility of engineering engraftable NSC clones into a
packaging/producer line for retroviral vectors in order to deliver the
env gene from a neurovirulent virus directly to host
microglial cells within the brain. In other words, we examined whether
NSCs, as packaging/producer cells, could serve as "platforms" for
the widespread dissemination of replication-incompetent foreign
gene-expressing viral vectors, just as they had for other diffusible
and nondiffusible factors.
The notion of transplanting packaging/producer cells (i.e., the cells
that provide the structural proteins for vector assembly) is not new;
however, their usefulness in vivo has been limited by the strong
propensity of the engineered fibroblast cell lines to become tumors in
transplant recipients (14, 52). That NSCs can participate in
the normal development of many regions at multiple stages along the
murine neuroaxis may provide the key to making an engraftable CNS gene
delivery system feasible.
Thus, the goals of our experimental approach were twofold. First, we
were interested in whether NSCs engineered into a retroviral packaging/producer line would make an efficient in vivo gene delivery system. Second, we wanted to test whether microglial expression of
env alone was sufficient to precipitate spongiform
neurodegeneration. We recognized that approaching this particular
problem as a proof of principle for a NSC-based strategy for
retrovirus-mediated transgene delivery was actually ideal. Because
microglia are not of neuroectodermal origin, nor can NSCs give rise to
microglia, expression of this index transgene (env) in these
cells would help rigorously prove host cell transduction via the
retrovirus and rule out misidentification of engineered donor-derived
cells as host cells.
We report that NSC-derived packaging cells can, indeed, act as in vivo
gene delivery systems. This technique helped demonstrate that
expression of env within microglia is insufficient to induce acute spongiform neurodegeneration, suggesting that defective retroviral assembly, rather than direct Env neurotoxicity, is the basis
for pathogenesis. In a broader context, we show that a clone of
migratory NSCs can be engineered ex vivo to package and release
replication-defective retroviral particles and that these engineered
NSCs, when transplanted into the brain, can serve as platforms for the
wider dissemination of these vectors in order to direct the transfer of
a desired gene to endogenous CNS host cells.
| |
MATERIALS AND METHODS |
Cells, plasmids, and viruses.
The C17-2 NSC line was derived
as previously described (47). In brief, neonatal mouse
cerebellar external germinal zone cells were infected with a defective
retrovirus vector encoding v-myc (transcribed from the long
terminal repeat [LTR]) and neo (transcribed from an
internal simian virus 40 [SV40] promoter) and selected in G418. The
resulting cells were then infected with a second retroviral vector,
BAG, encoding lacZ transcribed from the LTR and
neo transcribed from an internal SV40 promoter
(44). The first vector enabled these cells to grow in
culture indefinitely, while the second vector endowed the cells with
-galactosidase (-Gal) expression as a genetic cellular marker
(50). These stable lacZ-expressing NSCs are free
of helper virus (47, 50).
Mus dunni fibroblasts were used for virus focus assays as
outlined previously (8), and replication-competent virus
stocks were generated in Fisher rat embryo cells.
The replication-defective, CasBrE env-encoding
CasE virus was made in PA317 cells (37) as
previously outlined (32), with viral titers of
105 to 106 per ml of culture supernatant. No
helper virus is present within these stocks.
All cells were grown in Dulbecco's modified Eagle medium (high
glucose) supplemented with 10% fetal bovine serum, glutamine,
and
antibiotics (penicillin, streptomycin, and amphotericin B
[Fungizone]).
The amphotropic packaging plasmid pPAM3 (Fig.
1A; a gift from A. D. Miller, Fred
Hutchinson Cancer Research Center) has been
previously described
(
37). Plasmid pPGKPuro (a gift from P.
W. Laird,
University of Southern California) contains the puromycin
resistance
gene encoding puromycin
N-acetyltransferase under the
control of the phosphoglycerate kinase (PGK) promoter, followed
by PGK
polyadenylation sequences cloned into pBluescript SK+ (Stratagene)
(Fig.
1B). The structure of the pCas
E vector is shown in
Fig.
1C. The Cas
E vector was constructed by using the
defective Friend spleen focus-forming
virus (SFFV)-based retroviral
vector pSFF (
6) owing to the
exceptional capacity of pSFF to
be packaged into viral particles.
The Cas
E vector is also
efficiently packaged in retroviral particles and
readily spreads in
amphotropic packaging cell culture by ping-pong
transfer owing to the
presence of both an amphotropic
env (encoded
by pPAM3) and
an ecotropic
env (encoded by pCas
E)
(
32). Thus, Cas
E viral vector spread occurs by
using whichever cellular receptors
are not interfered with by
endogenous cellular
env expression
(
23,
28).
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FIG. 1.
Vectors used for converting C17-2 NSCs to packaging
cells capable of delivering CasBrE env to target cells.
C17-2 cells were converted to a packaging cell line by transfection
with the packaging plasmid pPAM3 (A) developed by Miller and Buttimore
(37). This plasmid was constructed using a Moloney MuLV
genome from which the  sequence had been deleted and the
pol-env region was exchanged for that of the 4070A
amphotropic virus (shaded) (38). In addition, the 3' LTR was
replaced with the SV40 polyadenylation site and viral sequences 5' of
the enhancers were deleted in the 5' LTR. The latter two modifications
were designed specifically reduce the likelihood that helper virus
would arise since it would require a minimum of two recombination
events. C17-2 cells containing pPAM3 were identified by selection for
puromycin resistance conferred by cotransfections with the selection
plasmid pPGKPuro (B), which encodes puromycin
N-acetyltransferase (dark rectangle) under control of the
PGK promoter followed by the PGK polyadenylation sequences. The CasBrE
env retroviral expression vector pCasE (C) was
constructed by introducing env into the pSFF vector
(6) at the multiple cloning site in order to maximize mRNA
packaging and protein expression. This vector, which is based on the
defective Friend SFFV, contains significant deletions (both engineered
and naturally occurring) in the gag, pol, and
env genes (6). These modifications result in a
failure to express normal viral proteins but allow both efficient
packaging of the vector RNA into particles and expression of exogenous
genes cloned into the polylinker site. (The possibility that the
 gag region gave rise to truncated Gag-like polyprotein,
as reported for the defective SFFV parent [46], was
not investigated in this study). Plasmid vector backgrounds (gray
diagonal stripes) for the expression constructs A, B, and C, were
pBR322, pBluescript (Stratagene), and pSP64, respectively. SD, splice
donor; SA, splice acceptor.
