MATERIALS AND METHODS FOR RIBOZYME TREATMENT OF RETINAL DISEASES

Abstract: The subject invention provides materials and methods for efficient, specific reduction or elimination of unwanted mRNA. These materials and methods can be used in therapies for retinal diseases. In one embodiment, ribozymes which degrade mutant rod opsin mRNA are used to treat retinitis pigmentosa or diabetic retinopathy. In a further embodiment, ribozymes which cleave RNAs involved in nitric oxide activity are used to treat blood-retinal dysfonction. The preferred targets include VEGF, eNOS, and iNOS.

Description: DESCRIPTION MATERIALS AND METHODS FOR RIBOZYME TREATMENT OF DISEASES The subject invention was made with government support under a research project supported by NIH Grant No. EY08571. The government has certain rights in 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 Ribozymes are biological catalysts consisting of only RNA. They promote a variety of reactions involving RNA and DNA molecules including site-specific cleavage, hgation, polymerization, and phosphoryl exchange (Cech, T.R. [1989] Biochem. Int. 18(1):7-14; Cech, T.R. [1990] Annu. Rev. Biochem 59:543-569). Ribozymes fall mto three broad classes: (1) RNAse P, (2) self-sphcmg mtrons, and (3) self-cleavmg viral agents. Self-cleavmg agents include hepatitis delta virus and components of plant virus satellite RNAs that sever the RNA genome as part of a rolling-circle mode of replication. Because of their small size and great specificity, ribozymes have the greatest potential for biotechnical applications. The ability of ribozymes to cleave other RNA molecules at specific sites m a catalytic manner has brought them into consideration as inhibitors of viral replication or of cell proliferation and gives them potential advantage over antisense RNA. Indeed, ribozymes have already been used to cleave viral targets and oncogene products in living cells (Koizumi, M., H. Kamiya, E. Ohtsuka [1992] Gene 117(2): 179-184; Kashani-Sabet, M., T. Funato, T Tone et al [1992] Antisense Res. Dev 2(1):3-15; Taylor, N.R., J.J. Rossi [1991] Antisense Res. Dev 1(2):173-186; von-Weizsacker, F., H.E. Blum, J.R. Wands [1992] Biochem Biophys. Res Commun 189(2):743-748; Ojwang, J.O., A. Hampel, D.J. Looney, F. Wong-Stall, J. Rappaport [1992] Proc Natl Acad. Sci USA 89(22):10802-10806; Stephenson, P., I. Gibson [1991] Antisense Res Dev l(3):261-268; Yu, M., J. Ojwang, O. Yamada et al [1993] Proc Natl. Acad Sci. USA 90(13):6340-6344; Xmg, Z., J.L. Whitton [1993] J Virol. 67(4): 1840- 1847; Yu, M., E. Poeschla, O. Yamada et al [1995] Virology 206(l):381-386; Little, E., A.S. Lee [1995] J Biol. Chem 270(16):9526-9534). Two kinds of πbozymes have been employed widely, hairpms and hammerheads. Both catalyze sequence-specific cleavage resulting in products with a 5' hydroxyl and a 2 ',3 '-cyclic phosphate. Hammerhead πbozymes have been used more commonly, because they impose few restrictions on the target site. Hairpm ribozymes are more stable and, consequently, function better than hammerheads at physiologic temperature and magnesium concentrations. A number of patents have issued describing various ribozymes and methods for designing ribozymes. See, for example, U.S. Patent Nos. 5,646,031; 5,646,020; 5,639,655, 5,093,246; 4,987,071; 5,116,742; and 5,037,746. However, the ability of ribozymes to provide therapeutic benefit in vivo has not yet been demonstrated. There are more than 200 mheπted diseases that lead to retinal degeneration in humans. Considerable progress has been made in identifying genes and mutations causing many forms of inherited retinal degeneration m humans and other animals. Diseases causing inheπted retinal degeneration in humans can be classified broadly into those that first affect peπpheral vision and the peπpheral retina, such as retinitis pigmentosa, and those that primarily affect central vision and the macula, such as macular dystrophy. The macula has the highest concentration of cones and the peπpheral retma is dominated by rods. Retinitis pigmentosa (RP) is a collection of heritable retinal degenerations caused by defects in one of several genes for proteins of photoreceptor (PR) cells. RP is characterized by progressive rod photoreceptor degeneration and eventual blindness. The exact molecular pathogenesis of RP is still unexplained. Ultrastructural observations suggest that the rod PRs are severely affected m the disease. Approximately 50,000 individuals in the United States are estimated to have RP. The clinical symptoms of retinitis pigmentosa include night blindness and loss of peπpheral vision. With time visual impairment progresses toward the center of the retina causing "tunnel-vision." Retinitis pigmentosa can be subdivided into several genetic categories: antosomal dominant (adRP), autosomal recessive (arRP), X-hnked (xIRP) or syndromic. There are also a number of clinical classes for retinitis pigmentosa. These classes have been condensed into two broad categories. Type 1 retinitis pigmentosa is characterized by rapid progression and diffuse, severe pigmentation; type 2 retinitis pigmentosa has a slower progression and more regional, less severe pigmentation. Macular degeneration is a deterioration 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. At least 500,000 individuals are estimated to suffer from macular degeneration currently in the United States Macular degeneration can have either a genetic basis or it may be an acquired disease. Approximately 10% of Americans over the age of 50 are afflicted with age-related macular degeneration, an acquired form of disease. The inherited forms of macular degeneration are much less common but usually more severe. Inheπted macular degeneration is characterized by early development of macular abnormalities such as yellowish deposits and atrophic or pigmented lesions, followed by progressive loss of central vision. There is currently no effective treatment for most forms of retinitis 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 retinitis pigmentosa Vitamin therapy does not treat the underlying cause of RP and is not a cure. There are many other inherited diseases that cause retinal degeneration in humans. Among these are gyrate atrophy, Nome disease, choroideremia and various cone-rod dystrophies. In addition there are numerous inherited systemic diseases, such as Bardet-Biedl, Charcot-Maπe-Tooth, and Refsum disease which include retinal degeneration among a multiplicity of other symptoms . Another important ocular disease is diabetic retinopathy. Diabetic retinopathy is the leading cause of blindness in adults between the ages of 18-72. Histological studies consistently implicate endothehal cell dysfunction in the pathology. Hyperglycemia directly contπbutes to the development of diabetic retinopathy, and early in the development of diabetic retinopathy there exists disruption of the blood-retmal barrier. NOS activity, as determined by conversion of argmine to citrullme, is significantly increased in diabetes Rosen, P., T.M. Danoff, A. DePiero, F.N. Ziyadeh [1995] Biochem Biophys Res Commun. 