WO1998048027A2 - Materials and methods for treatment of retinal diseases - Google Patents

Materials and methods for treatment of retinal diseases

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Publication number
WO1998048027A2
WO1998048027A2 PCT/US1998/008003 US9808003W WO9848027A2 WO 1998048027 A2 WO1998048027 A2 WO 1998048027A2 US 9808003 W US9808003 W US 9808003W WO 9848027 A2 WO9848027 A2 WO 9848027A2
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promoter
cells
construct
raav
retinal
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WO1998048027A3 (en
WO1998048027A9 (en
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William W. Hauswirth
John G. Flannery
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University of Florida
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University of Florida
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Priority to CA002287495A priority Critical patent/CA2287495A1/en
Priority to EP98922042A priority patent/EP0977880A2/en
Priority to AU74675/98A priority patent/AU7467598A/en
Priority to BR9809284-7A priority patent/BR9809284A/en
Priority to JP54626898A priority patent/JP2001527399A/en
Publication of WO1998048027A2 publication Critical patent/WO1998048027A2/en
Publication of WO1998048027A3 publication Critical patent/WO1998048027A3/en
Publication of WO1998048027A9 publication Critical patent/WO1998048027A9/en
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Ceased legal-status Critical Current

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Definitions

  • the subject invention was made with government support under a research project supported by NIH Grant Nos. EY07864 and EY11123 .
  • the government has certain ⁇ ghts m this invention.
  • Macular degeneration is a detenoration of the macula (the cone-rich center of vision) leading to gradual loss of central vision. Eventual loss of these cones leads to central vision loss and functional blindness. Macular degeneration may also have a genetic etiology or predisposition. Although a genetic etiology has not yet been established, based on pedigree studies it is very likely to exist. At least 500,000 individuals are estimated to suffer from macular degeneration currently in the United States.
  • the subject invention concerns materials and methods for achieving expression of proteins in retina cells.
  • the expression of proteins in retina cells can be used, for example, for the treatment of retinal diseases. More specifically, the subject invention provides polynucleotide sequences, and methods for using these sequences, to achieve highly specific expression of proteins in the retma. As described herein, the expression of these proteins can be used to treat a variety of retinal diseases.
  • the mate ⁇ als and methods of the subject invention can be used to treat autosomal dominant retmitis pigmentosa (ADRP).
  • ADRP autosomal dominant retmitis pigmentosa
  • the subject invention provides techniques for obtaining targeted, high level expression of any desired gene in the photoreceptor cells of the retina.
  • ribozymes can be highly and specifically expressed in the retina.
  • the ribozymes cleave the mutant forms of messenger RNA (mRNA) occurring in common forms of inherited retinal degeneration. This specificity makes these ⁇ bozymes able to destroy harmful mRNA while leaving normal mRNA mtact.
  • Ribozymes against other genetic forms of retmitis pigmentosa can be produced and used according to the subject invention.
  • Other polynucleotides encoding therapeutically useful products can also be selectively expressed in the eye using the teachings of the subject invention.
  • Figures la and lb show the construction of plasmids used according to the subject invention. These figures show a schematic diagram of the plasmid DNA constructs used to make rAAV viruses mOp-/ cZ (a) and Op-gfp (b).
  • TR 145 bp AAV terminal repeat sequence, mOp, 472 bp mu ⁇ ne rod opsm regulatory sequence from +86 to -388, SD/SA, 180 bp SV40 late viral protein gene 16S/19S splice donor and acceptor signal, lacZ; coding sequence for the bacterial lacZ gene; gfp, coding sequence for the synthetic green fluorescence gene; pA, pAl and pA2, polyadenylation signals; Epo, a tandem repeat of the polyoma virus enhancer region (bases 5210-5274); Ptk, thymidme kinase promoter of herpesvirus (bases 92-218); neo r , coding sequence of the neomycm resistance gene, Tn5 (bases 1555-2347) (Zolotukhin, S., M. Potter, W. Hauswirth, J. Guy, N. Muzyczka (1996) J. Virol. 70:4646-4654).
  • Ribozymes were: Hpl 1 hairpm ⁇ bozyme (filled circles), Hhl3 hammerhead ⁇ bozyme (filled triangles), Hpl "inactive” hairpin ⁇ bozyme (open circles), Hhl3 ⁇ "inactive” hammerhead ⁇ bozyme (open t ⁇ angle), or BOPS-g/p (X), all regulated by the same bovine opsm promoter. All injections were performed at P 14-15. The error bars were omitted if they fell withm the symbol, except for Hpl li at P75 and P90, where only one eye at each point was examined.
  • Rats Measurements of ONL thickness along the vertical me ⁇ dian of the eye from the optic nerve head (ONH) to the ora serrata (anterior margin of the retma) in rats at P90. Rats were either unmjected (open triangles) or injected at P14-15 with Hpl 1 hairpm ribozymes (filled circles) or Hhl3 hammerhead ribozymes (open circles).
  • the subject invention pertains to materials and methods for achieving highly specific expression of desired proteins in the retina. These proteins can be used as described herein to achieve a beneficial therapeutic affect.
  • the subject invention provides materials and methods which can be used to reduce or eliminate the symptoms of mhe ⁇ ted eye disease caused by mutations in genes for retinal proteins.
  • the subject invention provides materials and methods for achieving efficient and cell type-specific expression of exogenous genes in photoreceptor cells (PRs) of the mammalian retina.
  • PRs photoreceptor cells
  • rAAV recombmant Adeno-associated Virus
  • the subject invention provides a method for treating autosomal dominant retmitis pigmentosa (ADRP) at a molecular level.
  • ADRP autosomal dominant retmitis pigmentosa
  • Gene therapy for ADRP involves (1) an efficient and cell-type specific gene delivery/expression system, and (2) a selective means of inhibiting production of the mutant protein.
  • rAAV vectors are used to transfer the bacte ⁇ al lac gene or a synthetic green fluorescent protein gene (gfp) to mouse or rat retinas following injection into the subretmal space.
  • gfp synthetic green fluorescent protein gene
  • results demonstrate the surprising and advantageous ability to achieve highly specific expression of proteins m the mammalian retma.
  • reporter gene product was found exclusively in photoreceptors, not in any other retinal cell type or in the adjacent retinal pigment epithelium (RPE).
  • GFP-expressmg photoreceptors typically encompassed 10-20% of the total retinal area following a single 2 ⁇ injection. Both rod and cone photoreceptors were transduced with nearly 100% efficiency in the region directly surrounding the injection site. Approximately 2.5 million photoreceptors were transduced as a result of the single subretmal inoculation. The use of such proximal opsm promoters therefore can be used to substantially enhance the efficiency of expression in cases where rod and/or cone -specific expression of a potentially therapeutic gene is desired.
  • the gfp- contaimng rAAV stock was substantially free of both adenovirus and wild-type AAV, as judged by plaque assay and infectious center assay, respectively.
  • helper virus- free rAAV vectors can achieve high frequency tissue-specific transduction of terminally differentiated, postmitotic photoreceptor cells.
  • the human rod opsm, or cone opsm, promoter analogous to the mouse sequence would be used.
  • the appropriate regions of the human sequence can be readily identified and used by the skilled artisan having the benefit of the instant disclosure Assuming a 20 ⁇ l injection into the central human retma at the region of greatest rod density, approximately 8 million rods would be expected to be transduced m a focal region encompassing approximately 6% of the total retina. By transducing such an area of PRs it is possible to improve or delay retinal degenerations in a variety of mhe ⁇ ted retinal diseases.