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|
The marginally neurovirulent CasBrE virus, clone 15-1 (
41),
was made in FRE cells. This virus was used as a positive control
in the
virus titration and cell infection assays. The infection
of C17-2 NSCs
with the replication-competent CasBrE virus (clone
15-1) was performed
as previously outlined (
32). In some virus
titration
experiments, the 4070A amphotropic virus (a gift from
J. Cunningham,
Brigham and Women's Hospital and Harvard Medical
School) was used as a
positive control for detecting amphotropic
virus and morphology of
foci.
Generation and characterization of NSC packaging/producer
cells.
The isolation, cloning, propagation, maintenance, and
transplantation of engraftable multipotent murine NSC clone C17-2 has been previously detailed (32, 45, 48-50, 53). To convert them to a packaging/producer line, subclones of C17-2 NSCs were cotransfected by electroporation using 20 µg of the amphotropic packaging plasmid pPAM3 (Fig. 1) along with 0.5 µg of the puromycin selection plasmid pPGKPuro at a molar ratio of 20:1, respectively. After 48 h, transfected cells were selected in puromycin (2 µg/ml), and isolated colonies were analyzed for expression of plasmid pPAM3 by examining the cell clones for cell surface amphotropic envelope glycoprotein coat (clone 4070A) expression by
immunocytochemistry (29) and fluorescence-activated cell
sorting (32). The 4070A envelope was detected by using the
Env protein-specific monoclonal antibody 83A25 (13), which
recognizes an epitope common to many murine retroviral Env proteins.
83A25-positive colonies were then screened for packaging ability by
assessing which colonies had packaged lacZ into infectious
virus particles. (As noted above, a provirus containing lacZ
was already integrated into the C17-2 genome [50].)
This screen was performed by a virus focus assay evaluating -Gal
expression in target cells as previously reported [44]; however, in this instance, M. dunni
fibroblasts were used as naive targets. C17-2 clones indicative of
successful lacZ-viral vector packaging were then infected
with the CasBrE env gene encoding replication-defective
virus CasE at a multiplicity of infection of 1. CasBrE
env expression was assessed by immunohistochemistry using
monoclonal antibodies 667 and 697 (34).
env gene transfer by C17-2 packaging cells was assessed by
both an infectious center assay and a viral focus assay (see below).
For the infectious center assay, packaging cells were irradiated
with
2,000 rads and seeded at various dilutions along with
M. dunni target cells at 10
5 M. dunni cells
per well of a TC-6 plate in the absence of Polybrene.
Cells were grown
for 4 days, and cultures were scored for foci
after fixation and
immunostaining for CasBrE
env expression. Isolated
single
env+ cells, likely representing irradiated C17-2
packaging cells,
were not scored as foci. The number of foci detected
was compared
to the number of packaging cells originally seeded and
expressed
as a percentage of cells with gene transfer capability. This
number
does not account for packaging cell losses that may have
resulted
from the radiation treatment. Infectious center focus
morphology
was also examined as a means for evaluating whether
recombinant
helper virus appeared (see
below).
The C17-2 NSC packaging subclones, particularly BA6 and
BA6-Cas
E, were examined prior to transplantation for the
presence of several
neural cell type markers to assess grossly their
extent of differentiation
in vitro due to the introduction of the
packaging vector. For
immunocytochemical analyses performed as
previously described
(
29,
32,
48,
50), antibodies directed
against the following
markers were used: nestin, for immature neural
progenitors and
stem cells; 2',3'-cyclic nucleotide 3'-phosphohydrolase
(CNPase;
Sternberger (Moncolonals, Inc.) and galactocerebroside
(Chemicon),
for oligodendrocytes; neurofilament (Chemicon) and
neuron-specific
enolase (Polysciences), for neurons and neuroblasts,
respectively;
and glial fibrillary acidic protein (GFAP; Dako), for
astrocytes.
Tests for replication-defective and replication-competent virus
production.
For detecting and quantitating cell-free virus
production from packaging cells, both in culture supernatant and in
transplanted brains, a focus assay (8) was used. Briefly,
105 M. dunni cells were seeded into each well of
a TC-6 plate (Linbro) and exposed overnight to either filtered
(0.2-µm-pore-size filter) undiluted or serially diluted 48-h
supernatants taken from freshly confluent C17-2 packaging lines or
serial diluted brain extracts (see below), in the presence of 8 µg of
Polybrene. The medium was changed, and M. dunni cells were
grown to confluence and then examined immunohistochemically for viral
foci, using antibodies reactive to either the 4070A amphotropic viral
coat (monoclonal antibody 83A25 [13]), the CasBrE Env
protein, encoded by CasE (monoclonal antibodies 667 and 697 [34]), or -Gal (rabbit polyclonal antibody from
Cappel). Alternatively, BAG virus production was detected in a focus
assay using
5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal)
histochemistry (44). As performed, the sensitivity of this
assay is 0.5 focus-forming unit (FFU)/ml of cell supernatant/brain extract.
To detect the generation of recombinant helper virus, two methods were
used. First, we performed the focus assay described
above in duplicate,
with either monoclonal antibody 667/697 or
monoclonal antibody 83A25,
and examined the morphology of the
primary foci in detail. When
replication-competent virus is present,
Env protein staining in the
foci is contiguous because secondary
infection of surrounding
uninfected
M. dunni cells occurs efficiently
via
cell-to-cell contact as infected cells migrate. If
replication-defective
virus is present, the initial infectious event is
terminal and
the focus morphology is determined by the division and
cellular
migration of the originally infected target
M. dunni fibroblasts.
Typically,
M. dunni cells divide and
progeny cells migrate away
from one another. At the same time, other
uninfected cells divide
and move between them. Under these
circumstances, the foci are
noncontiguous (see Fig.