207(l):80-88). Gade and coworkers demonstrated that endothehal cell dysfunction correlated with elevated glucose in an in vitro wound model and was mediated by increased levels of NO (Gade, P.V., J.A. Andrades, M.E. Nemni et al [1997] J Vase. Surg. 26(2):319- 326). In rat cerebral arteπes acute glucose exposure dilates arteπes via an endothehum mediated mechanism that involves NO (Cipolla, M.J., J.M. Porter, G. Osol [1997] Stroke 28(2):405-411). Cosentmo demonstrated that prolonged exposure to high glucose increases eNOS gene expression, protein synthesis, and NO release Cosentmo, F., K. Hishikawa, Z.S. Katusic, T.F Luscher [1997] Circulation 96(l):25-28). Nitric oxide (NO) is a pleiotropic molecule with multiple physiological effects, neurotransmitter, component of the immune defense system, regulator of smooth muscle tone and blood pressure, inhibitor of platelet aggregation and a superoxide scavenger. NO is synthesized as a product of the conversion of L-argmine into L-citrullme by the so-called constitutive nitric oxide synthase (NOS), either neuronal (nNOS) or endothehal (eNOS) isoforms. NO regulates specific protein levels. NO increases mRNA levels for VEGF and iNOS. Although several studies on NO function in the retina have been published, very little information is available pertaining to its role in the diabetic retina (Chakravarthy, U., A.W. Stitt, J. McNally et al [1995] Curr. Eye Res 14(4):285-294; Goldstein, I.M., P. Ostwald, S. Roth [1996] Vision Res. 36(18):2979-2974). The iNOS isoform is expressed in the retina, as shown by RT-PCR and immunocytochemistry. It is believed to be involved in the development of diabetic retinopathy and in ischemia-reperfusion injury Hangai, M., N. Yoshimura, K. Hiπoi, M. Mandai, Y. Honda [1996] Exp Eye Res 63(5).501-509; Ostwald, P., I.M. Goldstein, A. Pachnanda, S. Roth [1995] Invest Ophthalmol Vis. Sci 36(12):2396-2403). Administering NOS inhibitors can ameliorate or prevent ischemia-reperfusion injury (Lam, T.T., M.O Tso [1996] Res Commun. Mol Pathol Pharmacol 92(3):329-340). Diabetic human retinal pigmented epithelial cells have augmented iNOS compared to non-diabetic cells. An increasing body of evidence indicates growth factors including vascular endothehal growth factor (VEGF) and msulin-hke growth factor-I (IGF-I) are involved in increased permeability of endothehum that leads to breakdown of the blood-retmal barπer in this microvascular disease. However, the mechanisms for growth factor action m disease progression remain elusive. Brief Summary of the Invention The subject invention concerns materials and methods for the treatment of diseases involving the expression of abnormal and/or excessive amounts of proteins. More specifically, the subject invention provides polynucleotide sequences, and methods for using these sequences, to achieve highly specific elimination or reduction of messenger RNA encoding abnormal and/or excess proteins in the retma. As descπbed herein, the materials and methods of the subject invention can be used to treat a vaπety of diseases. In a preferred embodiment, the disease which is treated is a disease of the retma. Specifically exemplified herein are materials and methods which can be used to treat autosomal dominant retinitis pigmentosa (ADRP). Also specifically exemplified are materials and methods for the treatment or prevention of diabetic retinopathy. In a specific embodiment of the subject invention, hammerhead and hairpin ribozymes have been designed to cleave mutant forms of messenger RNA (mRNA) occurring in various forms of inherited retinal degeneration. The catalytic domain of each πbozyme has been designed to have high stability. In a specific embodiment, the targeting domains are designed to cleave mRNA encoding the P23H mutation. This mutation leads to one of the most common forms of retinitis pigmentosa. These πbozymes have been shown to digest RNA containing the mutation, but not RNA containing the normal sequence. This specificity makes these πbozymes able to destroy harmful mRNA while leaving normal mRNA intact. Ribozymes against other genetic forms of retinitis pigmentosa can be produced and used according to the subject invention. A further aspect of the subject invention pertains to the reduction and/or elimination of pathological levels of proteins involved in endothehal cell nitric oxide (NO) regulation. This aspect of the subject invention provides materials and methods for the treatment and/or prevention of diabetic retinopathy. Increased inducible nitπc oxide synthase (iNOS), enhanced vascular endothehal growth factor levels, and disruption of the blood retinal barrier has been identified in the retinas of BBZ Wor diabetic rats compared to non-diabetic age-matched controls. Additionally, endothehal NOS (eNOS) has been identified in the plasmalemmal caveolae of retinal capillary endothehum from diabetic animals, and cytological evidence indicates translocation of the caveolae from the lumenal to the ablumenal surface of the endothehum. In high glucose environments, chronically increased NO activity results in endothehal cell dysfunction and impaired blood-retmal barrier integrity responsible for the development of diabetic retinopathy. A schematic diagram illustrating key factors in the cascading mechanisms responsible for damaged retinal endothehum, blood retinal barrier integrity, and diabetic retinopathy is shown m Figure 1. Specific embodiments of this aspect of the subject invention pertain to strategies designed to maintain blood retinal barπer integrity. Three mechanisms for increased NO activity in diabetic endothehal cells can be targeted: (a) growth factors which increase eNOS, (b) cytokines which increase iNOS; (c) glucose which directly increases growth factors, cytokines, and NOS isoforms. Three specific targets for the πbozymes of the subject invention are the messenger RNAs which encode VEGF, iNOS, and eNOS. The ribozymes of the subject invention can be delivered using any one of a variety of methods. In a preferred embodiment, recombmant Adeno-associated Virus (rAAV) vectors can be used to transfer the desired genes to retma cells with efficient and cell type-specific expression of the exogenous genes m photoreceptor cells. Other methods of delivery including the use of other viral vectors, hposomes, and naked DNA delivery can be utilized. With the benefit of the teachings provided herein, a person skilled in the art can readily identify, prepare, and use vectors which deliver the ribozymes of the subject invention to the desired location A major advantage of using AAV relative to retrovirus-based vectors include its lack of pathogenicity, its ability to mfect a broad variety of cells and tissues, and its ability to mfect growth-arrested cells. Recombmant AAV lacking its normal rep and cap genes are cuπently used for gene delivery. The small size of AAV (20 nm in diameter) makes it more permeable to retinal tissues than adenovirus. High titers of AAV are attainable in tissue culture, and the stable viπon can be concentrated to reasonably high titers (up