  • the mOp-gfp- rAAV plasmid DNA was made by first adding Not I linkers to the 472bp Bgl II/Xho I proximal opsm promoter fragment of pRG3 and inserting it into the Not I sites of pTRUF2 (Zolotukhin, S. M. Potter, W. Hauswirth, J. Guy, N. Muzyczka (1996) J Virol. 70:4646-4654) (Fig. lb) rAAV virus production and analysis.
  • human 293 cells were co-transfected with mOp-/ ⁇ cZ-rAAV or mOp-g/ >-rAAV plasmid DNA and the helper pIM45 plasmid DNA carrying the wtAAV genome without terminal repeats (Zolotukhm, S. M Potter, W. Hauswirth, J. Guy, N. Muzyczka (1996) J. Virol.
  • the RT-PCR employed a two buffer thermostable Tth polymerase system (Promega, Madison, WI) according to manufacturer's instructions and lacL sequence p ⁇ mers from nucleotides 105 to 124 (forward) and 303 to 286 (reverse). Rnase and Dnase digestions prior to the RT-PCR were performed as previously desc ⁇ bed (van Gmkel, P., W. Hauswirth (1994) J. Biol. Chem. 269:4986-4992).
  • Example 1 Design of rAAV Vectors for Gene Transfer to Photoreceptors
  • a 472bp of the proximal murme rod opsm promoter (+86 to -385) was linked to a lacZ-SV40 polyA reporter gene and then inserted this into pTR.
  • the gene construct was packaged into
  • AAV virus particles concentrated, tested for contaminating Adenovirus and titered for recombmant AAV by an infectious center assay.
  • the ⁇ ght eyes of 30 C57B1/6J mice were injected sub-retmally with l ⁇ l of mOp-/ ⁇ cZ virus (10 7 m per ml). After two weeks, the right
  • test and left (control) eyes of 12 animals were removed, fixed and stained with X-gal.
  • Test retina in 6 of 12 injected eyes exhibited a focal blue region consistent with a subretmal bleb of the injected virus creating a localized retinal detachment. All control eyes showed no X-gal reaction. Reporter gene expression was examined in mice sacrificed at later periods and was detected at 10 weeks post-mjection suggesting persistent reporter transgene expression.
  • lacZ gene product was analyzed at higher resolution by preparing se ⁇ al 50 ⁇ m transverse sections from the entire whole mounts.
  • the blue X-gal reaction product is observed p ⁇ manly in the PR inner segments.
  • Most of the PRs were filled with X-gal in this region.
  • X-gal staining was slightly above control levels in the PR synaptic termini m the outer plexiform layer.
  • PR outer segments, RPE and other retinal cells in this region did not reveal X- gal staining above baseline levels observed m identically treated, unmjected or PBS-mjected control returns from the contralateral eye.
  • GFP fluorescence typically extended over 10-20% of the retinal area m a radial pattern from the injection site. Immediately surrounding the point of infection, the transduction frequency, as judged by the intensity of GFP fluorescence, was very high, with a continuous positive signal. In transverse sections extending from the central retma to the pe ⁇ phery, beyond a region of apparently saturated GFP fluorescence, the percentage of transduced cells decreased radially with distance from the injection site. GFP-positive cells were easily identifiable as PRs by their specialized shape and location m the retma. Hence, only PR cells appeared to have been transduced, i.e., infected by the rAAV and expressing the gfp passenger gene.
  • Example 3 Opsm Promoter Confers Photoreceptor Cell Specificity
  • the PR-specific pattern of GFP expression was confirmed by laser confocal microscopy. GFP was not observed between the inner limiting membrane (vitreal face of the inner retma) and the outer plexiform layer (OPL) (junction of the inner retma with PR synaptic termini). This region contains all the non-PR retinal neuronal (bipolar, ho ⁇ zontal, amac ⁇ ne, and ganglion) and ghal (M ⁇ ller) cells. Virtually 100% of the PR inner segments, cell bodies, and synaptic terminals exhibited strong GFP fluorescence. In regions more peripheral to the injection site, the fraction of positive PRs was substantially reduced, consistent with the radial decline in fluorescence seen retinal whole mounts. It was established that all PR cell bodies contained
  • GFP signal by examining se ⁇ al optical sections (0.32 ⁇ m). Through-focus series demonstrated that occasional, dark regions in the ONL always contained a gfp-positive PR cell body in another plane of section. Therefore, all PRs, including both rods and cones, supported reporter gene expression. Outer segments demonstrated less fluorescence than other PR compartments, near the level of autofluorescence seen in control outer segments. No GFP signal was observed m the REP, choroid, or sclera.
  • GFP-positive PRs The area of GFP-positive PRs resulting from a typical injection from epifluorescence images of retinal whole mounts was established. GFP -positive areas were measured with NIH
  • Image software by segmenting the image into regions of GFP fluorescence and background on the basis of gray level. Area measurements were calibrated by imaging a 1000 ⁇ m reference scale on the film together with the whole mount.
  • the retinal area that contained 50% or more PR cells positive for GFP signal m whole mounts was measured. On average, the GFP-positive area covered -35% of the total retinal area of the rat retma.
  • the number of GFP -positive PRs resulting from a typical injection was estimated by examining se ⁇ al optical sections taken through the retma. Serial confocal images suggest 100% PR transduction m the region directly adjacent to the injection site, since we did not observe GFP-negative cell bodies within the outer nuclear layer m adjacent confocal optical sections. It is estimated that the whole rat retina contains 15.7 million PR cells. From these observations, a conservative estimate is that 2-3 million PRs were transduced by the gfp- containmg rAAV.
  • Example 5 Construction of Vectors and Expression in Target Cells rAAV- ⁇ bozyme constructs. Recombmant AAV constructs were based on the pTR-UF2 vector (Zolotukhm, S., M. Potter, W.W. Hauswirth et al. [1996] J Virol. 70:4646-4654). They resemble the vector used by Flannery et al. (Flannery, J.G., S. Zolotukhm, M.I. Vaquero et al [1997] Proc. Natl.
  • bovine promoter fragment contains three proximal promoter elements and the endogenous transc ⁇ ptional start site at its 3' end (DesJardm, L.E., W W. Hauswirth [1996] Inv Ophth. Vis. Sci. 37:154-165) and supports high efficiency, rat photoreceptor-specific expression in vivo.
  • ⁇ bozymes Active and inactive ⁇ bozymes were designed, tested and cloned. Each ⁇ bozyme gene was followed by an internally cleaving hairpm nbozyme derived from plasmid pHC (Althoffr, M., R. T ⁇ tz, A.A. Hampel [1992] Gene 122:85-90) resulting in ⁇ bozyme cassettes of 140-152 bp. Self cleavage at the internal cutting site m the primary nbozyme RNA leaves identical 3' ends on each mature nbozyme.
  • the ⁇ bozyme cassette was preceded by an intron derived from SV40 and followed by a polyadenylation signal in order to promote nuclear export of the nbozyme.