3A and B) because
the initial infectious
event has no means to spread to
M. dunni cells with which they
make contact in
culture.
Serial supernatant passage was used as a second measure for the release
of helper virus from the C17-2 packaging cell clones.
Specifically,
this assay used the supernatants taken from primary
M. dunni
cell targets (i.e., cells that were exposed to undiluted
BA6 and
BA6-CasE cell supernatants) once the target cells reach
confluence and
before they are stained for foci. These secondary
supernatants were
then incubated overnight with 10
5 naive
M. dunni
targets in the presence of Polybrene (8 µg/ml).
After a change of
medium, the secondary
M. dunni target cells
were cultured to
confluence in TC-6 plates and then examined for
cellular expression of
either the amphotropic coat, CasBrE Env
protein, or
-Gal or the
generation of neomycin-resistant colonies.
M. dunni cells
examined for neomycin-resistant colonies were grown
for 3 weeks in the
presence of 2 µg of G418 (approximately 50%
active) per ml. The
sensitivity of the assay is 0.5 FFU/ml of
secondary
supernatant.
NSC transplantation into and analysis of engrafted mouse
brains.
Transplantation of the above-mentioned NSC subclones
(including BA6 and BA6-CasE packaging NSCs and 15-1-infected C17-2
NSCs) was done as previously described (32, 50, 53). All
mice used were inbred Rocky Mountain Laboratories White strain (IRW) mice, which are highly susceptible to the neurodegenerative effects of
CasBrE virus infection. Briefly, NSCs, maintained in culture as
detailed elsewhere (29, 32, 48, 50, 53), were trypsinized from a 90% confluent dish and resuspended in phosphate-buffered saline
at 5 × 104 cells/µl. On the day of birth, the
lateral ventricles of cryoanesthetized pups were visualized by
transillumination of the head; 2 µl of the cellular suspension (to
which 0.08% trypan blue was added to assess cell viability as well as
to localize the inoculum) was gently expelled via a glass micropipette
inserted transcutaneously into each ventricle (gaining access to the
subventricular zone). Pups were returned to maternal care until weaning.
Donor-derived cells were tentatively identified by their expression of
-Gal either by X-Gal histochemistry (
50) or by an
antibody directed against
-Gal (
32). In addition, as
previously
described (
29,
32), engrafted brains were
analyzed immunocytochemically
with antibody (83A25) reactive against
the 4070A amphotropic coat
of the packaging vector (unique to
transplant-derived cells) and
with antibodies (667B or 697) against the
protein encoded by CasBrE
env (potentially detectable in
both donor-derived and host-derived
cells).
The number and phenotype of host cells (
lacZ negative)
expressing
env was assessed immunocytochemically by
double-label immunofluorescence
using antibodies directed against the
neural cell type-specific
markers listed above. Microglia were
identified by using rat monoclonal
antibodies directed to either Mac-1
(murine CD11b) (
55) or F4/80
(
2) or a rabbit
polyclonal antibody specifically directed to
the F4/80 protein (gift
from S. Gordon, Oxford University) as
described elsewhere
(
18).
Engrafted brains were tested for the production of both defective virus
and replication-competent helper virus as described
above. Brain
extracts were prepared by homogenizing freshly isolated
transplant
brains in 10 volumes of 150 mM NaCl-50 mM Tris-HCl
(pH 7.4)-0.1 mM
EDTA (pH 7.2) (TBS-EDTA) at 4°C (using 10 strokes
of a Wheaton glass
homogenizer). Extracts were centrifuged a 10,000
×
g
for 10 min at 4°C to remove nuclei and large cellular debris.
The
resulting supernatants were used in virus detection assays
as outlined
above. Alternatively, in some cases, serial 20-µm
frozen sections
were taken and stained for detection of cellular
engraftment, while
parallel sections were extracted in 50 µl of
TBS-EDTA and tested for
the presence of virus. The sensitivity
of these assay is 1 FFU/ml of
brain extract (approximately 4 FFU/brain),
or 1 FFU/tissue
section.
Brains were examined for histopathological changes by
hematoxylin-and-eosin staining of paraffin sections. In addition, Env
protein localization was carried out in paraffin sections as outlined
in reference
32 after antigen retrieval by steam
heating deparaffinized
slides immersed in 10 mM citrate buffer (pH 6.0)
for 20
min.
Clinical neurological signs were assessed as outlined previously
(
8-10,
31,
32,
41).
All animal experiments were carried out in accordance with federal
guidelines (
39a) and in accordance with the Animal Care
and
Use Committees at Harvard Medical School and Northeastern
Ohio
Universities College of
Medicine.
| |
RESULTS AND DISCUSSION |
Engineering NSCs into packaging lines.
One of the best-studied
models of NSC behavior is a clone designated C17-2. It is one of a
number of stable, self-renewing cellular clones originally derived from
neonatal mouse cerebellum but capable of participating in the normal
development of most CNS structures from fetus to adult upon
implantation into mouse CNS germinal zones. The cells differentiate
into neurons when transplanted into regions undergoing neurogenesis or
into glia where gliogenesis predominates (25, 32, 45, 48-50,
53). For detection, C17-2 cells constitutively and stably express
the lacZ reporter gene encoding -Gal introduced by a
retroviral vector. Having been used successfully in a number of animal
models of neurologic disease, C17-2 cells have been useful for
illustrating the range of developmental potential and therapeutic
possibilities of NSCs for gene therapy and repair (25, 33, 45, 48,
52-54).
