Download Adenovirus vector-mediated in vivo gene transfer into adult

Adenovirus Vector-Mediated In Vivo Gene Transfer
Into Adult Murine Retina
Jean Bennett,* James Wilson^ Dexue Sun* Brian Forbes* and Albert Maguire*
Purpose. To determine whether a reporter gene can be introduced into the adult mammalian
retina in vivo through means of a recombinant replication-deficient adenovirus.
Methods. A dilution series of purified Ad.CMV/acZ ranging from 105 to 10" pfu/ml was
prepared and microinjected into the subretinal space of adult CD-I mice. This virus contained
the cytomegalovirus (CMV)-promoted Escherichia coli reporter gene, lacZ. LacZ expression was
assessed in enucleated eyes from 0 to 95 days after injection by j8-galactosidase (/3-Gal) assay.
Results. The efficiency of transfection increased as a function of concentration of recombinant
virus injected. Eyes injected with greater than 107 pfu of Ad.CMVZacZ demonstrated /3-Gal
activity lasting at least 95 days. LacZ expression was apparent only in those cells directly exposed to the adenovirus. LacZ expression was observed in the retinal pigment epithelium
(RPE) at high efficiency at 48 hours after exposure. By 2 weeks after injection of > 107 pfu, lacZ
was also expressed in photoreceptors, but at lower density.
Conclusions. These results demonstrate that high efficiency stable transfer of functional genes
can be achieved in vivo in post-mitotic mammalian retina using recombinant adenoviral vectors. Adenovirus vectors appear to be a promising means for delivering therapeutic genes in
vivo to the mammalian neural retina and particularly to the RPE. Invest Ophthalmol Vis Sci.
1994;35:2535-2542.
vxene transfer offers the possibility of correcting mutations at the somatic level and may have application in
the treatment of such inherited retinal degenerations
as retinitis pigmentosa (RP). Numerous mutations
have been identified in RP involving genes expressed
specifically in retinal photoreceptors. The mutations
cause degeneration of these cells and thereby lead to
blindness. Although there are many avenues for delivering genes to actively dividing cells, there are few
techniques available to deliver genes to cells that have
completed their terminal mitosis, such as differentiated photoreceptors.
Methods have been developed that can achieve
successful transfer of exogenous genes into differenFrom tliu "Scheie Eye Institute, F. M. Kirby Center for Molecular Ophthalmology,
and 'fThe Institute for Human Gene Therapy, Wistar Institute, University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania.
Supported by a Research to Prevent Blindness Career Development Award (JB), a
Retinitis Pigmentosa Foundation Young Investigator Award (JB), and a University
of Pennsylvania Research Foundation Award (JB).
Submitted for publication September 21, 1993; revised November 3, 1993;
accepted November 12, 1993.
Proprietary interest category: N.
Reprint requests: Dr. Jean Bennett, Scheie Eye Institute, F. M. Kirby Center for
Molecular Ophthalmology, 51 North 39th Street, Philadelphia, PA. 19103.
tiated mammalian cells in vitro. Whereas some of the
physico-chemical techniques are impractical in vivo,
there are a few that have been successfully applied to
cells within the living animal and have resulted in prolonged expression. These include injection of naked
DNA alone12 and injection of DNA mixed with a
highly polar lipid vehicle, such as lipofectin.34 In addition, gene transfer accomplished through infection
with recombinant replication-deficient adenoviruses
has recently been demonstrated to result in stable
transfection with minimal toxicity in differentiated,
nonreplicating muscle,5"7 liver,8 lung,9 bronchial,1011
and neuronal12"15 cells. In all these cases, gene transfer
is most efficient in tissues in which high surface-tovolume ratios are present.
Administration of a gene transfer vehicle to the
subretinal space would allow for exposure of a large
number of RPE and photoreceptor cells due to the
high surface-to-volume ratio. Because the adenovirus
and lipofectin-mediated in vivo transfection techniques resulted in relatively stable transfection in neuroepithelial-derived tissues, we attempted to use them
to transfer genes into cells lining the subretinal space
of adult mice. The results of gene transfer using lipo-
Invcsligntive Ophthalmology & Visual Science, April 1994, Vol. 35, No. 5
Copyright © Association for Research in Vision and Ophthalmology
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Investigative Ophthalmology 8c Visual Science, April 1994, Vol. 35, No. 5
fectin as the transfer vehicle are described in detail
elsewhere16 (also Bennett et al, manuscript in preparation). In the present study, we used the recombinant
adenovirus, Ad.CMVlacZ, as the gene transfer vector.