  • Recombmant AAV titers were determined using both an infectious center assay (Flannery, J.G., Zolotukhm, S. Vaquero et al. [1997] Proc. Natl. Acad. Sci. USA 94:6916-6921) and a DNAse resistant physical particle assay employing a quantitative, competitive PCR of the ne gene contained within all rAAV- ⁇ bozyme particles (Zolotukhm, S., M. Potter, W.W. Hauswirth et al. [1996]
  • Each of the four rAAV- ⁇ bozyme virus preparations contained 10 10 to 10" DNASE resistant particles per ml and 10 8 to 10 9 infectious center units per ml. Contaminating helper adenovirus and wild-type AAV, assayed by serial dilution cytopathic effect or infectious center assay respectively, were less than five order of magnitude lower than rAAV.
  • RNA retma were isolated without fixation and total RNA immediately extracted using the
  • RNeasy Minikit Qiagen, Santa Clanta, CA. RT-PCR was performed using the Pharmacia First-Strand cDNA synthesis kit employing ohgo dT as the primer. Wild-type and transgene opsm cDNAs were amplified using a three p ⁇ mer system desc ⁇ bed above. P ⁇ mers specific for ⁇ -actm cDNA (Timmers, A.M., B.R. Newton, W.W. Hauswirth [1993] Exp Eye Res 56:251- 265) were included in each reaction for internal standardization.
  • Ribozymes were designed to recognize and cleave the unique transc ⁇ pt produced by the P23H transgene.
  • the mutant target sequence "5 '-UCGGAGUCUACUUCG-3 '" (SEQ ID NO. 17) contains two differences from the wild-type mRNA (indicated in bold). The hairpm nbozyme (Hpl l) cleaved 3' to the first adenosine residue (underlined) and the hammerhead ⁇ bozyme (Hhl3) cleaved 3' to the central cytosme residue (underlined).
  • Control nbozymes (Hpl li and Hhl3 ⁇ , respectively) retained the targeting domains but contained fatal flaws in their catalytic domains.
  • the active hammerhead nbozyme (Hhl3) was able to cleave 20% of the P23H target within 10 mm. of incubation and by 5 hours greater than 80% was converted to the expected products.
  • both nbozymes exhibited kinetic constants (K,-, and k- at ) similar to those of naturally occurnng ⁇ bozymes.
  • the two active ribozymes produced negligible cleavage of the wild-type transcnpt even in the presence of high MgCl 2 concentrations.
  • Control ⁇ bozymes (Hpl li and Hhl3 ⁇ ) containing inactivating mutations in their catalytic domains were without measurable activity on any substrate Using total RNA denved from retinas of P23H rats on P62, both the hairpm and the hammerhead ⁇ bozymes were able to cleave the mRNA product of the mutant transgene selectively.
  • TgN(P23H)3 (abbreviated P23H-3), that has a retinal degeneration phenotype similar to patients with retmitis pigmentosa (Steinberg, R.H., J.G. Flannery, M.I. Naash et al. [1996] Inv. Ophth Vis. Sci 37:S698) was used.
  • mutated opsm transgene begins at about postnatal day (P) 5 in rats, leading to a gradual death of photoreceptor cells. These rats develop an apparently normal retma up to PI 5, although there are somewhat more pyknotic photoreceptor nuclei m the outer nuclear layer (ONL) than m non-transgemc control rats. Thereafter, death of photoreceptor cells is almost linear until about P60, resulting m loss of about 40% of the photoreceptors. After P60, the rate of cell loss decreases, until by one year the retinas have less than a single row of photoreceptor nuclei.
  • the rAAV-nbozyme vector was injected into the mterphotoreceptor space between the photoreceptors and the adjacent retinal pigment epithelium at P14 or P15. Rats were sacnficed and eyes examined at 3 time points between P60-P90. At these ages in unmjected control eyes of P23H-3 rats, the ONL thickness, which is an index of photoreceptor cells number, was reduced to about 60% of normal.
  • Ribozyme-mjected eyes showed a modest but significant decrease in the accumulation of transcnpt denved from the P23H transgene. Control eyes exhibited little vanation in the level of transgene opsm mRNA. Eyes injected with either active ⁇ bozyme uniformly exhibited lowered transgene mRNA levels relative to total opsm mRNA in the same eye. Retinas receiving the hairpm ⁇ bozyme Hpl 1 showed a 15.3 ⁇ 3.3% decrease m transgene expression, and those with the hammerhead nbozyme Hhl3 showed a decrease of 11.1+5.1% decrease
  • the upper bound denves from the DNAse resistant particle assay, indicating that 2 ⁇ l of the rAAV- ⁇ bozyme virus preparation contained 2 x 10 7 to 10 8 rAAV This is an upper bound because not all particles counted are expected to be infectious.
  • the lower bound for rAAV titer is generated by the infectious center assay, indicating 10 6 to 10 7 rAAV per ⁇ l.
  • rAAV retinal pigment epithelium
  • the initial volume of extracellular space approximately 0.5 ⁇ l, increases greatly with the 2 ⁇ l injection.
  • the fluid transport function of the RPE dehydrates this space, reapposmg the photoreceptors and RPE and concentrating the rAAV
  • the detachment of the photoreceptors from the RPE resolves withm several hours Du ⁇ ng the reattachment process, viral particles are spread laterally through the subretmal space.
  • RIS the unmjected control retinas had RIS that were about 90% of normal. Both the active and inactive ⁇ bozymes resulted m RIS lengths of 98% or greater of the normal length, and about 10- 15% longer than unmjected controls.

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Abstract

The subject invention provides materials and methods for efficient, specific expression of proteins in retinal photoreceptor cells. Specifically, the constructs are composed of an adeno-associated vital vector containing a rod or cone-opsin promoter. These materials and methods can be used in therapies for retinal diseases. In one embodiment, ribozymes which degrade mutant mRNA are used to treat retinitis pigmentosa.

Description

DESCRIPTION
MATERIALS AND METHODS FOR TREATMENT OF RETINAL DISEASES
The subject invention was made with government support under a research project supported by NIH Grant Nos. EY07864 and EY11123 . The government has certain πghts m this invention.
Cross-Reference to Related Applications
This application claims priority from provisional applications USSN 60/046,147, filed May 9, 1997; and USSN 60/044,492, filed April 21, 1997.
Background of the Invention Retmitis pigmentosa (RP) is a collection of heπtable retinal degenerations caused by defects in one of several genes for proteins of photoreceptor (PR) cells. RP is a clinically and genetically heterogeneous group of conditions characteπzed by progressive rod photoreceptor degeneration and eventual blindness. Of the nearly 100 causative gene defects currently known, all are either directly or indirectly involved in the rod cell specific visual response. The exact molecular pathogenesis of RP is still unexplained. Ultrastructural observations suggest that the rod PRs are severely affected m the disease. RP families have been documented with dominant, recessive, X-linked, and digemc patterns of inheritance, and more than fifteen separate loci have been implicated by linkage studies. Currently, the mutations identified to date all occur in genes exhibiting a PR-specific pattern of expression. Approximately 50,000 individuals m the United States are estimated to have RP.
Macular degeneration is a detenoration of the macula (the cone-rich center of vision) leading to gradual loss of central vision. Eventual loss of these cones leads to central vision loss and functional blindness. Macular degeneration may also have a genetic etiology or predisposition. Although a genetic etiology has not yet been established, based on pedigree studies it is very likely to exist. At least 500,000 individuals are estimated to suffer from macular degeneration currently in the United States.
There is currently no effective treatment for most forms of retmitis pigmentosa or macular degeneration. Treatment with a massive supplement (15,000 I.U. per day) of vitamin A often retards the course of retinal degeneration in retmitis pigmentosa. Vitamin therapy does not treat the underlying cause of RP and is not a cure.