It had been previously established that clone C17-2 NSCs could be
infected in vitro with replication-defective retroviral
vectors (e.g.,
for transducing genes for
-Gal, enzymes, and neurotrophins
[
25,
33,
49,
53]) as well as with
replication-competent
retroviruses (
32). These infections
did not diminish the ability
of these NSCs to engraft and integrate
into the CNS parenchyma.
Therefore, it seemed feasible to convert these
cells into engraftable
in situ packaging/producer
lines.
In the discussions that follow, to avoid confusion, we will be careful
to distinguish between procedures relevant to characterizing
a
retroviral vector producer line and those that coincidentally
happen to
look at
env (the CasBrE retroviral surface glycoprotein)
as
the foreign gene of interest in these
studies.
An NSC-based retroviral packaging line was produced as outlined in
Materials and Methods by introducing the amphotropic packaging
plasmid
pPAM3 (used previously to generate the PA317 fibroblast
packaging cell
line [
37]) (Fig.
1A) along with the selection
plasmid
pPGKPuro (Fig.
1B) into C17-2 NSCs. Colonies were selected
in
puromycin, and 48 clones that were immunopositive for the amphotropic
coat by immunostaining with monoclonal antibody 83A25 (
13)
were
isolated. No significant difference in the level of coat
expression
among clones was detected by fluorescence-activated cell
sorting
(not
shown).
These clones were then screened for the ability to package the
endogenous
lacZ gene, which was originally introduced into
NSCs by a retroviral vector at the time of their derivation from
the
cerebellum (
50). Eighteen pPAM3-transfected clones with
packaging ability were identified. These clones expressed very
low
-Gal virus titers of 10
0 to 10
2 FFU/ml. To
identify whether any of these clones could more efficiently
package a
pSFF expression vector, the 18 clones were infected
with
replication-defective Cas
E virus, which encodes the CasBrE
Env protein (Fig.
1C). The Cas
E vector was observed to
spread throughout the 18 C17-2 packaging
cultures, most likely because
of incorporation of pCas
E RNA into virions expressing both
amphotropic (4070A) and ecotropic
(CasBrE) coats. Assessment of the
C17-2 NSC packaging cell clones
ability to package and transfer the
gene of interest (
env) to
naive targets indicated that all
18 subclones packaged
env into
infectious viral particles
with titers of 10
2 to 10
4 FFU/ml. In
particular, one subclone, BA6, consistently and reproducibly
transduced
CasBrE
env with high efficiency, producing titers of
10
4 to 10
5 FFU/ml, even after extensive
passaging. Therefore, we concentrated
our efforts on this subclone,
designated BA6-Cas
E.
To evaluate the ability of BA6-Cas
E to deliver
env by cell-to-cell contact (a more sensitive means for
identifying a cell's
infectious potential), irradiated
BA6-Cas
E cells were evaluated by an infectious center assay
(see Materials
and Methods). Of the BA6-Cas
E cells seeded,
31% ± 11% (
n = 4) generated distinguishable foci.
This result was indicative of successful delivery of virus to
surrounding susceptible fibroblasts and suggested the potential
of the
line to deliver genes in vivo. Attempts to subclone cells
from the
BA6-Cas
E population did not yield lines with a
higher-efficiency
env transduction.
Both BA6 and
BA6-Cas
E cells were negative for the production of
replication-competent
amphotrophic or recombinant CasBrE helper virus
when assayed by
serial supernatant
passage.
Interestingly, the BA6 and BA6-Cas
E cells packaged
lacZ at 10
0 to 10
2 FFU/ml even
though 80 to 100% of these cells constitutively express
-Gal from
an integrated provirus. This situation is akin to that
noted previously
where C17-2 cells, infected with replication-competent
virus, had
lacZ viral titers which were 2 to 3 logs lower than
the
infecting virus titers (
32). This phenomenon may reflects
the relative levels of message for each vector. However, it may
also
relate to (i) the ability of the Cas
E vector to more
efficiently ping-pong through the cultures as
outlined previously
(
28,
32) and/or (ii) the presence of
gag sequences present in the Cas
E vector, which could increase
the efficiency of packaging (
5).
It is also possible that
over the extended passing of the C17-2
cells, the packaging sequences
in the integrated
lacZ-encoding
provirus mutated, rendering
the
lacZ mRNA less suitable for viral
packaging.
Fortuitously, this ability of the BA6 packaging line
to efficiently
express
-Gal but inefficiently package
lacZ-containing
viral vectors allowed us to continue using this genetic marker
for
tentatively identifying transplanted C17-2 cells, since the
likelihood
that host target cells will be infected by
lacZ-expressing
virus, versus the
env-containing virus Cas
E, is
comparatively low (i.e., 2 to 3 orders of magnitude
minimally).
To assess whether the cellular and molecular derivation of BA6 and
BA6-Cas
E cells from C17-2 NSCs resulted in an alteration of
their progenitor-like
phenotype, cells were examined
immunohistochemically for nestin
expression. Both BA6 and
BA6-Cas
E cells expressed nestin at the same levels as the
parent C17-2
NSCs. In addition, proliferating cells were not
immunoreactive
to antibodies against GFAP, galactocerebroside,
neurofilament,
neuron-specific enolase, and CNPase, suggesting that
they maintained
an immature, undifferentiated NSC/progenitor-like
phenotype.
It should also be noted that the packaging NSC subclone BA6, into which
the
env-encoding vector was transduced to make
BA6-Cas
E cells, was phenotypically indistinguishable from
BA6-Cas
E except for lacking the
env-encoded
protein. In other words, BA6
packaging NSCs could in the future be
transfected with vectors
encoding other cDNAs for different research
purposes. BA6 and
BA6-Cas
E, as the best packaging and
transducing NSC subclones, were expanded
and used for transplantation
into neonatal IRW mouse brains as
outlined
below.
Transplantation of packaging NSCs into the brains of IRW mice.