to 10 12 infectious virus per ml) Even though up to 90% of the human population has been exposed to AAV, there is no association with human disease, making AAV inherently safer than herpesvirus or adenovirus based vectors. Brief Summary of the Drawings Figure 1 shows a schematic diagram illustrating key factors m the cascading mechanisms responsible for damaged retinal endothehum, blood retinal barπer integrity, and diabetic retinopathy. Figures 2a and 2b 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-/αcZ (a) and mOp-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, thymidine kmase 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). Figure 3 shows the structure of a πbozyme useful according to the subject invention. Figure 4 shows the structure of a πbozyme useful according to the subject invention Figure 5 shows the structure of a nbozyme useful according to the subject invention. Figure 6 shows the structure of a nbozyme useful according to the subject invention Figures 7A and 7B show the secondary structure of the P23H and S334Ter hammerhead ribozymes (6A) and the P23H hairpin nbozyme (6B) The uppercase letters represent the πbozyme sequences, and the lowercase letters represent the target RNA sequences. Substrate sequences diffeπng from the wild-type opsin are in bold type. Roman numerals label the helices. Helix IV of the hairpm has been extended by 4 base pairs and loop C converted to a GNRA tetraloop. Arrows indicate the site of cleavage. Figures 8a-8b show outer retinal layer with P23H ribozymes. 7a, measurements of ONL thickness (left), RIS length (middle), and ROS length (right) in rats killed at different ages. Filled squares denote normal, non-rransgenic animals. P23H-3 rats were either uninjected (open squares), injected subretmally with PBS (open diamonds), or injected with AAV vectors carrying one of five ribozymes or controls. Ribozymes were: Hpl 1 hairpm nbozyme (filled circles), Hhl3 hammerhead nbozyme (filled tnangles), Hpl li "inactive" hairpin πbozyme (open circles), Hhl3ι "inactive" hammerhead πbozyme (open tπangle), or BOFS-gfp (X), all regulated by the same bovme opsm promoter. All injections were performed at PI 4- 15. The eπor bars were omitted if they fell within the symbol, except for Hpl li at P75 and P90, where only one eye at each point was examined. 7b, 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 retina) in rats at P90. Rats were either uninjected (open triangles) or injected at P14-15 with H l 1 hairpm ribozymes (filled circles) or Hhl3 hammerhead ribozymes (open circles). Brief Description of the Sequences SEQ ID NO. 1 is the constructed ohgonucleotide sequence designated S334Ter. SEQ ID NO. 2 is the constructed ohgonucleotide sequence designated P23H. SEQ ID NO. 3 is the wild-type ohgonucleotide sequence S334. SEQ ID NO. 4 is the wild-type oligonucleotide sequence P23H. SEQ ID NO. 5 is an overlapping ohgonucleotide for the S334Ter hammerhead nbozyme. SEQ ID NO. 6 is an overlapping ohgonucleotide for the S334Ter hammerhead nbozyme. SEQ ID NO. 7 is an overlapping oligonucleotide for the P23H hammerhead nbozyme. SEQ ID NO. 8 is an overlapping oligonucleotides for the P23H hammerhead πbozyme. SEQ ID NO. 9 is an overlapping oligonucleotides for the P23H hairpm nbozyme. SEQ ID NO. 10 is an overlapping oligonucleotides for the P23H hairpm nbozyme. SEQ ID NO. 11 is a downstream P23H pπmer for both mutant and wild-type genes used according to the subject invention. SEQ ID NO. 12 is an upstream P23H pπmer for both mutant and wild-type genes used according to the subject invention. SEQ ID NO. 13 is an upstream P23 primer for wild-type genes used according to the subject invention. SEQ ID NO. 14 is a downstream S334Ter pπmer for both mutant and wild-type genes used according to the subject invention. SEQ ID NO. 15 is an upstream S334Ter primer for both mutant and wild-type genes used according to the subject invention. SEQ ID NO. 16 is an upstream S334 pπmer for wild-type genes used according to the subject invention. SEQ ID NO. 17 is a mutant target sequence used according to the subject invention. Detailed Disclosure of the Invention The subject invention pertains to methods for achieving highly specific elimination and/or reduction of mutant and/or excess proteins associated with pathological conditions Specifically exemplified herein is the use of ribozymes to treat and/or prevent diseases in the retma. In one aspect, the subject invention provides materials and methods which can be used to reduce or eliminate the symptoms of inherited eye disease caused by mutations in genes for retinal proteins. In a specific embodiment, the subject invention provides a method for treating autosomal dominant retinitis pigmentosa (ADRP) at a molecular level. Twelve percent of Ameπcan patients with autosomal dominant retinitis pigmentosa carry a substitution of histidme for prolme at codon 23 (P23H) in their rhodopsm gene, resulting in photoreceptor cell death from the synthesis of the abnormal gene product. One aspect of the current invention pertains to the use of ribozymes to discriminate and catalyze the destruction of P23H mutant mRNAs Expression of either a hammerhead or hairpm πbozyme in a rat model markedly slows the rate of photoreceptor degeneration for at least 15 months. Catalytically inactive control πbozymes had significantly lesser effect on retarding the retinal degeneration. Intracellular production of ribozymes m photoreceptors can be achieved, for example, by transduction with a recombmant Adeno- Associated Virus (rAAV) incorporating a rhodopsm promoter. The subject invention utilizes the catalytic properties of πbozymes. Ribozymes are enzymes compπsed of nbonucleic acid (RNA). In nature, ribozymes conduct a variety of reactions involving RNA, including cleavage and hgation of polynucleotide strands The specificity of πbozymes is determined by base pairing (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 ribozymes, the part that actually performs the biochemical work, can also be changed in order to increase activity or stability of the nbozyme. Ribozymes, if delivered as descπbed herein to photoreceptor cells of the retina by a gene delivery vector such as a specially designed virus, provide a long-term, even permanent treatment for retinitis pigmentosa, macular degeneration, or other pathological retina condition. Viral vectors, such as rAAV, are well known and readily available to those skilled in the art Utilizing the techniques of the subject invention, πbozymes can be continuously produced m the retinal cells from a copy of the πbozyme integrated in the patient's DNA. In one embodiment the subject invention concerns synthetic genes for several πbozymes. These πbozymes recognize the nucleotide change causing the P23H mutation m one form of ADRP and the S334ter mutation in another. Genes have been constructed which encode several πbozymes having the ability to specifically destroy target RNAs for mutant retma proteins. Specifically, Figure 3 shows a nbozyme for the destruction of RNA having the S334 mutation. Figures 4, 5, and 