Recombmant AAV vectors have been reported to efficiently transduce central nervous system (Kaplitt, M., P. Leone, R. Samulski, X. Kigo, D. Pfaff, K. O'Mally, M. During (1993) Nature Genetics 8:148-154), lung (Aflone, S., C. Conrad, W. Kearns, S. Chunduru, R. Adams,
T. Reynolds, W. Guggino, G. Cutting, B. Carter, T. Flotte (1996) 7 Virol. 70:3235-3241; Flotte,
T., S. Afione, C. Conrad, S.A. McGrath, R. Solow, H. Oka, P.R. Zeitlm, W.B. Guggino, C. Bi
(1993) Proc. Natl. Acad. Sci USA 90: 10613-10617), and muscle (Xiao, X, J. Li, R. Samulski
(1996) J Virol. 70:8098-8108; Kessler, P., G. Podsakoff, X. Chen, S. McQulstron, P. Colosi, L. Matehs, G. Kurtzman, B. Byrne (1996) Proc. Natl Acad. Sci. USA 93:14082-14087).
Although a recent report (Kido, M., K. Rich, G. Lang, E. Barron, D. Kohn, M. Al-Ubaidi, J
Blanks (1996) Curr Eye Res. 15:333-344) describes using an opsm promoter in a recombmant retrovirus for ex vivo transduction of cultured cells, dislocated retinal cells and fetal mouse retinal explants, the efficiency was very low (ca. < 0.1%).
Bnef Summary of the Invention
The subject invention concerns materials and methods for achieving expression of proteins in retina cells. The expression of proteins in retina cells can be used, for example, for the treatment of retinal diseases. More specifically, the subject invention provides polynucleotide sequences, and methods for using these sequences, to achieve highly specific expression of proteins in the retma. As described herein, the expression of these proteins can be used to treat a variety of retinal diseases. In an embodiment specifically exemplified herein, the mateπals and methods of the subject invention can be used to treat autosomal dominant retmitis pigmentosa (ADRP). In one embodiment, the subject invention provides techniques for obtaining targeted, high level expression of any desired gene in the photoreceptor cells of the retina. These methods involve the use of specific sets of promoter sequences that allow RNA transcription of the delivered gene exclusively m retinal rod and/or cone photoreceptor cells. Rods and cones are the pnncipal cell types affected in retmitis pigmentosa and macular degeneration respectively, hence the ability to target expression of therapeutic genes to these cells without altering unaffected cell types m the retma provides a genetic therapy approach of high specificity and low risk.
In a specific embodiment of the subject invention, ribozymes can be highly and specifically expressed in the retina. The ribozymes cleave the mutant forms of messenger RNA (mRNA) occurring in common forms of inherited retinal degeneration. This specificity makes these πbozymes able to destroy harmful mRNA while leaving normal mRNA mtact. Ribozymes against other genetic forms of retmitis pigmentosa can be produced and used according to the subject invention. Other polynucleotides encoding therapeutically useful products can also be selectively expressed in the eye using the teachings of the subject invention.
Brief Summary of the Drawings Figures la and lb show the construction of plasmids used according to the subject invention. These figures show a schematic diagram of the plasmid DNA constructs used to make rAAV viruses mOp-/ cZ (a) and Op-gfp (b). TR, 145 bp AAV terminal repeat sequence, mOp, 472 bp muπne rod opsm regulatory sequence from +86 to -388, SD/SA, 180 bp SV40 late viral protein gene 16S/19S splice donor and acceptor signal, lacZ; coding sequence for the bacterial lacZ gene; gfp, coding sequence for the synthetic green fluorescence gene; pA, pAl and pA2, polyadenylation signals; Epo, a tandem repeat of the polyoma virus enhancer region (bases 5210-5274); Ptk, thymidme kinase promoter of herpesvirus (bases 92-218); neor, coding sequence of the neomycm resistance gene, Tn5 (bases 1555-2347) (Zolotukhin, S., M. Potter, W. Hauswirth, J. Guy, N. Muzyczka (1996) J. Virol. 70:4646-4654).
Figures 2a-2b show outer retinal layer with P23H ribozymes. 2a, measurements of ONL thickness (left), RIS length (middle), and ROS length (πght) m rats killed at different ages. Filled squares denote normal, non-transgemc animals. P23H-3 rats were either unmjected (open squares), injected subretmally with PBS (open diamonds), or injected with AAV vectors carrying one of five πbozymes or controls. Ribozymes were: Hpl 1 hairpm πbozyme (filled circles), Hhl3 hammerhead πbozyme (filled triangles), Hpl "inactive" hairpin πbozyme (open circles), Hhl3ι "inactive" hammerhead πbozyme (open tπangle), or BOPS-g/p (X), all regulated by the same bovine opsm promoter. All injections were performed at P 14-15. The error bars were omitted if they fell withm the symbol, except for Hpl li at P75 and P90, where only one eye at each point was examined. 2b, Measurements of ONL thickness along the vertical meπdian of the eye from the optic nerve head (ONH) to the ora serrata (anterior margin of the retma) in rats at P90. Rats were either unmjected (open triangles) or injected at P14-15 with Hpl 1 hairpm ribozymes (filled circles) or Hhl3 hammerhead ribozymes (open circles).
Detailed Disclosure of the Invention The subject invention pertains to materials and methods for achieving highly specific expression of desired proteins in the retina. These proteins can be used as described herein to achieve a beneficial therapeutic affect. In one aspect, the subject invention provides materials and methods which can be used to reduce or eliminate the symptoms of mheπted eye disease caused by mutations in genes for retinal proteins. To this end, the subject invention provides materials and methods for achieving efficient and cell type-specific expression of exogenous genes in photoreceptor cells (PRs) of the mammalian retina. In a specific embodiment of the subject invention, recombmant Adeno-associated Virus (rAAV) vectors can be used to transfer the desired genes to retma cells.
In a specific embodiment, the subject invention provides a method for treating autosomal dominant retmitis pigmentosa (ADRP) at a molecular level. Gene therapy for ADRP according to the subject invention involves (1) an efficient and cell-type specific gene delivery/expression system, and (2) a selective means of inhibiting production of the mutant protein.
Provided herein are the results of expeπments wherein rAAV vectors are used to transfer the bacteπal lac gene or a synthetic green fluorescent protein gene (gfp) to mouse or rat retinas following injection into the subretmal space. These results demonstrate the surprising and advantageous ability to achieve highly specific expression of proteins m the mammalian retma. For example, employing a proximal muπne rod opsin promoter (+86 to -385) to drive expression, reporter gene product was found exclusively in photoreceptors, not in any other retinal cell type or in the adjacent retinal pigment epithelium (RPE). GFP-expressmg photoreceptors typically encompassed 10-20% of the total retinal area following a single 2 μ\ injection. Both rod and cone photoreceptors were transduced with nearly 100% efficiency in the region directly surrounding the injection site. Approximately 2.5 million photoreceptors were transduced as a result of the single subretmal inoculation. The use of such proximal opsm promoters therefore can be used to substantially enhance the efficiency of expression in cases where rod and/or cone -specific expression of a potentially therapeutic gene is desired. The gfp- contaimng rAAV stock was substantially free of both adenovirus and wild-type AAV, as judged by plaque assay and infectious center assay, respectively. Thus, highly purified, helper virus- free rAAV vectors can achieve high frequency tissue-specific transduction of terminally differentiated, postmitotic photoreceptor cells. These methods can be used as described herein for gene therapy to treat retinal diseases.