To control for the possibility that the BA6 NSC packaging cell line
(NSCs without the CasBrE env) could cause neuropathologic changes when engrafted into the brain, BA6 cells were introduced into
the lateral ventricles of newborn IRW mouse brains. The brains of 16 animals were examined for engraftment and for the induction of
spongiform neurodegeneration. Areas showing the most reproducible levels of engraftment in all mice examined were the forebrains and
olfactory bulbs (OBs) (Fig. 2A and B).
(Therefore, as in prior studies [32], for uniformity
across experiments, analyses focused on these regions.) To evaluate
whether there were any untoward effects in response to engrafted BA6
cells only, we examined transplanted mice daily for clinical neurologic
signs and examined brains for histopathology at 15, 18, 25, and 28 days
postimplantation (dpi) (n = 4 at each time point). No
clinical signs were noted through the course of these experiments, and
histopathologic analysis revealed no spongiform neurodegenerative
changes (not shown). Furthermore, no evidence of inflammation was
observed, nor was overt microglial or astrocytic activation detectable
by immunohistochemical staining for F4/80 or GFAP, respectively (not
shown). These results were in agreement with previous studies in our
laboratory which demonstrated that CNS expression of
replication-restricted retrovirus from C17-2 NSCs does not induce acute
neuropathologic changes in the brain (32).
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FIG. 2.
Engraftment of BA6 and BA6-CasE retroviral
vector packaging NSCs and associated expression in vivo of the foreign
gene of interest (env). (A and B) Low-magnification
micrographs of representative sections through the forebrain and OBs of
mice transplanted with either BA6 packaging NSCs (A, at 25 dpi) or
BA6-CasE packaging NSCs (B, at 31 dpi), the latter a
subclone of BA6 specifically engineered to package into
replication-incompetent retroviral vectors the index gene of interest,
env. The sections were processed with X-Gal histochemistry
to show donor-derived lacZ-expressing cells as blue
(arrowheads). (C and E to G) Immunostaining for the gene product
encoded by CasE (env) in the brains of mice
transplanted with BA6-CasE cells. (C) A frozen section from
the OB immunostained for env, using aminoethyl carbazole as
the chromogen (red, arrowheads), is representative of the intense and
widespread env expression observed in the OBs and forebrains
of BA6-CasE-transplanted animals. To assess the likelihood
that transplanted NSCs are delivering env to endogenous CNS
cells, panels D and E directly compare the distribution of
-Gal+ cells (32) with
env-expressing cells in adjacent paraffin sections from the
OB (subjected to antigen retrieval prior to immunohistochemistry using
antibody 697). Panel D demonstrates a rather restricted number of
-Gal+ cells (aminoethyl carbazole; red, arrowheads)
across the multiple layers of the OB. In comparison, env
expression (diaminobenzidine; brown-stained cells, arrowheads) in panel
E appears as intense immunoreactivity in practically all cells of the
glomerular layer (Gl), as well as a large number of cells in the
external plexiform (Epl) and granule layers (GrO), implying that the
engrafted NSC-derived packaging cells may successfully deliver the
index foreign gene, env, to host cells. Representative
env expression is also detected in cells of deep cortical
layers and corpus callosum in panel F and at higher magnification in
the septum in panel G (red, arrows), two CNS regions documented to be
susceptible to the neurodegenerative effects of the CasBrE virus
(10, 32) (see also Fig. 3). As seen particularly well in
panel G, the env-expressing cells in these regions are
highly ramified, a morphology characteristic of microglia, a cell type
of nonneuroectodermal origin. Immunostaining of sections corresponding
to those in panels F and G for -Gal expression failed to detect the
presence of -Gal+ donor derived NSCs (not shown).
Furthermore, most env+ tissue sections examined
in the septum and deep cortex actually had few if any detectable
-Gal+ cells (donor derived), further suggesting that the
env+ cells were of host rather than donor
origin. Magnifications: A and B, ×25; C, ×125; D to G, ×250.
|
|
BA6-Cas
E packaging NSCs were next similarly implanted into
neonatal mouse brains to determine whether they could deliver
significant
levels of the retroviral vector-transduced gene
env to endogenous
dividing cells. Therefore, the
distribution and phenotype of host
(
-Gal-negative) cells expressing
env was assessed; alternatively
stated, infected
env+ cells were examined to determine whether
they were
-Gal negative.
In addition, we examined the animals
clinically and analyzed brain
sections histopathologically as described
above. No clinical neurological
signs were noted in any
BA6-Cas
E NSC-transplanted animals through the course of
these experiments
(
n = 20).
Examination of the brains at 14, 22, and 31 dpi (
n = 4,
4, and 12, respectively) revealed X-Gal-positive, blue donor-derived
cells in the rostral forebrain and OBs of all mice (Fig.
2B).
In
contrast to the somewhat restricted distribution of X-Gal reactivity,
immunostaining for the protein encoded by
env was much more
widespread,
including significant expression in the OB (Fig.
2C and E),
deep
layers of the cerebral cortex and the corpus callosum (Fig.
2F),
striatum, and septum (Fig.
2G).
When colocalization of
env expression with
-Gal was
attempted via double immunostaining, very little
-Gal-positive
reactivity
was observed in
env+ cells,
suggesting that engrafted NSC-derived packaging cells
may, indeed, have
successfully delivered the virally transduced
foreign gene
env to host cells. This assessment was supported
by the
representative data presented in Fig.
2 which demonstrated
that the
extensive distribution of
env expression was significantly
broader than, and not restricted to, the typical distribution
of
engrafted
-Gal-expressing (
-Gal
+) packaging NSCs; for
example, in the OB, comparison can be made
between the extent of
env immunoreactivity in Fig.
2E with the
distribution of
donor-derived
-Gal immunoreactivity in Fig.
2D.
In addition, certain
env+ tissue sections actually had few if any
detectable donor-derived
cells; for example, sections corresponding to
those in Fig.
2F
and G were immunonegative for
-Gal
+ cells.