6 show πbozymes which destroy RNA having the P23H mutation The πbozyme in Figure 6 is a hammerhead πbozyme (Rz23) which is directed against the human P23H mRNA. With the benefit of the teachings provided herein, the skilled artisan can construct genes encoding πbozymes which destroy mutant RNA molecules associated with human RP or other retinal diseases. Using a recombmant Adeno-associated virus (rAAV) in which expression is driven by a portion of the rod opsm promoter, photoreceptor-specific expression of reporter genes can be achieved by ocular injection. Transgenic rat lines carrying the P23H or S334ter mutation m the rod opsm gene under control of the opsin promoter exhibit a course of retinal disease remarkably similar to that observed in humans beaπng such mutations. rAAV-πbozymes in P23H and S334ter can be tested in transgenic rats to confirm that the course of the RP-like disease can be ameliorated with a minimum of pathogenic side effects. Assays for activity include morphological analysis of retinal degeneration, quantitative mRNA studies, and electroretmography. Ribozymes can also be used according to the subject invention as a partial treatment for recessive or semi-dominant genetic diseases of the eye as a supplement to gene replacement therapy. The delivery-expression matenals and methods of the subject invention can be used to replace any gene responsible for recessive photoreceptor disease. Specific examples include the genes responsible for retinitis pigmentosa or macular degeneration. Additionally, πbozymes can be used accordmg to the subject invention to treat RP-like disease resulting from the numerous known mutations in the rhodopsm gene. Examples of such mutations are well known to those skilled in the art. See, for example, Daiger, S.P., L.S. Sullivan, J.A. Rodriguez (1995) Behavioral Brain Sci. 18:452-467. A further aspect of the current invention pertains to therapeutic strategies that can retard or block the effects of high glucose on progression of diabetic retinopathy High glucose environments can result in chronically increased nitπc oxide (NO) activity which leads to endothehal cell dysfunction and impaired blood retinal barner mtegπty characteπstic of diabetic retinopathy. Reducing the synthesis of NOS using nbozymes can be used to retard or eliminate the damage to the blood retinal barrier. For example, ribozymes which reduce mRNA for VEGF, iNOS, or eNOS can be used. In specific embodiments, to inhibit the expression of iNOS and eNOS, hammerhead ribozymes that contain one long (46 nt) targeting arm 3' to the catalytic domain and a short (5 nt) targeting sequence 5' to the catalytic domain can be used. The long targeting arm permits rapid association with the target sequence. Keeping one arm short permits rapid dissociation of product necessary for catalytic turnover. Messenger RNA molecules have a complex pattern of intramolecular hydrogen bonds that reduce the portion of the molecule available for nbozyme attack. Sites in the iNOS and eNOS mRNAs accessible to nbozyme binding can be determined using synthetic transcnpts of iNOS and eNOS cDNA clones Ribozyme cleavage can be tested on short oligonucleotides identical to sequences of accessible regions containing hammerhead target sites. The most active nbozymes can then be tested on synthetic transcripts of the entire cDNA clone and on total mRNA extracted from endothehal cells to identify the most preferred ribozymes. Genes encoding ribozymes can be cloned in the AAV vector or other suitable vector High-potency πbozymes that cleave eNOS, iNOS, and/or VEGF mRNA can be constructed by those skilled in the art having the benefit of the instant disclosure. Delivering these to retinal endothehal cells can be done to reduce expression of iNOS, eNOS, or VEGF and, ultimately, to reduce the production of nitric oxide Reduction of NO production will, in turn, reduce or delay retinal permeability dysfunction. Mateπals and Methods rAAV plasmid construction The mOp-/αcZ -rAAV plasmid DNA was made by first inserting the 4 3 kbp BglWBamHI fragment containing the proximal murme 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 BglΩ. sites of pTR which contains the AAV TR sequences and a SV40 polyadenylation sequence (Figure 2a). The mOp- gfp-rAAV plasmid DNA was made by first adding Notl linkers to the 472 bp BgRllXhol proximal opsin promoter fragment of pRG3 and inserting it into the Notl sites of pTRUF2 (Zolotukhm, S. M. Potter, W. Hauswirth, J. Guy, N. Muzyczka [1996] J Virol. 70:4646-4654) (Fig. 2b). rAAV virus production and analysis. To generate recombmant virus, human 293 cells were co-transfected with mOp-/αcZ-rAAV or mOp-g/p-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 centei 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-ZαcZ-rAAV. Adenovirus was not detectible in either of the r AAΛ preparations. Subretmal injection of rAAV. Thirty adult C57BL/6I (Jackson Laboratories, Bar Harbor, ME) pigmented mice between 3 and 6 months of age and 27 adult albmo Sprague- Dawley rats between 3 and 4 months of age were used. Animals were anesthetized b> ketamme/xylazme injection, eyes were dilated (2.5% phenylephnne and 0.5% tropicamide) and a local anesthetic (proparacam HC1) was applied. Injections (1 μl in mice and 2 μl in rats) were made into the right eye with blunt 32 gauge needle through an opening in the pars-plana delivering the rAAV suspension into the superior subretmal space. Control injections wei e made m the contralateral eye with PBS only. Injections were performed with an operating microscope and the subretmal location of the injected volume was confirmed 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 posterior eyecup placed m primary fixative. For β-galactosidase staining, eyecups were fixed m 0.5% glutaraldehyde in 0.1M Cacodylate buffer pH 7.5 for 15 minutes at room temperature. Following a 10 mmute wash m PBS, the eyecups were incubated m an iron-based X-gel staining solution (Sanes, J., J Rubenstem, J. Nicolas [1986] EMBO J. 5:3133-3142) m 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 in isotomc PBS on a vibratome at 50-100 μm. Bnght field and phase-contrast micrographs of whole mounts and β-galactosidase-stamed sections were made with a Zeiss Axiophot. GFP fluorescence was examined in retinal whole mounts and agarose embedded sections. Tissue fixation was minimized to reduce retinal autofluorescence. Retinas were detached from eyecups, fixed for 15 minutes at room temperature in 4% formaldehyde, 0.1 M PO 4 buffer pH 7.5, and rmsed three times in PBS. Whole mounts were photographed with epifluorescence using Zeiss filter set 09 (ex. 450-490 nm, barπer 510 nm, emission 520 nm) and an AttoArc (Carl Zeiss, Inc., New York) variable output UV lamp to minimize GFP bleaching Whole mount retinas were then embedded m 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 NIH Image analysis software (Rasband, W. D. Bright [1995] Microbeam Analysis Society Journal 4: 137-149). Expression of the lacZ reporter gene in muπne retinal cells was analyzed by reverse transcπptase PCR (RT-PCR). Pieces