In a preferred embodiment the human rod opsm, or cone opsm, promoter analogous to the mouse sequence would be used. The appropriate regions of the human sequence can be readily identified and used by the skilled artisan having the benefit of the instant disclosure Assuming a 20 μl injection into the central human retma at the region of greatest rod density, approximately 8 million rods would be expected to be transduced m a focal region encompassing approximately 6% of the total retina. By transducing such an area of PRs it is possible to improve or delay retinal degenerations in a variety of mheπted retinal diseases. Currently, a number of PR genes of therapeutic potential have been identified experimentally by virtue of their involvement in recessive human retinal disease and by their ability at least to delay the course of recessive RP- ke disease animal models. These include PDE-B (Bennett, J., T. Tanabe, D. Sun, Y. Zeng, H. Kjeldbye, P. Gouras, A.M. Maguire (1996) Nature Med. 2:649-654; Lem, J., J. Flannery, T. Li, M. Applebury, D. Forber, M. Simon (1992) Proc. Natl Acad. Sci. USA 89:4422-4426) and peπpheπn/rds (Travis, G.H., K.R. Groshan, M.
Lloyd, D. Bok (1992) Neuron 33.113-119). Also included m this list are more general cell survival-promoting factors such as bcl-2 (Chen, J., J. Flannery, M. LaVail, R Steinberg, J. Xu, M.I. Simon (1996) Proc. Natl. Acad. Sci USA 93:7042-7047) and a variety of growth factors and neurotrophins (Faktorovitch, E., R.H. Steinberg, D. Yasumura, M. Matthes, M.M. LaVail (1990) 347.83-86). Thus, all forms of RP (Daiger, S., L. Sullivan, J. Rodriguez (1995) Behav
Brain Sci. 18:452-467) descnbed to date are potential candidates for therapy m this context.
In one specific embodiment, the subject invention utilizes the catalytic properties of πbozymes. Ribozymes are enzymes compπsed of πbonucleic acid (RNA). In nature, πbozymes conduct a vaπety of reactions involving RNA, including cleavage and hgation of polynucleotide strands. The specificity of πbozymes is determined by base paiπng (hydrogen bonding) between the targeting domain of the πbozyme and the substrate RNA. This specificity can be modified by alteπng the nucleotide sequence of the targeting domain. The catalytic domain of πbozymes, the part that actually performs the biochemical work, can also be changed in order to increase activity or stability of the πbozyme. Utilizing the techniques of the subject invention, πbozymes are continuously produced in the retinal cells from a copy of the πbozyme integrated m the patient's DNA. In a preferred embodiment patients require a single mtra-ocular injection and do not require hospitahzation. Long term (more than 15 months) unattenuated expression of proteins has been observed in cells transformed as described herein. In one embodiment the subject invention concerns synthetic genes for several ribozymes. These ribozymes can recognize, for example, the nucleotide change causing the P23H mutation in one form of ADRP and the S334ter mutation in another. Genes can be constructed which encode πbozymes having the ability to specifically destroy target RNA's for mutant retina proteins. With the benefit of the teachings provided herein, the skilled artisan can construct genes encoding ribozymes which destroy mutant RNA molecules associated with human RP or other genetic retinal diseases.
Using a recombmant Adeno-associated virus (rAAV) in which expression is dπven by a portion of the rod opsm promoter, we have achieved photoreceptor-specific expression of reporter genes m mouse and rat by ocular injection. The cone opsm promoter can also be used to dπve expression selectively m photoreceptor cells. The delivery-expression materials and methods of the subject invention can be used to replace any gene responsible for photoreceptor disease. Specific examples include the genes responsible for retmitis pigmentosa or macular degeneration. General survival-promoting genes such as growth factor and neurotrophm genes are also candidates for both recessive and dominant forms of retinal disease. Finally, genes for agents such as πbozymes or tπplex-forming o gonucleotides that can be designed to eliminate specific genetic defects are candidates for treating retinal disease using this technique. Assays for activity include morphological analysis of retinal degeneration, quantitative mRNA studies, and electroretmography.
Matenals and Methods rAAV plasmid construction. The mOp-/αcZ -rAAV plasmid DNA was made by first inserting the 4.3 kbp Bgl II/Bam HI fragment containing the proximal muπne rod opsm promoter (+86 to -385) and the entire lacZ gene of clone pRG3 (Lem, J., M. Applebury, J. Falk, J. Flannery, M. Simon (1991) J Biol Chem. 266:9667-9672) into the Bgl II sites of pTR which contains the AAV TR sequences and a SV40 polyadenylation sequence (Fig. la). The mOp-gfp- rAAV plasmid DNA was made by first adding Not I linkers to the 472bp Bgl II/Xho I proximal opsm promoter fragment of pRG3 and inserting it into the Not I sites of pTRUF2 (Zolotukhin, S. M. Potter, W. Hauswirth, J. Guy, N. Muzyczka (1996) J Virol. 70:4646-4654) (Fig. lb) rAAV virus production and analysis. To generate recombmant virus, human 293 cells were co-transfected with mOp-/αcZ-rAAV or mOp-g/ >-rAAV plasmid DNA and the helper pIM45 plasmid DNA carrying the wtAAV genome without terminal repeats (Zolotukhm, S. M Potter, W. Hauswirth, J. Guy, N. Muzyczka (1996) J. Virol. 70-4646-4654) Cultures were then infected with helper Adenovirus, Ad-tsl49 for the lacL virus or with Ad5 for the gfp virus, at a multiplicity of infection of 10 rAAV and wtAAV titers were determined by infectious center assay (McLaughlin, S. P. Colhs, P. Hermonat, N. Muzyczka (1988) J Virol 62:1963-1973), which is independent of the transgene or opsm promoter used. Titers of contaminating adenovirus were determined by plaque assay for mOp-g p-rAAV and by serial dilution cytopathic effect for mOp-/αcZ-rAAV. Adenovirus was not detectible in either of the rAAV preparations.
Subretmal injection of rAAV. Thirty adult C57BL/6I (Jackson Laboratoπes, Bar Harbor, ME) pigmented mice between 3 and 6 months of age and 27 adult albino Sprague- Dawley rats between 3 and 4 months of age were used. Animals were anesthetized by ketamme/xylazine injection, eyes were dilated (2.5% phenylephπne and 0.5% tropicamide) and a local anesthetic (proparacam Hcl) was applied. Injections (1 μl m mice and 2 μl m rats) were made into the right eye with blunt 32 gauge needle through an opening m the pars-plana, dehveπng the rAAV suspension into the supeπor subretmal space. Control injections were made in the contralateral eye with PBS only. Injections were performed with an operating microscope and the subretmal location of the injected volume was confirmed by ophthalmoscopy.
Tissue analysis. Animals were euthanized by intramuscular injection of ketamme, followed by phenobarbital overdose. The eyes were immediately enucleated and the site of virus injection marked. The cornea, lens and vitreous of each eye were removed and the posteπor eyecup placed m primary fixative.