This virus contains the cytomegalovirus (CMV)-promoted Escherichia coli reporter gene, lacZ, and is deleted of viral El sequences, making it replication defective.11 The studies reported herein describe techniques with which to introduce therapeutic genes
specifically into the adult neural retina in vivo.
MATERIALS AND METHODS
Preparation of Adenovirus-Containing
Solutions for Injection
Adenovirus vectors were prepared, purified, and titered to 1013 particles/ml as described.11 The same
stock of Ad.CMV/acZ was used for all experiments.
Serial dilutions of the titered adenovirus were made
(ranging from 105 to 1011 plaque-forming units (pfu)/
ml) in 20% sucrose in phosphate-buffered saline
(PBS). All manipulations of adenoviral vectors were
done in accordance with institutional and national
biosafety restrictions.
Surgical Approach
All animals were cared for in accordance with the
ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and with federal, state, and
local regulations.
CD-I mice (ages 3 weeks to 3 months) were anesthetized with avertin, and their pupils were dilated
with 0.5% tropicamide. A 30-gauge cannula was inserted into the subretinal space of the peripheral retina via an external transscleral transchoroidal approach. The cannula was advanced and secured in the
subretinal space. One to 10 ^1 of transfection solution
was injected, thereby raising a dome-shaped retinal
detachment. Excess solution escaped from around the
injection cannula and was discarded.
All injections were monitored by indirect ophthalmoscopy and by direct visualization through the operating microscope. Visualization of the detachment
confirmed that the transfection solution was injected
into the targeted location. Exposure of RPE and photoreceptor cells to the subretinal material was confirmed histologically with injection of PBS containing
0.01 /urn rhodamine-labeled latex microspheres (Molecular Probes, Eugene, OR). Control solutions consisted of a CsCl-purified CMV-promoted lacZ construct, pIC400 DNA, 1 mg/ml (generously contributed by I. Chiou) in either 20% sucrose in PBS or PBS
alone. In addition to pharmacologic controls, surgical
controls were performed with injections into the vitreous and with subretinal injections of solutions lacking
DNA or viral particles. The latter solutions included
20% sucrose/PBS and PBS alone. All injections in
which blebs were not successfully raised were noted
and analyzed separately.
Histochemical Analysis
Animals were sacrificed from 2 hours to 94 days after
injection, and eyes were enucleated. Eyes were either
immediately fixed for 1 hour at 4°C in PBS containing
4% paraformaldehyde or immersed in OCT compound (Baxter, McGaw Park, IL) and frozen in liquid
N2. The fixed eyes were washed with PBS and reacted
for 12 hours with 1 mg/ml of X-Gal (5-bromo-4chloro-3-indolyl galactopyranoside; Sigma Chemical
Co., St. Louis, MO) in a solution containing 10 mM
K3Fe(CN)6, 10 mM K4Fe(CN)6-3H2O, 2 mM MgCl2,
0.01% sodium deoxycholate, 0.02% NP40 in PBS. XGal reacted eyes were then post-fixed in 4% paraformaldehyde for 24 hours, washed with PBS, embedded in OCT, and frozen in liquid N2.
Both fixed and unfixed frozen eyes were sectioned
at 5 to 16 jwn at —24°C. Unfixed frozen sections were
immersed in 4% paraformaldehyde for 1 minute and
then reacted with X-Gal for 2 hours as described
above. Specific cell types were identified on the basis
of their location within the well-defined cell layers of
the retina, determined by examination of serial sections. Cell counts were made only in sections where
the /3-Gal reaction product had not diffused into
surrounding cells. Photoreceptors were identified by
determining the presence of staining localized to the
layers occupied by photoreceptors: the outer plexiform layer (OPL), outer nuclear layer (ONL), and inner segment layer (ISL) of individual cells. The outer
segments of /acZ-expressing photoreceptors were not
stained (also observed in mice transgenic for lacZ constructs—Bennett, unpublished data). Retinal cells that
possessed staining in any of the layers not occupied by
photoreceptors (the inner nuclear layer (INL), inner
plexiform layer (IPL), or ganglion cell layer (GCL)),
were classified according to their location and identified by cytologic features.