Although we believe that the dissociation between
env and
-Gal expression represents endogenous CNS cells (defined by their
-Gal negativity) that were infected by the
env-containing
vector
released from transplanted NSCs, it is also possible that the
appearance of such cells was due simply to down regulation of
lacZ expression in engrafted BA6-Cas
E cells.
Although most (
80%) BA6-Cas
E packaging NSCs are
-gal
+, it was still possible for some donor-derived
cells to be incorrectly
identified as host cells if they were among the
few cells that
failed to express
-Gal. Even looking at the
particular neural
cell types expressing
env would not be
compelling in this regard
(colocalization of
env with
various neuronal and glial markers
was, indeed, detected). For example,
distinguishing between cells
of host and donor origin in even an
extensively
env-expressing
region like the OB (Fig.
2C and
E) where postnatal neurogenesis
persists is difficult because NSCs can
give rise to the neural
cell types comprising this region
(
49).
However, there was one instance where observing
env
expression in a particular cell type would compellingly prove the
env transduction phenomenon. As seen particularly well in
Fig.
2G,
the
env-expressing cells in most regions are highly
ramified cells
with a morphology and size characteristic of microglia.
Because
microglia are not of neuroectodermal origin and C17-2 NSCs
cannot
give rise to this bone marrow-derived cell type, expression of
the transgene
env in these cells would strongly suggest host
cell
transduction by the transplanted BA6-CasE packaging NSCs and not
misidentification of engineered donor-derived cells as host
cells.
To prove unambiguously that this strategy for delivering foreign genes
to, and genetically manipulating, endogenous host cells
in the CNS via
infection by transplanted packaging NSCs, we carried
out rigorous cell
type analysis to determine whether the
env+
cells, as seen in Fig.
2C and E to G, were microglia, a cell
type which
must be host and not donor derived. Therefore, brain
sections were
double immunostained for the protein encoded by
env and the
microglial marker F4/80 (
18,
40). The results
(Fig.
3) clearly indicate the coincidence of
the two markers,
indicating that
env was transmitted to
microglia by the transplanted
BA6-CasE packaging NSCs. In fact, outside
of the OB, we observed
few
env+ cells that did
not also stain with the F4/80 microglial marker.
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FIG. 3.
Localization of env expression within
microglia demonstrates that NSCs are capable of retrovirus-mediated
transgene delivery to host CNS cells. (A) Indirect immunofluorescent
staining for Env protein within the deep layers of the cerebral cortex
viewed through the fluorescein filter set (using biotinylated antibody
667). Note the highly arborized nature of these cells, characteristic
of a microglial phenotype. (B) The same section as in panel A stained
with the microglia-specific antibody rabbit anti-F4/80 as outlined
elsewhere (18) and viewed through the rhodamine filter set.
Note the colocalization of env and F4/80 in panels A and B,
respectively (arrowheads). (C) Region of the septum in which Env
protein (green) colocalizes with the microglial marker F4/80 (red) to
yield yellow cells (arrowheads), further suggesting that the
transplanted NSC packaging cells delivered the env gene to
endogenous host cells. Magnification bars: 50 µm.
|
|
This result interestingly also highlights the particular susceptibility
of microglia to in vivo infection by murine retroviruses.
What accounts
for this particular viral tropism is not known.
However, in assessing
the CNS response to this microglial
env expression we noted
that in the local regions where microglia
were demonstrated to be
expressing
env, there was an elevated
expression of the
microglial antigens F4/80 and Mac-1 (CD11b)
(Fig.
4) compared with control engrafted (BA6)
or unengrafted
animals. These results suggest that CasBrE
env expression in microglia
was inducing microglial
activation, a phenomenon believed by some
to play a contributory role
in neuropathogenesis (
12,
16,
35). However, we have
previously noted a dissociation between
glial activation and the acute
induction of spongiform myeloencephalopathy
by the highly neurovirulent
chimeric CasBrE virus FrCas
E (
10,
29,
31,
32).
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FIG. 4.
env expression in host microglia results in
local microglial activation. Microglial activation was compared between
BA6-CasE-transplanted (A and C) and control uninoculated (B
and D) mice at 31 days of age by immunostaining brain sections from the
striatum for the microglial markers F4/80 (A and B) and Mac-1 (CD11b)
(C and D), using rat monoclonal antibodies (2, 56). Note
that expression of both antigens is greater in this region of
engraftment than in control sections from the same brain region. This
elevated staining was observed only in areas where microglial
env expression was noted (see also Fig. 3B), as areas in the
BA6-CasE brains closely adjacent to those expressing
env showed no increased staining for F4/80 and Mac-1
compared with controls (not shown). Magnification bars: 40 µm.
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|
To rule out the possibility that microglial infection resulted from the
in vivo generation of a replication-competent virus,
homogenates from
serial frozen engrafted brain sections or whole
brain homogenates were
evaluated for the presence of replication-competent
virus on
M. dunni fibroblasts by serial supernatant passage (see
Materials and
Methods). When the target cells were exposed to
antibodies to detect
virus infection, only colonies with patterns
consistent with defective
infection (noncontiguous cell staining)
were evident (Fig.
5). Furthermore, passage of undiluted
supernatants
from cultures like that shown in Fig.
5A onto additional
naive
M. dunni targets failed to reveal the presence of
viral foci either
by staining for viral Env antigens or
-Gal or by
selection for
neomycin. These results support the conclusion that the
microglial
expression of
env was due to the delivery of the
env-encoding
vector by transplanted BA6-CasE packaging NSCs
and not as a result
of the emergence of a recombinant virus.
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FIG. 5.