For β-galactosidase staining, eyecups were fixed in 0.5% glutaraldehyde in 0.1M Cacodylate buffer pH 7.5 for 15 mm. At room temperature. Following a 10 mm. Wash in PBS, the eyecups were incubated in an iron-based X-gel staining solution (Sanes, J., J. Rubenstem, J. Nicolas (1986) EMBO J. 5:3133-3142) in a shaking water bath at 35°C for 12 hours. For agarose embedment, retinas were detached from the RPE, submerged without dehydration in molten 5% agarose and cooled to 25 °C. Retinas were sectioned in the transverse axis m lsotomc PBS on a vibratome at 50-100 μm. Bπght field and phase-contrast micrographs of whole mounts and β-galactosidase-stamed sections were made with a Zeiss Axiophot. GFP fluorescence was examined m retinal whole mounts and agarose embedded sections. Tissue fixation was minimized to reduce retinal autofluorescence. Retinas were detached from eyecups, fixed for 15 mm. At room temperature in 4% formaldehyde, 0.1 M P04 buffer pH 7.5, and πnsed three times m PBS. Whole mounts were photographed with epifiuorescence using Zeiss filter set 09 (ex. 450-490 nm, barπer 510 nm, emission 520 nm) and an AttoArc (Carl Zeiss, Inc., New York) vaπable output UV lamp to minimize GFP bleaching
Whole mount retinas were then embedded in agarose as above for 100 μm transverse vibratome sections, and fluorescence was documented as for the whole mount. Higher resolution images were collected with a Molecular Dynamics confocal microscope (Nikon 40X or 60X 1.4 n.a. oil objectives; argon laser excitation at 514 nm, emission at 520-560 nm). Optical sections were made m 0.32 μm steps. Full frame (768 x 512) 8-bit images were collected and processed with Adobe Photoshop. Area measurements were made with NTH Image analysis software (Rasband, W. D. Bπght (1995) Microbeam Analysis Society Journal 4:137-149).
Expression of the lacL reporter gene in murme retinal cells was analyzed by reverse ranscπptase PCR (RT-PCR). Pieces of retina (1mm2), were detached from unfixed eyecups and dissected free of RPE, homogenized with a pestle fitted to a 1.5 ml tube and total RNA isolated using the tπzol reagent (phenol-guanidme isothiocyanate, Gibco-BRL, Gaitherberg, MND) according to the manufacturer's recommendations. The RNA was additionally puπfied over an RNA-easy spin column (Qiagen, Chatsworth, CA). The RT-PCR employed a two buffer thermostable Tth polymerase system (Promega, Madison, WI) according to manufacturer's instructions and lacL sequence pπmers from nucleotides 105 to 124 (forward) and 303 to 286 (reverse). Rnase and Dnase digestions prior to the RT-PCR were performed as previously descπbed (van Gmkel, P., W. Hauswirth (1994) J. Biol. Chem. 269:4986-4992).
Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
Example 1 — Design of rAAV Vectors for Gene Transfer to Photoreceptors To express a foreign gene specifically m the mammalian PR by AAV-mediated delivery, a 472bp of the proximal murme rod opsm promoter (+86 to -385) was linked to a lacZ-SV40 polyA reporter gene and then inserted this into pTR. The gene construct was packaged into
AAV virus particles, concentrated, tested for contaminating Adenovirus and titered for recombmant AAV by an infectious center assay. The πght eyes of 30 C57B1/6J mice were injected sub-retmally with lμl of mOp-/αcZ virus (107 m per ml). After two weeks, the right
(test) and left (control) eyes of 12 animals were removed, fixed and stained with X-gal. Test retina in 6 of 12 injected eyes exhibited a focal blue region consistent with a subretmal bleb of the injected virus creating a localized retinal detachment. All control eyes showed no X-gal reaction. Reporter gene expression was examined in mice sacrificed at later periods and was detected at 10 weeks post-mjection suggesting persistent reporter transgene expression.
Example 2 - Lac-Z and GFP Reporter Genes are Expressed Exclusively in Photoreceptors
The distribution of lacZ gene product was analyzed at higher resolution by preparing seπal 50 μm transverse sections from the entire whole mounts. The blue X-gal reaction product is observed pπmanly in the PR inner segments. Most of the PRs were filled with X-gal in this region. X-gal staining was slightly above control levels in the PR synaptic termini m the outer plexiform layer. PR outer segments, RPE and other retinal cells in this region did not reveal X- gal staining above baseline levels observed m identically treated, unmjected or PBS-mjected control returns from the contralateral eye. Examination of additional transverse sections confirmed that the region of positive staining radiated outward from the injection site m a progressively reducing fraction of PR inner segments until baseline levels were seen. The area of X-gal positive PRs was consistent with the blue area m the whole-mount view. Neural retma and RPE were separated and analyzed independently to control for the possibility that the β- galactosidase enzyme or its X-gal reaction product was transferred from transduced RPE cells to PRs. Total mRNA was extracted from neural retma, and RPE from injected animals and tested for the presence oϊlacZ mRNA by RT-PCR. The 199bp amplification product diagnostic for lacZ RNA (nucleotides 105 to 303) can be seen when total RNA from a portion of a mouse retma sacrificed at 2 weeks post-mjection is amplified. The amplification template was a cellular RNA because of its resistance to Dnase pretreatment and sensitivity to Rnase pretreatment. The remaining RPE tissue was negative for this RT-PCR product. This demonstrates that the observed X-gal product was denved from β- galactosidase expression within PR cells and not denved from RPE expression.
A second reporter gene, a synthetic version of the A victoπa green fluorescent gene (gfp) (Zolotukhin, S. M. Potter, W. Hauswirth, J. Guy, N. Muzyczka (1996) J Virol 70:4646-
4654) was used to independently confirm the apparent cell-type specificity of transduction. The same muπne rod opsm promoter was used as well as an analogous rAAV vector to construct the mOp-gfp virus (Fig. lb). Two μl of gfp-contaming rAAV was injected into the subretmal space of 8 Sprague-Dawley rats. Rats were used in place of mice because the larger eye allowed more reproducible subretmal inoculations. Retinal whole mounts prepared from all eight rat eyes that were injected contained a fluorescent region of superior retma surrounding the site of inoculation. GFP fluorescence typically extended over 10-20% of the retinal area m a radial pattern from the injection site. Immediately surrounding the point of infection, the transduction frequency, as judged by the intensity of GFP fluorescence, was very high, with a continuous positive signal. In transverse sections extending from the central retma to the peπphery, beyond a region of apparently saturated GFP fluorescence, the percentage of transduced cells decreased radially with distance from the injection site. GFP-positive cells were easily identifiable as PRs by their specialized shape and location m the retma. Hence, only PR cells appeared to have been transduced, i.e., infected by the rAAV and expressing the gfp passenger gene. Example 3 — Opsm Promoter Confers Photoreceptor Cell Specificity
The PR-specific pattern of GFP expression was confirmed by laser confocal microscopy. GFP was not observed between the inner limiting membrane (vitreal face of the inner retma) and the outer plexiform layer (OPL) (junction of the inner retma with PR synaptic termini). This region contains all the non-PR retinal neuronal (bipolar, hoπzontal, amacπne, and ganglion) and ghal (Mϋller) cells. Virtually 100% of the PR inner segments, cell bodies, and synaptic terminals exhibited strong GFP fluorescence. In regions more peripheral to the injection site, the fraction of positive PRs was substantially reduced, consistent with the radial decline in fluorescence seen retinal whole mounts. It was established that all PR cell bodies contained
GFP signal by examining seπal optical sections (0.32 μm). Through-focus series demonstrated that occasional, dark regions in the ONL always contained a gfp-positive PR cell body in another plane of section. Therefore, all PRs, including both rods and cones, supported reporter gene expression. Outer segments demonstrated less fluorescence than other PR compartments, near the level of autofluorescence seen in control outer segments. No GFP signal was observed m the REP, choroid, or sclera.