To screen for potential toxic systemic effects of
the recombinant virus, selected organs (brain, lungs,
heart, liver, spleen, kidneys) from experimental animals, uninjected littermates, and offspring were also
fixed and reacted with X-Gal in whole-mount fashion
as described above.
RESULTS
Lack of /?-Gal Activity in Cells Bordering
Subretinal Space of Control Animals
There was no /3-Gal activity after subretinal injections
of control solutions containing CMV-promoted lacZ.
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In Vivo Adenovirus-Mediated Retinal Gene Transfer
(3-Gal activity was not evident in cells bordering the
subretinal space when any injection (including injections of adenovirus) had not raised a subretinal bleb.
Injections of PBS with rhodamine-labeled latex microspheres confirmed the subretinal location of injected
material. There was no /3-Gal activity after injections of
rhodamine microspheres (Fig. 1A). In all instances,
rhodamine spheres were found localized within the
subretinal space, with the highest density near the injection site (Fig. 1). The fluorescent beads also outlined the needle penetration site (Fig. IB, arrow) and
extended over the entire region that had been experimentally detached (Fig. IB, hollow arrow). Auto-
fluorescence in the lens and in the surrounding connective tissue was apparent.
There was no RPE or outer retina staining of ocular tissue after intravitreal injection of 109 pfu of
Ad.CMVkcZ (data not shown). In a few cases (13%, n
= 16), 0-Gal activity was localized to occasional ganglion cell bodies.
Efficiency of Transgene Expression Is
Dependent Upon Viral Titer
The injected material was rapidly absorbed from the
injection site so that, by 24 hours after injection, the
retina had reattached as judged by indirect ophthal-
l. No /S-Gal activity is present 48 hours after injection of rhodamine-labeled latex
microspheres. Solid arrow represents the needle entry site where the RPE and photoreceptors have not yet reattached. (A) X-Gal-reacted section viewed with Hoffman modulation
optics. (B) Rhodamine-labeled spheres viewed with fluorescence outline the subretinal space
that was the original site of retinal detachment. Hollow arrow represents reattached retina.
FIGURE
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Investigative Ophthalmology & Visual Science, April 1994, Vol. 35, No. 5
moscopv. Between 24 to 18 hours after injection,
whole-mount preparations of mouse1 eyes injected
with 1 (>•* (or higher) pin of Ad.CMVIarZ and reacted
with the lurZ substrate. \-(ial, revealed a blue circular
region coi responding to the retinal detachment site.
The intensity of staining al the detachment site appealed to he a function of viral dose from JO' to 10s
plii (Kig. 2). In those eves injected with the lower virus
concentrations, the staining pattern within the injection area was punctate, suggesting localization of the
jtf-Gal activity to individual cells. Staining was localized
to the RPE (and none was observed in the neural retina) after injections of less than 1 ()'' pfu/ml (or 10'
pfu) of virus.
FIGL-'RE 2. Dose-response relationship ol 0-G;d activity after injection of Ad.CM\7<7fZ. Eves
wen- enucleated and reacted with \-()al IS hours after injection. The number of pfu ol
injected virus appears in the upper right corner. Regions of blue staining, visible through the
sclera, correspond with the areas of snbretinal injection. /rf-Gal activity correlates with the
concentration of Acl.CMX lac/..
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In Vivo Adenovirus-Mediated Retinal Gene Transfer
B
3. Cells in the neural retina and RPE cells express j8-Gal 2 weeks after subretinal
injection with 107 pfu of Ad.CMVlacZ. (A) Section (16 ^m) demonstrating near 100% expression of /3-Gal by the RPE cells. Arrows represent portions of individual /acZ-expressing photoreceptors. (B) Eosin-stained section (8 fivn) from a separate eye. Solid arrows represent
lacZ-express'mg photoreceptors. As seen in mice transgenic for human rhodopsin-promoted
lacZ (Bennett, unpublished data, 1993), staining in photoreceptors is confined to the ISL,
ONL (location of solid arrows), and the OPL, corresponding to the photoreceptor inner
segment, cell body, and synaptic projections. A Miiller cell possesses /3-Gal activity in the
inner nuclear layer (hollow arrow), an area not occupied by photoreceptors, as well as in the
outer plexiform and outer nuclear layers. Large arrowheads represent RPE cells.
FIGURE
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Investigative Ophthalmology 8c Visual Science, April 1994, Vol. 35, No. 5
LacZ was expressed at low levels in other ocular
tissues that came in contact with the transfection solution. Such tissues included localized regions of conjunctiva and sclera where the injection cannula penetrated the eye, the endothelial surface of the cornea,
and the iris.