of retma (1 mm 2 ), 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-guamdme isothiocyanate, Gibco-BRL, Gaithersberg. MD) according to the manufacturer's recommendations. The RNA was additionally purified 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 lacZ sequence primers 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 such as a gene encoding a nbozyme specifically in photoreceptor (PR) cells, it is possible to utilize a specifically adapted viral vector. To demonstrate this capability 472 bp of the proximal muπne rod opsm promoter (+86 to -385) were 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 right eyes of 30 C57B1/6J mice were injected sub-retmally with lμl of mOp-lacZ virus (10 7 IU per ml). After two weeks, the πght (test) and left (control) eyes of 12 animals were removed, fixed and stained with X-gal. Test retma 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 senal 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 in the outer plexiform layer. PR outer segments, RPE and other retinal cells this region did not reveal X- gal staining above baseline levels observed m identically treated, uninjected or PBS-mjected control retinas from the contralateral eye. Examination of additional transverse sections confirmed that the region of positive staining radiated outward from the injection site in 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 oflacZ mRNA by RT-PCR. The 199-bp 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-injection 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 derived from β- galactosidase expression within PR cells and not derived from RPE expression. A second reporter gene, a synthetic version of the A victoria green fluorescent gene (gfp) (Zolotukhm, 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 munne rod opsin promoter was used as well as an analogous rAAV vector to construct the mOp-gfp virus (Figure 2b). Two μl of gfp-contammg rAAV was injected into the subretmal space of 8 Sprague-Dawley rats. Rats were used m 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 in 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 in 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 peπpheral to the injection site, the fraction of positive PRs was substantially reduced, consistent with the radial decline in fluorescence seen m retinal whole mounts. We 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 in the REP, choroid, or sclera. Example 4 - Construction of Plasmids Encoding the Substrate RNA and Ribozymes Two duplex synthetic DNA oligonucleotides containing the target coding sequences and flanked by Pstl and BamΑl restriction sites were ligated into the plasmid pT7/T3-19 (Life Technologies, Gaithersburg, MD) downstream of a T7 RNA polymerase promoter. The sequences of the target oligonucleotides were S334Ter: 5'-GCCCTGCAGG ACGACGCCTA AGCCACCGCT TCCGGATCCG GC-3' (SEQ ID NO. 1); and P23H: 5'-GCCCTGCAGG GTCGGAGTCA CTTCGAGCAG GGATCCGGC-3' (SEQ ID NO. 2). The wild-type target " 5 ohgonucleotide sequences were S334: 5'-GCCCTGCAGG ACGACGCCTC TGCCACCGCT TCCGGATCCG GC-3' (SEQ ID NO. 3); and P23H: 5'-GCCCTGCAGG GTCGGAGCCC CTTCGAGCAG GGATCCGGC-3' (SEQ ID NO. 4). Ribozyme were generated by extension of two overlapping synthetic DNA oligonucleotides flanked by Kpnl and Mlul restriction sites. The large fragment of DNA pol I 10 (Klenow; NEB, Beverly, MA) was used as follows to fill out the DNA duplexes: Overlapping oligonucleotides were heated to 65 °C for 2 minutes and annealed by slow cooling to room temperature for 30 minutes. The annealed oligonucleotides pnmed each other and were mutually extended by DNA polymerase in the presence of 5 mM deoxynucleoside tnphosphates and polymerase buffer (10 mM Tπs-HCI [pH 7.5], 5 mM MgCl 2 , and 7.5 mM dithiothreitol) for 15 1 hour at 37°C. The fully duplex fragments were digested and ligated into the T7 RNA polymerase expression plasmid pHC 40 at the Kpnl and Mlul restriction sites. Ligated plasmids were transformed into Eschenchia coli DH5-α cells. Clones were screened by hybridization analysis and were verified by sequencing. In the S334Ter hammerhead, sequences of the overlapping oligonucleotides for each 20 πbozyme were : 5 ' -GCGCGGTACC GTGGCTCTG.ATGAGCC GCTTCGGC-3 ' (SEQ ID NO 5) and 5'-GCGCACGCGT GACGCCTTTC GCCGCCGA AGCGGC-3' (SEQ ID NO. 6); m the P23H hammerhead: 5 '-GCGCGGTACC CGAAGTCTG ATGAGCCGCT TCGGC-3' (SEQ ID NO. 7) and 5'-GCGCACGCGT TCGGAGTTTC GCCGCCGAAG CGGC-3' (SEQ ID NO. 8); and m the P23H hairpm: 5 '-GCGCGGTACC GAAGTAGAAC CGAACCAGAG AAACA-3' 25 (SEQ ID NO 9) and 5 ' -GCGCACGCGT ACCAGGJAAT ATACCACGCT CTTACGAGCG TGTGTTTCTC TGGTT-3 ' (SEQ ID NO. 10). Underlined nucleotides were mutated in a second set of oligonucleotides (hammerhead: A-C, G-C, G-T; hairpm: T-A, C-G) to create inactive ribozymes as controls. In vitro transcription. Plasmids containing target sequences were linearized with 30 BamΑl, and plasmids containing πbozyme sequences with Mlul. All transcπpts were generated with T7 RNA polymerase and were labeled by incorporation of [α- 32 P] uπdme tπphosphate (ICN; Costa Mesa, CA). Transcnption reactions were brought to 0.5% sodium dodecyl sulfate, extracted with phenol-chloroform-isoamyl alcohol (50:50:1), precipitated with ethanol, washed twice with 70% ethanol, and resuspended m water. For hairpm ribozymes, ethanol precipitation was replaced by gel filtration Sephadex G-50 (Pharmacia; Uppsala, Sweden) columns. RNA extraction. Total RNA was extracted from the retinas of the P23H and S334Ter transgenic rats (RNeasy Mini-preps; Qiagen, Santa Clara, CA) according to the manufacturer's recommendations. Retinas were snap frozen m liquid nitrogen before RNA extraction and stored at -70°C. A typical preparation resulted m 100 μg total RNA. Ribozyme cleavage reactions. Specific radioactivity of the [α- 32 P] undine tπphosphate and the base composition of each molecule were used to calculate the concentration of target and ribozyme molecules. Standard cleavage conditions consisted of 50 nM substrate RNA and 20 nM πbozyme, 20 mM MgCl 2 , 40 mM Tns-HCl (pH 7.5), and incubation at 37°C. Hammerhead πbozymes were renatured by incubation in 10 mM MgCl 2 at 37 °C for 4 to 10 hours. Hairpm ribozymes were denatured at 90°C for 2 minutes in 0.1 mM EDTA and renatured at room temperature for 5 minutes. Cleavage reactions were started with the addition of MgCl 2 . Reactions were stopped by addition of 50 mM EDTA, than an equal volume of 10 M urea, 0 002% bromphenol blue, and 0.002% xylene cyanol. Experimental conditions, such as time of incubation, magnesium concentration, and πbozyme concentration, were vaπed individually All reactions were performed in a final volume of 15 μl. Cleavage assays with the retinal RNAs as target substrates contained 0.1 μg total RNA extract and 50 nM nbozyme. Cleavage products were analyzed by electrophoresis on 8 M urea 10% acrylamide sequencing gels (run in 0.089 M Tns-borate, 0.089 M bone acid, and 0.002 M EDTA [pH 8]). Cleavage of transgenic opsm RNA was determined by reverse transcription of all opsin RNA into cDNA and polymerase chain