Transplanted BA6-CasE packaging NSCs release defective
virus encoding env without helper virus. (A) Example of the
viral foci obtained after exposing naive M. dunni
fibroblasts to 10% CNS homogenate extract from a mouse transplanted
with the BA6-CasE packaging NSCs 7 days
posttransplantation. Note that abundant env+
cells (black) are not in contiguous distinct foci, like that observed
for a replication-competent 4070A amphotropic virus (C). Upon infection
with a replication-defective virus, dividing, immunoreactive cells
become separated due to cellular migration. Were these cells infected
by a replication-competent virus, they would deliver virus to naive
cells with which they make contact, as they migrate, to generate a
contiguous focus (C). Dilution of a BA6-CasE brain extracts reveals the
nature of a single virus focus (B). Again, note that the individual
env+ cells are separated from one another
(arrowheads) by uninfected cells, suggesting that the original
infectious event did not result from a replication-competent virus and
thus could not spread to adjacent dividing cells. Passage of
supernatants from cultures such as that shown in panel A onto
additional naive M. dunni fibroblasts failed to result in
the appearance of detectable foci. These data support the conclusion
that in vivo, NSCs continue to produce viral particles carrying the
transgene of interest without producing helper virus. Magnification
bars: A and C, 500 µm; B, 200 µm.
|
|
Previous reports on retrovirus-induced spongiform neurodegeneration
suggested that microglial infection was directly associated
with this
pathologic process (
3,
4,
19,
28,
29,
31,
32) and that
env expression alone in the CNS might be sufficient
for
inducing neurodegenerative changes (
21,
22,
43,
61,
62).
Given that significant
env expression was observed in
microglia
after transplantation with BA6-Cas
E packaging
NSCs (Fig.
2F and G and 3), we evaluated engrafted
mice for
histopathologic changes indicative of status spongiosis.
Mice examined
at 22 (
n = 4) and 31 (
n = 12) dpi
showed no evidence
of spongiform neurodegeneration in regions with
microglial
env expression (Fig.
6A and
B). Whether this was influenced by the
microglial activation noted is not known. However, this picture
contrasted with the spongiosis noted in positive control mice
transplanted with the complete neurovirulent, replication-competent
15-1 clone of the CasBrE virus (
32,
41) wherein extensive
pathology was noted by 14 days after transplantation of C17-2
cells
(Fig.
6C and D), and microglial activation was not detected
(not
shown).
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FIG. 6.
env expression within host microglia as
mediated by engraftment of BA6-CasE packaging NSCs is not sufficient to
induce CNS spongiosis. (A) Representative hematoxylin-and-eosin-stained
section from the deep cerebral cortex of a BA6-CasE
packaging NSC-engrafted mouse. This section was taken from a region
where significant microglial expression of env was observed.
(B) Paraffin section from the cerebral cortex immunostained for
env-encoding protein after antigen retrieval (using antibody
697) and counterstained with hematoxylin. Note that no spongiform
neurodegenerative changes are observed by 31 days after engraftment of
NSCs in panel A or B despite abundant env expression in
microglia (B; arrows). (C and D) positive controls, demonstrating that
NSC-mediated infection with the complete, replication-competent
neurovirulent virus is capable of inducing neurodegeneration. A
hematoxylin-and-eosin-stained section (C) and Env-immunostained (brown)
and hematoxylin-counterstained section (D) show the same CNS regions
presented in panels A and B but from a mouse transplanted with C17-2
NSCs containing replication-competent CasBrE virus (clone 15-1) 17 days
posttransplantation. Note the abundant vacuolation (arrowheads)
associated with the presence of env expression within
microglia (arrows) (see also references 29, 32, and
43). Magnification bars: 50 µm.
|
|
Therefore, with respect to our novel approach for exploring the
pathogenesis of spongiform myeloencephalopathy, we suggest
that
expression of the CasBrE
env gene alone within microglia
is
not sufficient to mimic the neuropathogenic effects of the
complete
neurovirulent retrovirus. It appears that
env-encoded
protein requires interaction with additional viral structural
components to cause acute vacuolar neurodegeneration. Taken together
with our previous studies, which demonstrated that in vivo CasBrE
virus
binding and entry are also not sufficient to induce disease
(
32), the data suggest that retrovirus-induced CNS
pathogenesis
involves an interaction between the
env-encoded
protein and structural
proteins from the late stages of viral
replication, perhaps in
the virus assembly and maturation
process.
In this regard, it has been demonstrated that assembly and release are,
indeed, defective in CasBrE-infected microglia in
culture
(
28). Specifically, we have observed that infected microglia
fail to process the gpr85 envelope precursor protein to gp70 and
p15E.
Associated with this defective processing, viral particles
are observed
to bud errantly into intracellular compartments rather
than from the
cell surface. It may be that the process of trying
to assemble
retroviral particles in microglia by using improperly
processed or
defective components alters microglial physiology
to the point where
microglia either (i) release a neurotoxic factor
or (ii) can no longer
adequately provide the support required
for maintaining local neuronal
homeostasis.
The results presented herein and previously (
32) indicate
that CasBrE
env expression alone is not sufficient to induce
acute
spongiform neurodegeneration regardless of the cell type in which
env is expressed. These results are in seeming contrast to
those
generated in studies examining CasBrE (
22) and
ts1
env expression
in transgenic animals. In
these studies it was observed that animals
harboring and expressing
mRNA from the respective
env genes correlated
with mild
clinical and neuropathological changes. However, it
should be noted
that disease incidence was markedly reduced, and
when it did appear it
had a time course much longer than that
noted for infection with virus.
While it is possible that the
mild phenotype observed in the transgenic
animals was the direct
result of the limited
env gene
expression noted in these animals,
it is also possible that the lack of
acute disease was due to
the lack of additional retroviral proteins. In
fact, given the
extended time course of the transgenic experiments, it
is possible
that endogenous expression of
gag and
pol transcripts and protein
could complement the transgenic
env expression without resulting
in recombinant virus. Such
a possibility was not formally excluded
in these experiments. Moreover,
we have previously documented
that CasBrE-induced spongiform
neuropathology can arise acutely
after postnatal day 10 (
10,
30), and this pathology is directly
associated with a small
number of virus infected microglial cells,
in the absence of other
cellular infection (
31). In the experiments
described
herein, CasBrE env protein expression in microglia was
not
significantly different from that noted in mice transplanted
with the
"slow" 15-1 clone of CasBrE and was consistent with that
previously
noted in microglial transplant experiments (
31).