In consecutive experiments using the same viral
stock, the efficiency of gene transfer to all cells decreased after each freeze-thaw cycle.
Histologic Effects of Retinal Detachment
At 24 hours after injection, histologic analysis revealed
regions of focal detachment (see Fig. IB, arrow),
disruption of outer segments, and folding of the
neural retina. These features persisted for 1 week after
injection and were present after injections of all concentrations of virus or control solutions into the subretinal space. There was minimal evidence of inflammation, however, as judged by the lack of polymorphonuclear or round cell infiltrate. Much of the
histologic disorganization resolved by 2 weeks after
injection. The retina appeared histologically normal
by light microscopy by 6 weeks after injection.
Alterations in LacZ-Expressing Retinal Cell
Types as a Function of Time
LacZ was expressed in the RPE of mice injected with
107 pfu of Ad.CMVlacZ within 48 hours after injection. Concentration of the /?-Gal reaction product was
so great in areas that it diffused into the outer segments of adjacent photoreceptors (see Fig. 3A). Over
the next 2 weeks, the percentage of /?-Gal-expressing
RPE cells increased from 79% ± 11% to 92% ± 9% (P
< 0.027, Student's J-test). There was no lacZ expression in the neural retina of such eyes until 2 weeks
after injection. When comparing different eyes, there
was a high amount of variability in the cell types expressing lacZ at 2 weeks after injection. In 100% of
eyes (n = 6) observed at 2 weeks after injection, many
cells of the outer retina (including photoreceptors) expressed lacZ (Fig. 3). There were also occasional lacZexpressing cells that traversed the neural retina. These
were identified as Miiller cells. RPE cells and photoreceptors continued to demonstrate /3-Gal activity 3
months after injection (n = 2). There did not appear to
be any alterations in the percentages of stained cells at
this point, although there was a decrease in the intensity of the staining. Throughout this time, there was no
evidence of toxic or cytologically disruptive effects
due to the virus in 72 eyes injected with Ad.CMVlacZ.
All the animals maintained a healthy appearance and
had high rates of fertility. All offspring were normal.
None of the injected animals, uninjected littermates,
or offspring of the injected animals displayed any histochemical evidence of virally transmitted jS-Gal activ-
ity in heart, lungs, liver, or spleen (or eyes, in the cases
of uninjected littermates and offspring).
DISCUSSION
The results presented here demonstrate that recombinant El-deleted adenoviruses can deliver functional
exogenous genes into cells bordering the subretinal
space—photoreceptor cells, Miiller cells, and RPE
cells. Gene transfer into these cells occurred only
when the adenovirus was injected into the subretinal
space. Transgene product was present up to 13 weeks
after infection. This is in sharp contrast to results from
lipofectin-mediated transfection of the retina, where
histochemically detectable lacZ expression lasted only
4 days after injection.16 Expression of the adenovirally
transferred reporter gene was localized to ocular tissues contiguous with the injection, and there was no
evidence of systemic infection.
Though the surgical technique used was simple
and reproducible, short-term disruption of the outer
retina was observed. Outer retinal injury is a recognized effect of retinal detachment17 and was probably
not caused by toxicity of the injected material. The
histologic effects of subretinal injection appeared to
be transient, with evidence of recovery apparent
within 2 weeks. Ultrastructural studies have demonstrated recovery of photoreceptors, with regeneration
and reorientation of outer segments evident after a
2-week reattachment period.17
The delay in photoreceptor-specific expression of
the reporter gene could be due to the immediate structural damage incurred on these cells by the physical act
of the retinal detachment. Around 2 weeks, when
much of the structural damage has been resolved, the
rates of gene expression may increase enough to allow
histochemical visualization of /3-Gal activity. Alternatively, the delay in expression by these cells may be due
to physical obstructions that prevent the immediate
infection of their cell bodies by adenoviruses. The
barrier between the subretinal space and the photoreceptor cell bodies imposed by the outer segments and
the tenacious extracellular matrix that encases them
might be expected to restrict the access of the adenovirus. Over a course of 2 weeks, enough virus particles
may have traversed the structural barriers, by slow replication, transfer from RPE cells, or diffusion, to infect
the photoreceptors.