reaction amplification by pnmers that distinguished the mutant transgene from the normal chromosomal genes. To detect reverse transcπption-polymerase chain reaction (RT-PCR) products, [α- 32 P]deoxyadenosme tπphosphate was included during the final PCR cycle. The fragments were analyzed by electrophoresis on 4% or 5% nondenaturmg polyacrylamide gels Radioactivity of product, substrate, and ribozyme bands, or of PCR fragments was quantitated by scintillation counting of excised bands or by radio-analytic scanning (Phosphorlmager, Molecular Dynamics, Durham, NC). Kinetic analysis. Analyses to determine multiple-turnover kinetic constants were earned out 20 mM MgCl 2 , 40 mM Tns-HCl (pH 7.5), at 37°C for 15 minutes. Samples were premcubated at 37 °C and reactions were initiated by addition of ribozyme to substrate RNA Samples contained increasing concentrations of substrate RNA, holding πbozyme concentration constant Values for maximum velocity (F mαv ), K M , and k aφ were obtained by double reciprocal plots of velocity versus substrate concentration. Reverse transcπption-polymerase chain reaction (RT-PCR.. RNAs were subjected to reverse transcπption using a first strand synthesis kit (Pharmacia: Uppsala, Sweden) primed by ohgo-deoxythymidme. The P23H, S334Ter, and wild-type opsm cDNAs were amplified by PCR, using a three-pπmer system (the downstream pπmer anneals to the mutant and wild-type ' 5 cDNAs; two upstream primers anneal to the mutant or wild-type cDNAs) resulting in PCR products of different lengths. Because the mutant transgene was derived from the mouse rhodopsm gene, wild-type (rat) mRNA was distinguished from mutant (mouse) mRNA The P23H product was 195 nucleotides, the P23 wild-type product was 208 nucleotides, the S334Ter product was 298 nucleotides, and the S334 wild-type product was 315 nucleotides. 10 The downstream P23H pπmer (for mutant and wild-type genes) was 5 ' -CAGCCACGGC CAAGTTGAG (SEQ ID NO. 11) The upstream P23H pπmer was 5'-GCGTGGGTCG GAGTCACTTC (SEQ ID NO. 12) The upstream P23 primer for wild-type genes was 5'- TCCAACATCA CGGGCGTGGT (SEQ ID NO. 13). The downstream S334Ter primer (for mutant and wild-type genes) was 5'-TGGGGAGCCT CATTTTG (SEQ ID NO. 14) The 15 upstream S334Ter pnmer was 5'-CTCTTCCATC TATAACCCGG (SEQ ID NO. 15). The upstream S334 pπmer for wild-type genes was 5'-TTTCTTTGCT AAGACCGCC (SEQ ID NO 16). In all control reactions without active ribozyme, the ratio of mutant to wild-type transcπpt was relatively mvanant, 60.6+3.2% for S334TER and 56.5+1.2% for P23H. Because 20 the appropriate combination of mutant and wild-type upstream primers was used for RT-PCR analysis of each transgenic retina, this baseline value reflects a combination of the true mutant:wιld-type transcπpt ratio and any inherent difference in amplification efficiency caused by the two distinct upstream pπmers. Therefore, any change in this ratio when the ribozyme is added documents an internally corrected measure of allele-specific, πbozyme-mediated RNA 25 cleavage. Example 5 - Ribozyme Destruction of an mRNA Causing Retinitis Pigmentosis To test the activity and specificity of ribozymes as a tool for combating autosomal dominant retinitis pigmentosa, two hammerhead ribozymes were designed, one targeting the C 30 to A transversion leading to the P23H mutation (P23H-Hh) and the other targeting a UCA to UAA sequence change at codon 334 (S334Ter-Hh; Figure 7A). Both transgenes contain 2 nucleotide changes relative to the wild-type (rat) sequences in the targeted regions. In the case of P23H, these changes are a C to A transversion in codon 23 and a silent transition mutation in codon 22 (AGC-AGT). The S334ter mutation was created by altenng codon 334 from TCC to TAA. A hairpin πbozyme was also created to cleave the P23H mRNA (P23H-Hp) at the same sequence as the hammerhead (Figure 7B). These modifications were tested to determine the most active structure in vitro. The U39:C mutation increased cleavage activity two-fold compared with that m the naturally occurπng hairpm structure, and the extension of helix IV to 9 base pairs, capped by a GUAA tefraloop, increased cleavage activity fourfold. The effect of these changes were not additive in the double mutant, however. We also strengthened helix II of the hammerhead ribozyme compared with that of the naturally occurring structure by substituting G:C base pairs for A:T pairs (Figure 7A). Using these activity-increasing modifications, three πbozymes were tested for sequence specificity of cleavage. In addition to the three active ribozymes, corresponding inactive ribozymes were used as a control for antisense effects. These inactive ribozymes contained mutations at residues known to be cπtical in the catalytic activity of the hairpm and hammerhead ribozymes. For each active ribozyme, the cleavage reaction resulted m two discrete products: a larger 5' fragment and a smaller 3' fragment, which resulted from the breakage of the targeted phosphodiester bond. In the case of P23H-Hh and P23H-Hp the 5 ' products were 26 and 29 nucleotides, respectively. For the S334Ter hammerhead ribozyme, the 5' product was 39 nucleotides and the 3' product was 18 nucleotides. The S334Ter-Hh ribozyme appeared less active than did the ribozymes directed against the P23H target. No cleavage products were detected after incubation of the active ribozymes with the wild-type opsin target RNAs. The cleavage site m the P23H and S334Ter (mutant) targets differ in two nucleotide positions from their respective wild-type sequences. Similarly, there was no digestion of a target after incubation with the inactive ribozymes. This result confirms that appearance of the smaller band depends on the catalytic domains. Example 6 — Cleavage is Dependent on Magnesium Concentration Because of the key role of Mg 2+ in RNA structure and in the breakage of phosphodiester bonds, the cleavage assay was performed with each of the πbozymes as a function of magnesium concentration Although all the ribozymes showed greater activity m higher concentrations of magnesium, the greatest increase was seen between 0 mM and 20 mM. Improvement of cleavage reached a plateau at 80 mM magnesium, but little difference was seen beyond that point All πbozymes cleaved at magnesium concentrations of 5 mM or less, indicating that cleavage will occur under physiological conditions. Example 7 — Ribozymes Target and Cleave Intact Retinal RNA Although the active πbozymes specifically cleaved synthetic target RNAs, an important question relevant to their application in vivo is whether they exhibit similar specificity on full- length mRNA m the context of all RNAs found m retinal photoreceptors. These πbozymes were assayed using total RNA isolated from the retinas of transgenic rats. These Sprague-Dawley rats contain the P23H mutant rhodopsm transgene or the S334Ter mutant rhodopsm transgene, in addition to their normal endogenous rhodopsm alleles. Both mutant fransgenes were derived from the mouse rod opsm gene. The retinal RNA extracts were used as target substrates in place of the synthetic targets in the standard