Example 4 — rAAV Transduces PRs with High Efficiency
The area of GFP-positive PRs resulting from a typical injection from epifluorescence images of retinal whole mounts was established. GFP -positive areas were measured with NIH
Image software by segmenting the image into regions of GFP fluorescence and background on the basis of gray level. Area measurements were calibrated by imaging a 1000 μm reference scale on the film together with the whole mount.
The retinal area that contained 50% or more PR cells positive for GFP signal m whole mounts was measured. On average, the GFP-positive area covered -35% of the total retinal area of the rat retma. The number of GFP -positive PRs resulting from a typical injection was estimated by examining seπal optical sections taken through the retma. Serial confocal images suggest 100% PR transduction m the region directly adjacent to the injection site, since we did not observe GFP-negative cell bodies within the outer nuclear layer m adjacent confocal optical sections. It is estimated that the whole rat retina contains 15.7 million PR cells. From these observations, a conservative estimate is that 2-3 million PRs were transduced by the gfp- containmg rAAV. Since there were 9.2 million infectious rAAV particles in the 2-μl injection volume, one PR cell was transduced for every 3-4 rAAV injected, a 25% transduction efficiency. Example 5 — Construction of Vectors and Expression in Target Cells rAAV-πbozyme constructs. Recombmant AAV constructs were based on the pTR-UF2 vector (Zolotukhm, S., M. Potter, W.W. Hauswirth et al. [1996] J Virol. 70:4646-4654). They resemble the vector used by Flannery et al. (Flannery, J.G., S. Zolotukhm, M.I. Vaquero et al [1997] Proc. Natl. Acad. Sci. USA 94:6916-6921) to direct GFP expression to rat photoreceptors except that a 691 bp fragment of the proximal bovine rod opsm promoter replaced the 472 bp muπne rod opsin promoter and the πbozyme gene replaced the gfp gene. The bovine promoter fragment contains three proximal promoter elements and the endogenous transcπptional start site at its 3' end (DesJardm, L.E., W W. Hauswirth [1996] Inv Ophth. Vis. Sci. 37:154-165) and supports high efficiency, rat photoreceptor-specific expression in vivo. Active and inactive πbozymes were designed, tested and cloned. Each πbozyme gene was followed by an internally cleaving hairpm nbozyme derived from plasmid pHC (Altschuler, M., R. Tπtz, A.A. Hampel [1992] Gene 122:85-90) resulting in πbozyme cassettes of 140-152 bp. Self cleavage at the internal cutting site m the primary nbozyme RNA leaves identical 3' ends on each mature nbozyme. The πbozyme cassette was preceded by an intron derived from SV40 and followed by a polyadenylation signal in order to promote nuclear export of the nbozyme. Recombmant AAV titers were determined using both an infectious center assay (Flannery, J.G., Zolotukhm, S. Vaquero et al. [1997] Proc. Natl. Acad. Sci. USA 94:6916-6921) and a DNAse resistant physical particle assay employing a quantitative, competitive PCR of the ne gene contained within all rAAV-πbozyme particles (Zolotukhm, S., M. Potter, W.W. Hauswirth et al. [1996]
J Virol 70:4646-4654). Each of the four rAAV-πbozyme virus preparations contained 1010 to 10" DNASE resistant particles per ml and 108 to 109 infectious center units per ml. Contaminating helper adenovirus and wild-type AAV, assayed by serial dilution cytopathic effect or infectious center assay respectively, were less than five order of magnitude lower than rAAV.
Subretmal miection of rAAV. Line 3 albino transgenic rats (P23H-3) on an albino Sprague-Dawley background (produced by Chrysalis DNX Transgenic Sciences, Pπnceton, NJ) were injected at the ages of P14 or P15. Animals were anesthetized by ketamme/xylazine injection, and a direction, and b-waves were measured from the cornea-negative peak to the major cornea-positive peak. For quantitative comparison of differences between the two eyes of rats, the values from all the stimulus intensities were averaged for a given animal
Retinal tissue analysis. The rats were euthanized by overdose of carbon dioxide inhalation and immediately perfused mtracardially with a mixture of mixed aldehydes (2% formaldehyde and 2.5% glutar ldehyde). Eyes were removed and embedded m epoxy resm, and 1 μm thick histological sections were made along the vertical meridian (26). Tissue sections were aligned so that the ROS and Mϋller cell processes crossing the inner plexiform layer were continuous throughout the plane of section to assure that the sections were not oblique, and the thickness of the ONL and lengths of RIS and ROS were measured as described by Faktorovich et al. (Faktorovich, E.G., R.H. Steinberg, D. Yasamura et al [1990] Nature 347.83-86). Bπefly,
54 measurements of each layer or structure were made at set points around the entire retinal section. These data were either averaged to provide a single value for the retina, or plotted as a distribution of thickness or length across the retma. The greatest 3 contiguous values for ONL thickness m each retma were also compared to determine if any region of retma (e g., nearest the injection site) showed proportionally greater rescue; although most of these values were slightly greater than the overall mean of all 54 values, they were no different from control values than the overall mean. Thus, the overall mean was used in the data cited, since it was based on a much larger number of measurements.
RT-PCR. For quantification of opsm mRNA retma from nbozyme injected or control eyes, retma were isolated without fixation and total RNA immediately extracted using the
RNeasy Minikit (Qiagen, Santa Clanta, CA). RT-PCR was performed using the Pharmacia First-Strand cDNA synthesis kit employing ohgo dT as the primer. Wild-type and transgene opsm cDNAs were amplified using a three pπmer system descπbed above. Pπmers specific for β-actm cDNA (Timmers, A.M., B.R. Newton, W.W. Hauswirth [1993] Exp Eye Res 56:251- 265) were included in each reaction for internal standardization.
Such constructs result m persistent photoreceptor expression of the passenger gene of greater than 15 months. Ribozymes were designed to recognize and cleave the unique transcπpt produced by the P23H transgene. The mutant target sequence "5 '-UCGGAGUCUACUUCG-3 '" (SEQ ID NO. 17) contains two differences from the wild-type mRNA (indicated in bold). The hairpm nbozyme (Hpl l) cleaved 3' to the first adenosine residue (underlined) and the hammerhead πbozyme (Hhl3) cleaved 3' to the central cytosme residue (underlined). Control nbozymes (Hpl li and Hhl3ι, respectively) retained the targeting domains but contained fatal flaws in their catalytic domains. In vitro, the active hammerhead nbozyme (Hhl3) was able to cleave 20% of the P23H target within 10 mm. of incubation and by 5 hours greater than 80% was converted to the expected products. In multiturnover expenments, both nbozymes exhibited kinetic constants (K,-, and k-at) similar to those of naturally occurnng πbozymes. The two active ribozymes produced negligible cleavage of the wild-type transcnpt even in the presence of high MgCl2 concentrations. Control πbozymes (Hpl li and Hhl3ι) containing inactivating mutations in their catalytic domains were without measurable activity on any substrate Using total RNA denved from retinas of P23H rats on P62, both the hairpm and the hammerhead πbozymes were able to cleave the mRNA product of the mutant transgene selectively.
For expeπments in vivo, a line of transgenic rats, TgN(P23H)3 (abbreviated P23H-3), that has a retinal degeneration phenotype similar to patients with retmitis pigmentosa (Steinberg, R.H., J.G. Flannery, M.I. Naash et al. [1996] Inv. Ophth Vis. Sci 37:S698) was used.