A significant advantage of introducing the recombinant adenovirus into the subretinal space is that the
epithelial-derived tissues bordering this space have a
high surface-to-volume ratio. A high percentage of
RPE and photoreceptor cells can therefore be exposed to therapeutic genes in vivo in an efficient manner. Although both cell types expressed the recombinant genes in a highly stable fashion, the efficiency of
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In Vivo Adenovirus-Mediated Retinal Gene Transfer
gene transfer to the two cell types differed substantially. The extremely high efficiency of gene transfer to
the RPE cells within 48 hours after injection of the
recombinant adenovirus may be due to the high rates
of active transport and phagocytosis normally maintained by these cells. In contrast, few if any of the
photoreceptors expressed the transfected gene at this
time. However, 2 weeks after injection, the number of
/3-Gal-expressing photoreceptors increased significantly.
These experiments provide a promising step in the
development of techniques to introduce therapeutic
genes into the retina. In the adult retina, photoreceptor cells cannot reenter the cell cycle, thus limiting the
use of conventional gene transfer techniques that depend on cell division. The high efficiency and stability
of adenoviral-mediated gene transfer to RPE cells may
allow localized delivery of therapeutic molecules to
diseased photoreceptors. Although the efficiency of
gene transfer to normal photoreceptors is low under
the conditions described here, there may be a selective
increase in efficiency of transfer to degenerating photoreceptors. Most retinal degenerative diseases result
in the pathologic loss of outer segments and the
surrounding matrices. It is possible that, in degenerating retinas, the photoreceptors will be more accessible
to reagents introduced into the subretinal space.
Several different inherited forms of RP are associated with mutations in genes that are primarily expressed in retinal photoreceptor cells. Such mutations
have been identified in the rhodopsin gene,1819 the
peripherin RDS gene,20"22 and in the gene encoding
the /3-subunit of cGMP phosphodiesterase.23 These
mutant genes may decrease photoreceptor cell viability by disrupting cellular metabolism or by interfering
with the visual transduction cascade.24
Gene transfer therapy for different forms of RP
would require a tailored strategy based on the underlying molecular defect. The introduction of wild-type
genes to photoreceptors may be useful in recessive
diseases where gene products are absent or nonfunctional. Even though introduced gene expression levels
may be less than the endogenous mutant gene levels, a
small amount of the material expressed appropriately
by the introduced genes may be sufficient to slow or
halt the progression of the disease. This effect has
been demonstrated with transgenic rescues of the rd
and of the rds phenotypes.25'26 Such an approach may
not be useful in the treatment of diseases caused by
dominant genes where abnormal gene products are
present in relative excess. In those cases, the mutant
genes would have to be replaced by their wild-type
counterparts.
Alternative strategies might include introduction
of growth factor genes into photoreceptors or retinal
pigment epithelium cells. Localized expression of spe-
2541
cific growth factors might ameliorate the deleterious
effects of photoreceptor-specific mutant genes. The
appropriate choice of regulatory sequences should allow exogenous genes to be expressed as desired in the
relevant cells. The ability to introduce stable exogenous genes into the adult mammalian retina could ultimately pave the way for genetic therapy as a treatment
for blinding, and currently untreatable, inherited retinal dystrophies such as RP.
Key Words
adenovirus, gene transfer, photoreceptors, subretinal injection, retinal pigment epithelial cells
Acknowledgments
The authors thank Virginia Harris and Aki Maraishi for help
in purification of the virus and Judy Swain for providing
space and an environment conducive for these experiments.
References
1. Acsadi G, Dickson G, Love DR, et al. Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs. Nature. 1991;352:815-818.
2. Wolff JA, Malone RW, Williams P, et al. Direct gene
transfer into mouse muscle in vivo. Science.
1990;247:1465-1468.
3. Nabel E, Plautz G, Nabel G. Site-specific gene expression in vivo by direct gene transfer into the arterial
wall. Science. 1990:249:1285-1288.
4. Zhu N, Liggitt D, Liu Y, Debs R. Systemic gene expression after intravenous DNA delivery into adult
mice. Science. 1993; 261:209-211.
5. Quantin B, Perricaudet LD, Tajbakhsh S, Mandel JL.
Adenovirus as an expression vector in muscle cells in
vivo. Proc Natl Acad Sci USA. 1992; 89:2581-1584.
6. Stratford-Perricaudet LD, Makeh I, Perricaudet M,
Briand P. Widespread long-term gene transfer to
mouse skeletal muscles and heart. J Clin Invest.
1992;90:626-630.