Yet the
differences in the induction of pathology are striking.
The possibility
that BA6-CasE C17-2 transplant recipients can
develop neuropathological
changes at extended time points is moot
given the caveats associated
with endogenous retroviral gene expression
for which there are no
adequate controls. To clearly address the
differences noted between the
transplant and transgenic studies,
experiments involving microglial
coexpression of neurovirulent
env and additional viral
structural constituents will need to
be undertaken in order to
understand what precipitates acute
disease.
We expect that dissecting the precise CasBrE viral assembly events that
occur in microglia and precipitate neural spongiform
changes will
provide insight into understanding the molecular
pathogenesis of other
CNS diseases of viral and nonviral origin.
Of particular note are human
immunodeficiency virus-induced cognitive-motor
complex, where
microglial infection appears to be critical for
disease induction, and
prion diseases, where microglia have been
implicated in the vacuolar
myeloencephalopathy associated with
abnormal protease-resistant protein
assemblies (
7). Analyses
of these pathologies at the
molecular level should be facilitated
by future experiments wherein
transplantable packaging NSCs are
used to deliver to microglia,
individual or multiple retroviral
or prion components, in a stepwise
fashion, to selectively reconstitute
the infectious process in the
brain.
In a much broader sense, particular note should be made of the approach
used to address these questions regarding neuropathogenesis.
These data
support the hypothesis that engraftable, migratory
NSCs may be
engineered ex vivo to serve as novel platforms for
the effective
dissemination in vivo of replication-defective viral
vectors and their
encoded genes directly to endogenous CNS host
cells. The generation of
a viral packaging/producer cell line
that can engraft and intercalate
into the CNS parenchyma provides
a novel avenue for delivering genes
(therapeutic or otherwise)
to relatively inaccessible sites within the
CNS. Certainly this
strategy magnifies the often limited distribution
of retroviral
vector-mediated gene transfer to the CNS and therefore
extends
their applicability and efficacy for a much broader range of
research
and clinical applications. Certainly, microglia and cells of
mesenchymal
origin in the CNS can be targeted. This is of great
significance
given that microglia appear to play a significant role in
a variety
of genetic, sporadic, and infectious diseases (
7,
12,
17,
35) and have been rather refractory to genetic manipulation
due to their limited turnover in the mature CNS (
24,
26,
27).
Observations in the OB (a region of persistent postnatal
neurogenesis
where
env expression extended beyond donor cell
engraftment and
colocalized with neuronal and glial markers) suggest
that dividing
cells of neuroectodermal origin may also be targeted
(Fig.
2C
and E). (Note in particular the intense
env
immunoreactivity in
all OB neuronal layers in Fig.
2C.) Thus, the
potential exists
for manipulating multiple CNS cell types via the NSC
packaging
cell
strategy.
Regarding the genes of interest for transfer to the CNS, while
env was the index gene explored in these
feasibility/proof-of-principle
experiments, the approach for making
packaging NSC lines transducing
other types of genes (e.g., those
encoding neurotrophic or cytotoxic
agents) to manipulate host cells for
other research or clinical
demands would follow similar procedures,
with anticipated similar
success. Although this work was done in the
postnatal mouse cerebrum,
we anticipate that the same approach can be
applied where mitotic
cells are prevalent in a given region of the
developing or adult
CNS. In the fetus, such cells could be neuroblasts
in most regions
of CNS; in the postnatal or adult brain, such cells
could include
glia, microglia, endothelium, choroid, persistent centers
of neurogenesis,
and even neoplastic cells. Therefore, the strategy
presented in
this report may potentially be applicable to a wide
variety of
therapies for both genetic and acquired disorders of the
CNS.
Given that in the present study we used a second-generation murine
retrovirus packaging vector, pPAM3, as a starting point,
it is likely
that the application of newer packaging plasmids
will only further
enhance gene transfer efficacy to CNS cells.
In particular, the
marriage of lentivirus-based vectors to NSCs
should provide a means to
extensively target genes to nondividing
as well as to dividing cells of
the CNS. Furthermore, NSCs may
be amenable to delivering other types of
viral vectors that previously
used producer cells of nonneural origin
(adeno-associated viral
vectors, herpesvirus amplicon-augmented
vectors, etc.). In short,
this approach may represent the interface
between two gene therapy
strategies, virus-mediated and cell-mediated
gene delivery, maximizing
the advantages of each (
14,
49,
52,
58) and providing
yet another strategy by which NSC biology may
be harnessed for
genetically manipulating the
CNS.
| |
ACKNOWLEDGMENTS |
Support for this work was provided in part by a grant from the
Amyotrophic Lateral Sclerosis Association and NINDS grant NS37614 to
W.P.L., NINDS grant NS31065 to A.H.S., and grants from NINDS (NS34247),
the American Paralysis Association, and the Paralyzed Veterans of
America to E.Y.S. Mental Retardation Research Center grant
NIH-P30-HD18655 to Children's Hospital also provided support for this project.
| |
FOOTNOTES |
*
Corresponding author. Mailing address for William P. Lynch: Department of Microbiology/Immunology, NEOUCOM, P.O. Box 95, 4209 State Rte. 44, Rootstown, OH 44272. Phone: (330) 325-6137. Fax: (330) 325-5914. E-mail: wonk{at}riker.neoucom.edu. Mailing
address for Evan Y. Snyder: Departments of Neurology and Pediatrics,
Children's Hospital, 300 Longwood Ave., 248 Enders Bldg.,
Boston, MA 02115. Phone: (617) 355-6277. Fax: (617) 738-1542. E-mail:
Snyder{at}A1.TCH.Harvard.Edu.
| |
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Journal of Virology, August 1999, p. 6841-6851, Vol. 73, No. 8
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