Expression of the mutated opsm transgene begins at about postnatal day (P) 5 in rats, leading to a gradual death of photoreceptor cells. These rats develop an apparently normal retma up to PI 5, although there are somewhat more pyknotic photoreceptor nuclei m the outer nuclear layer (ONL) than m non-transgemc control rats. Thereafter, death of photoreceptor cells is almost linear until about P60, resulting m loss of about 40% of the photoreceptors. After P60, the rate of cell loss decreases, until by one year the retinas have less than a single row of photoreceptor nuclei. The rAAV-nbozyme vector was injected into the mterphotoreceptor space between the photoreceptors and the adjacent retinal pigment epithelium at P14 or P15. Rats were sacnficed and eyes examined at 3 time points between P60-P90. At these ages in unmjected control eyes of P23H-3 rats, the ONL thickness, which is an index of photoreceptor cells number, was reduced to about 60% of normal.
Ribozyme-mjected eyes showed a modest but significant decrease in the accumulation of transcnpt denved from the P23H transgene. Control eyes exhibited little vanation in the level of transgene opsm mRNA. Eyes injected with either active πbozyme uniformly exhibited lowered transgene mRNA levels relative to total opsm mRNA in the same eye. Retinas receiving the hairpm πbozyme Hpl 1 showed a 15.3±3.3% decrease m transgene expression, and those with the hammerhead nbozyme Hhl3 showed a decrease of 11.1+5.1% decrease
Histologically, eyes injected with the nbozymes retained significantly more photoreceptors at P60, P75 and P90 than unmjected contralateral control eyes. Retinas receiving a subretmal injection of Hhl3 at P14-15 retained 88% of the normal ONL thickness, compared to about 60% m the unmjected controls (Figure 2a). Thus, the ONL thickness after Hhl3 expression was 40-43% greater than that of unmjected P23H-3 controls (Figure 2b), a highly significant difference (p=0.001 or less at P60 and P90). Injection of the Hpl 1 nbozyme also resulted m significant rescue when compared to controls, with preservation of 77-83% of normal ONL thickness (Figure 2a). Thus, the ONL thickness after Hpl 1 expression was 30-39% greater than that of unmjected P23H-3 controls (Figure 2b), a highly significant difference (p<0.0005 at all ages).
There was little or no rescue m PBS-mjected control eyes (p>0.169 in all cases) as shown in Figure 2a. As a control for possible rescue by the expression of the bovme opsm promoter (BOPS), AAV-BOPS-g/ was injected at a titer of 1.75 x 108, similar to the titer used with the AAV-nbozymes. The injection of AAV-BOPS-g//. did not rescue photoreceptors (Figure 2a). The inactive Hpl li did yield ONL thickness measures greater than unmjected control values, but they were consistently less than that resulting from the active Hpl 1 and Hhl3 ribozymes (Figure 2a).
The pan-retinal extent of photoreceptor rescue that resulted from a single 2-μl injection of the rAAV suspension was surpnsmg (Figure 2b). From photoreceptor counts, it is estimated that there are approximately 107 photoreceptors m the rat retma. Recombmant AAV titers were estimated using both an infectious center assay and a physical particle assay Together they permit construction of upper and lower bounds for the number of functional rAAV particles in a single 2 μl injection. The upper bound denves from the DNAse resistant particle assay, indicating that 2 μl of the rAAV-πbozyme virus preparation contained 2 x 107 to 108 rAAV This is an upper bound because not all particles counted are expected to be infectious. The lower bound for rAAV titer is generated by the infectious center assay, indicating 106 to 107 rAAV per μl.
The lateral extent of rescue resulting from a single injection may also be explained by the unique nature of the retinal tissue. For in vivo delivery to the photoreceptors, rAAV is injected into extracellular space separating the photoreceptor and retinal pigment epithelium (RPE) layers. The initial volume of extracellular space, approximately 0.5 μl, increases greatly with the 2 μl injection. Following injection, the fluid transport function of the RPE dehydrates this space, reapposmg the photoreceptors and RPE and concentrating the rAAV The detachment of the photoreceptors from the RPE resolves withm several hours Duπng the reattachment process, viral particles are spread laterally through the subretmal space.
Along with the survival of more photoreceptor cells, injection of the πbozymes resulted in greater lengths of rod inner segments (RIS) and rod outer segments (ROS) In the case of
RIS, the unmjected control retinas had RIS that were about 90% of normal. Both the active and inactive πbozymes resulted m RIS lengths of 98% or greater of the normal length, and about 10- 15% longer than unmjected controls. The PBS and AAV-BOPS-g^. were indistinguishable from unmjected eyes. The ROS lengths were about 15-25% longer m the ribozyme-mjected eyes compared to those m the unmjected control eyes. However, ROS in the nbozyme-mjected eyes were, at greatest, only 65-75% of normal, compared to the virtually normal RIS lengths. The ROS of the active nbozymes differed significantly from the unmjected controls (p<0005 for all, except <0.02 for Hpl 1 at P90), as did the inactive Hpl li (p<0.05). The finding that nbozyme-targeted destruction of P23H mutant RNA markedly slows the rate of retinal degeneration in P23H transgenic rats, along with functional preservation of the retma, is the first demonstration of this therapeutic approach in an animal model of a dommantly inheπted human disease. Furthermore, because complete removal of mutant rRNA is not necessary to achieve phenotypic rescue, this approach can be applied to other dommantly inherited diseases as well.
It should be understood that the examples and embodiments descnbed herein are for illustrative purposes only and that vanous modifications or changes in light thereof will be suggested to persons skilled the art and are to be included within the spmt and purview of this application and the scope of the appended claims.

Claims

Claims
1. A method for expressing a polynucleotide at high levels specifically m photoreceptor cells wherein said method compnses administenng to said photoreceptor cells a construct compπsing said polynucleotide wherein said polynucleotide is under the control of a promoter sequence which directs expression only in said photoreceptor cells.
2. The method, according to claim 1, wherein said cells are rod cells.
3. The method, according to claim 1, wherein said cells are cone cells.
4. The method, according to claim 1, wherein the promoter is a rod opsm promoter.
5. The method, according to claim 1 , wherein the promoter is a cone opsm promoter.
6. The method, according to claim 1 , wherein said polynucleotide sequence is delivered to said retina cells by subretmal injection.
7. The method, according to claim 1, wherein said polynucleotide sequence is delivered using a recombmant Adeno-Associated Virus (rAAV).
8. The method, according tro claim 1, which is used to treat retinal disease.
9. The method, according to claim 8, wherein said retinal disease is selected from the group consisting of Retmitis Pigmentosa and Macular Degeneration.
10. A construct for expressing a polynucleotide sequence selectively in photoreceptor cells wherein said construct compnses said polynucleotide sequence under the control of a promoter which directs expression selectively m photoreceptor cells.
11. The construct, according to claim 10, wherein said promoter is a rod opsm promoter.
12. The construct, according to claim 10, which comprises a recombmant Adeno- Associated Virus (rAAV).
13. The construct, according to claim 10, wherein said polynucleotide sequence is for the treatment of a retinal disease.
14. The construct, according to claim 13, wherein said retinal disease is selected from the group consisting of Retinitis Pigmentosa and Macular Degeneration.
15. The construct, according to claim 10, wherein said promoter is a rod opsin promoter.
16. The construct, according to claim 10, wherein said promoter is a cone opsin promoter.
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