7. Ragot T, Vincent N, Chafey P, et al. Efficient adenovirus-mediated transfer of a human minidystrophin
gene to skeletal muscle of mdx mice. Nature.
1993;361:647-650.
8. Jaffe HA, Danel C, Longenecker G, et al. Adenovirusmediated in vivo gene transfer and expression in normal rat liver. Nature Genet. 1992; 1:372-378.
9. Rosenfeld MA, Siegfried W, Yoshimura K, et al. Adenovirus-mediated transfer of a recombinant 1-antitrypsin gene to the lung epithelium in vivo. Science.
1991;252:431-434.
10. Rosenfeld MA, Yoshimura K, Trapnell BC, et al. In
vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium. Cell. 1992; 68:143-155.
11. Engelhardt JF, Yang Y, Stratford-Perricaudet LD, et
al. Direct gene transfer of human CFTR into human
bronchial epithelia of xenografts with El-deleted adenoviruses. Nature Genet. 1993;4:27-33.
12. Le Gal La Salle G, Robert JJ, Berrard S, et al. An
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933178/ on 08/03/2017
2542
13.
14.
15.
16.
17.
18.
19.
20.
Investigative Ophthalmology & Visual Science, April 1994, Vol. 35, No. 5
adenovirus vector for gene transfer into neurons and
glia in the brain. Science. 1993;259:988-990.
Davidson BL, Allen ED, Kozarsky KF, Wilson JM,
Roessler BJ. A model system for in vivo gene transfer
into the central nervous system using an adenoviral
vector. Nature Genet. 1993;3:219-223.
Akli S, Caillaud C, Vigne E, et al. Transfer of a foreign
gene into the brain using adenovirus vectors. Nature
Genet. 1993; 3:224-228.
Bajocchi G, Feldman SH, Crystal RG, Mastrangeli A.
Direct in vivo gene transfer to ependymal cells in the
central nervous system using recombinant adenovirus
vectors. Nature Genet. 1993; 3:229-234.
Maguire AM, Sun D, Zack DJ, Bennett J. In vivo gene
transfer into adult mammalian retina. ARVO Abstracts. Invest Ophthalmol Vis Sci. 1993;34:1455.
Guerin CJ, Lewis GP, Fisher SK, Anderson DH. Recovery of photoreceptor outer segment length and
analysis of membrane assembly rates in regenerating
primate photoreceptor outer segments. Invest Ophthalmol Vis Sci. 1993;34:175-183.
Humphries P, Kenna P, Farrar GJ. On the molecular
genetics of retinitis pigmentosa. Science. 1992; 256:
804-808.
Mclnnes RR, Bascom RA. Retinal genetics: A nullifying effect for rhodopsin. Nature Genet. 1992; 1:155157.
Farrar GJ, Kenna P, Jordan SA, et al. A three-base-
21.
22.
23.
24.
25.
26.
pair deletion in the peripherin-/?D5 gene in one form
of retinitis pigmentosa. Nature. 1991; 354:478-480.
Kajiwara K, Hahan LB, Mukai S, Travis GH, Berson
EL, Dryja TP. Mutations in the human retinal degeneration slow gene in autosomal dominant retinitis pigmentosa. Nature. 1991; 354:480-483.
Wells J, Wroblewski J, Keen J, et al. Mutations in the
human retinal degeneration slow (RDS) gene can
cause either retinitis pigmentosa or macular dystrophy. Nature Genet. 1993; 3:213-218.
McLaughlin ME, Sandberg MA, Berson EL, Dryja TP.
Recessive mutations in the gene encoding the /3-subunit of rod phosphodiesterase in patients with retinis
pigmentosa. Nature Genet. 1993;4:130-134.
Sung C-H, Schneider B, Agarwal N, Papermaster DS,
Nathans J. Functional heterogeneity of mutant rhodopsin responsible for autosomal dominant retinitis
pigmentosa. Proc Natl Acad Sci USA. 1991;88:88408844.
I .em J, FlanneryJG, Li T, Applebury ML, Farber DB,
Simon MI. Retinal degeneration is rescued in transgenic rd mice by expression of the cGMP phosphodiesterase beta subunit. Proc Natl Acad Sci USA.
1992;89:4422-4426.
Travis GH, Groshan KR, Lloyd M, Bok D. Complete
rescue of photoreceptor dysplasia and degeneration
in transgenic retinal degeneration-slow (rds) mice. Neuron. 1992;9:113-119.
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