ribozyme cleavage assay to test the activity of the πbozymes against full-length mRNA. In this case, RT-PCR was used to detect the wild-type and mutant targets. Cleavage of the mutant mRNA derived from the transgene should reduce the level of this RT-PCR product compared with that deπved from the endogenous (wild-type) gene The active nbozymes specifically cleaved the transgenic opsm RNA molecules during an extended (12-hour) incubation. The relative concentrations of mutant and wild-type mRNA was determined by RT-PCR analysis specific for the two messages. In the S334Ter transgenic rats, the mutant mRNA was determined by RT-PCR analysis specific for the two messages. In the S334Ter transgenic rats, the mutant mRNA was 60.6% that of wild-type opsin mRNA before treatment. After incubation with S334Ter-Hh ribozyme, the amount of transgenic RNA was reduced by 17% compared with that of the wild-type. A more dramatic drop was observed when the hammerhead ribozyme was directed against the P23H animals; the mutant transgene was expressed at 56.5% of the wild-type level. After incubation with P23H-Hh, the level was reduced by approximately 50%. The hairpin πbozyme cleaved the P23H RNA to a lesser extent, resulting m a reduction of 19% compared with that of the wild-type standard. Thus, these ribozymes bind and cleave their respective targets, effectively distinguishing among the cellular mRNAs. Example 8 - Construction of Vectors and Expression in Target Cells rAAV-nbozyme 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). The> 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 opsin promoter replaced the 472 bp munne rod opsin promoter and the πbozyme gene replaced the gfp gene. The bovme promoter fragment contains three proximal promoter elements and the endogenous transcriptional 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 ribozymes were designed, tested and cloned as described above. Each ribozyme gene was followed by an internally cleaving hairpm πbozyme deπved from plasmid pHC (Altschuler, M., R. Tπtz, A.A. Hampel [1992] Gene 122:85-90) resulting in ribozyme cassettes of 140-152 bp Self cleavage at the internal cutting site in the primary ribozyme RNA leaves identical 3' ends on each mature πbozyme. The πbozyme cassette was preceded by an mtron deπved from SV40 and followed by a polyadenylation signal in order to promote nuclear export of the ribozyme 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 neo τ gene contained withm 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 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 senal dilution cytopathic effect or infectious center assay respectively, were less than five order of magnitude lower than rAAV Subretmal injection 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/xylazme 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% glutaraldehyde). 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 Muller 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). Bnefly, 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 retma, or plotted as a distnbution 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 m the data cited, since it was based on a much larger number of measurements. RT-PCR. For quantification of opsm mRNA retina from ribozyme injected or control eyes, retina were isolated without fixation and total RNA immediately extracted using the RNeasy Mmikit (Qiagen, Santa Claπta, CA). RT-PCR was performed using the Pharmacia First-Strand cDNA synthesis kit employing oligo dT as the primer. Wild-type and transgene opsin cDNAs were amplified using a three pπmer system descnbed 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 m 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 hairpin ribozyme (Hpl l) cleaved 3' to the first adenosme residue (underlined) and the hammerhead nbozyme (Hhl3) cleaved 3' to the central cytosme residue (underlined) Control ribozymes (Hpl li and Hhl3ι, respectively) retained the targeting domains but contained fatal flaws in their catalytic domains. In vitro, the active hammerhead πbozyme (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 expeπments, both ribozymes exhibited kinetic constants (!__„, and k^,,) similar to those of naturally occurnng πbozymes. The two active ribozymes produced negligible cleavage of the wild-type transcπpt even in the presence of high MgCl 2 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 ribozymes were able to cleave the mRNA product of the mutant transgene selectively For experiments in vivo, a line of transgenic rats, TgN(P23H)3 (abbreviated P23H-3), that has a retinal degeneration phenotype similar to patients with retinitis 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 in non-transgenic control rats. Thereafter, death of photoreceptor cells is almost linear until about P60, resulting in 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-ribozyme vector was injected into the interphotoreceptor space between the photoreceptors and the adjacent retinal pigment epithelium at P14 or P15. Rats were sacrificed and eyes examined at 3 time points between P60-P90. At these ages in uninjected 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-injected eyes showed a modest but significant decrease in the accumulation of transcript derived from the P23H transgene. Control eyes exhibited little variation in the level of transgene opsin mRNA. Eyes injected with either active ribozyme uniformly exhibited lowered transgene mRNA levels relative to total opsin mRNA in the same eye. Retinas receiving the hairpin ribozyme Hpl 1 showed a 15.3+3.3% decrease in transgene expression, and those with the hammerhead ribozyme Hhl3 showed a decrease of 11.1+5.1% decrease. Histologically, eyes injected with the ribozymes retained significantly more photoreceptors at P60, P75 and P90 than uninjected contralateral control eyes. Retinas receiving a subretinal injection of Hhl3 at P14-15 retained 88% of the normal ONL thickness, compared to about 60% in the uninjected controls (Figure 8a). Thus, the ONL thickness after Hhl3 expression was 40-43% greater than that of uninjected P23H-3 controls (Figure 8b), a highly significant difference (p=0.001 or less at P60 and P90). Injection of the Hpl 1 ribozyme also resulted in significant rescue when compared to controls, with preservation of 77-83%> of normal ONL thickness (Figure 8a). Thus, the ONL thickness after Hpl 1 expression was 30-39% greater than that of uninjected P23H-3 controls (Figure 8b), a highly significant difference (p<0.0005 at all ages). There was little or no rescue in PBS-injected control eyes (p>0.169 in all cases) as shown in Figure 8a. As a control for possible rescue by the expression of the bovine opsin promoter (BOPS), AAV-BOVS-gfp was injected at a titer of 1.75 x 10 8 , similar to the titer used with the AAV-ribozymes. The injection of AAV-BOPS-g/p did not rescue photoreceptors (Figure 8a). The inactive Hpl li did yield ONL thickness measures greater than uninjected control values, but they were consistently less than that resulting from the active Hpl 1 and Hhl 3 ribozymes (Figure 8a). The pan-retinal extent of photoreceptor rescue that resulted from a single 2-μl injection of the rAAV suspension was surprising (Figure 8b). From photoreceptor counts, it is estimated that there are approximately 10 7 photoreceptors in the rat retina. Recombinant 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 m a single 2 μl injection. The upper bound derives from the DNAse resistant particle assay, indicating that 2 μl of the rAAV-πbozyme 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 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 nbozymes resulted in greater lengths of rod inner segments (RIS) and rod outer segments (ROS). In the case of RIS, the uninjected control retinas had RIS that were about 90% of normal. Both the active and inactive πbozymes resulted in RIS lengths of 98% or greater of the normal length, and about 10- 15% longer than uninjected controls. The PBS and AAV-BOPS-g/p were indistinguishable from uninjected eyes. The ROS lengths were about 15-25% longer in the πbozyme-mjected eyes compared to those m the uninjected control eyes. However, ROS m the πbozyme-mjected eyes were, at greatest, only 65-75% of normal, compared to the virtually normal RIS lengths. The ROS of the active πbozymes differed significantly from the uninjected controls (p<0.005 for all, except <0.02 for Hpl 1 at P90), as did the inactive Hpl li (p<0.05). The finding that πbozyme-targeted destruction of P23H mutant RNA markedly slows the rate of retinal degeneration m 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 lnhented 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 described herein are for illustrative purposes only and that various modifications or changes m light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (ii) TITLE OF INVENTION: (iii) NUMBER OF SEQUENCES: 17 (iv) CORRESPONDENCE ADDRESS : (A) ADDRESSEE: Saliwanchik, Lloyd & Saliwanchik (B) STREET: 2421 N.W. 41st Street, Suite A-l (C) CITY: Gainesville (D) STATE: FL (E) COUNTRY: USA (F) ZIP: 32606 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 60/046,147 (B) FILING DATE: 09-MAY-1997 (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 60/044,492 (B) FILING DATE: 21-APR-1997 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Saliwanchik, David R. (B) REGISTRATION NUMBER: 31,794 (C) REFERENCE/DOCKET NUMBER: UF-191A (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 352-375-8100 (B) TELEFAX: 352-372-5800 (2) INFORMATION FOR SEQ ID NO : 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 : GCCCTGCAGG ACGACGCCTA AGCCACCGCT TCCGGATCCG GC 42 (2) INFORMATION FOR SEQ ID NO : 2 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 : GCCCTGCAGG GTCGGAGTCA CTTCGAGCAG GGATCCGGC 39 (2) INFORMATION FOR SEQ ID NO : 3 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 : GCCCTGCAGG ACGACGCCTC TGCCACCGCT TCCGGATCCG GC 42 (2) INFORMATION FOR SEQ ID NO : 4 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 : GCCCTGCAGG GTCGGAGCCC CTTCGAGCAG GGATCCGGC 39 ( 2 ) INFORMATION FOR SEQ ID NO : 5 : ( i ) SEQUENCE CHARACTERISTICS : (A) LENGTH : 34 base pairs (B ) TYPE : nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 : GCGCGGTACC GTGGCTCTGA TGAGCCGCTT CGGC 34 (2) INFORMATION FOR SEQ ID NO : 6 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 : GCGCACGCGT GACGCCTTTC GCCGCCGAAG CGGC 34 (2) INFORMATION FOR SEQ ID NO : 7 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 : GCGCGGTACC CGAAGTCTGA TGAGCCGCTT CGGC 34 (2) INFORMATION FOR SEQ ID NO : 8 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 : GCGCACGCGT TCGGAGTTTC GCCGCCGAAG CGGC 34 (2) INFORMATION FOR SEQ ID NO : 9 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 : GCGCGGTACC GAAGTAGAAC CGAACCAGAG AAACA 35 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GCGCACGCGT ACCAGGTAAT ATACCACGCT CTTACGAGCG TGTGTTTCTC TGGTT 35 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: CAGCCACGGC CAAGTTGAG 19 (2) INFORMATION FOR SEQ ID NO : 12 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GCGTGGGTCG GAGTCACTTC 20 (2) INFORMATION FOR SEQ ID NO : 13 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: TCCAACATCA CGGGCGTGGT 20 (2) INFORMATION FOR SEQ ID NO : 14 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: TGGGGAGCCT CATTTTG 17 (2) INFORMATION FOR SEQ ID NO : 15 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 15 : CTCTTCCATC TATAACCCGG 20 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: TTTCTTTGCT AAGACCGCC 19 (2) INFORMATION FOR SEQ ID NO : 17 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: UCGGAGUCUA CUUCG 15

Claims: Claims 1. A method for reducing the amount of a protein in the cell of a mammal, wherein said method comprises administering to said mammal an effective amount of a ribozyme wherein said ribozyme cleaves RNA encoding said protein. 2. The method, according to claim 1, wherein said cell is a retina cell. 3. The method, according to claim 2, wherein said method is used in the treatment of a pathological condition selected from the group consisting of autosomal dominant retinitis and diabetic retinopathy. 4. The method, according to claim 3, wherein said ribozyme cleaves mRNA encoding a mutant form of rod opsin. 5. The method, according to claim 4, wherein said ribozyme cleaves mRNA encoding rod opsin having the P23H mutation. 6. The method, according to claim 5, wherein said ribozyme is selected from the group consisting of Rp 11 , Rp 13 , and Rz23. 7. The method, according to claim 6, wherein said ribozyme is Rz23. 8. The method, according to claim 4, wherein said ribozyme cleaves mRNA encoding rod opsin having the S334 mutation. 9. The method, according to claim 8, wherein said ribozyme is Rp9. 10. The method, according to claim 3, wherein the cleavage of RNA by said ribozyme is used to treat blood-retinal barrier dysfunction. 11. The method, according to claim 10, wherein the cleavage of RNA by said ribozyme reduces nitric oxide activity in said cell. 12. The method, according to claim 11, wherein said ribozyme cleaves mRNA encoding a protein selected from the group consisting of VEGF, eNOS, and iNOS. 13. The method, according to claim 1, wherein said ribozyme is delivered by a viral vector. 14. The method, according to claim 13, wherein said viral vector is an adeno- associated virus. 15. The method, according to claim 14, wherein said adeno-associated virus comprises regulatory elements which direct the expression of said ribozyme in retina cells. 16. A ribozyme which cleaves mRNA encoding a protein selected from the group consisting of mutant rod opsin proteins, VEGF, eNOS, and iNOS. 17. The ribozyme, according to claim 16, wherein said ribozyme is selected from the group consisting of Rp9, Rp 11 , Rp 13 , and Rz23. 18. The ribozyme, according to claim 17, wherein said ribozyme is Rz23.

Inventors: DRENSER KIMBERLY, HAUSWIRTH WILLIAM W, LEWIN ALFRED S, DRENSER, KIMBERLY, HAUSWIRTH, WILLIAM, W., LEWIN, ALFRED, S.
Applicants: UNIV FLORIDA, UNIVERSITY OF FLORIDA
Application year: 1998
Main classification: C12N15
IPC codes: A61K31, A61K35, A61K38, A61K48, A61P9, A61P27, C12N9, C12N15