Link:  Pharm/Biotech Resources

Title:  Use of recombinant gene delivery vectors for treating or preventing diseases of the eye

United States Patent:  6,943,153

Issued:  September 13, 2005

Inventors:  Manning, Jr.; William C. (Redwood City, CA); Dwarki; Varavani J. (Pittstown, NJ); Rendahl; Katherine (Berkeley, CA); Zhou; Shangzhen (Alameda, CA); Miller; Sheldon S. (Berkeley, CA); Wang; Fei (Albany, CA)

Assignee:  The Regents of the University of California (Oakland, CA); Chiron Corporation (Emeryville, CA)

Appl. No.:  665493

Filed:  September 20, 2000

Gene delivery vectors, such as, for example, recombinant adeno-associated viral vectors, and methods of using such vectors are provided for use in treating or preventing diseases of the eye.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides compositions and methods for treating, preventing, or, inhibiting diseases of the eye. Within one aspect of the present invention, methods are provided for treating or preventing diseases of the eye comprising the step of intraocularly administering a gene delivery vector which directs the expression of one or more neurotrophic factors, or, anti-angiogenic factors, such that the disease of the eye is treated or prevented. Within related aspects of the present invention, gene delivery vectors are provided which direct the expression of one or more neurotrophic factors such as FGF, as well as gene therapy vectors which direct the expression of one or more anti-angiogenic factors. Within certain embodiments of the invention, a viral promoter (e.g., CMV) or an inducible promoter (e.g., tet) is utilized to drive the expression of the neurotrophic factor.

Representative examples of gene delivery vectors suitable for use within the present invention may be generated from viruses such as retroviruses (e.g., FIV or HIV), herpesviruses, adenoviruses, adeno-associated viruses, and alphaviruses, or from non-viral vectors.

Utilizing the methods and gene delivery vectors provided herein a wide variety of diseases of the eye may be readily treated or prevented, including for example, glaucoma, macular degeneration, diabetic retinopathies, inherited retinal degeneration such as retinitis pigmentosa, retinal detachment or injury and retinopathies (whether inherited, induced by surgery, trauma, a toxic compound or agent, or, photically). Similarly, a wide variety of neurotrophic factors may be utilized (either alone or in combination) within the context of the present invention, including for example, NGF, BDNF, CNTF, NT-3, NT-4, FGF-2, FGF-5, FGF-18, FGF-20 and FGF-21.

Within certain embodiments of the invention, it is preferred that the gene delivery vector be utilized to deliver and express an anti-angiogenic factor for the treatment, prevention, or, inhibition of diabetic retinopathy, wet AMD, and other neovascular diseases of the eye (e.g., ROP). Within other embodiments it is desirable that the gene delivery vector be utilized to deliver and express a neurotrophic growth factor to treat, prevent, or, inhibit diseases of the eye, such as, for example, glaucoma, retinitis pigmentosa, and dry AMD. Within yet other embodiments, it may be desirable to utilize either a gene delivery vector which expresses both an anti-angiogenic molecule and a neurotrophic growth factor, or two separate vectors which independently express such factors, in the treatment, prevention, or inhibition of an eye disease (e.g., for diabetic retinopathy).

Within further embodiments of the invention, the above-mentioned methods utilizing gene delivery vectors may be administered along with other methods or therapeutic regimens, including for example, photodynamic therapy (e.g., for wet AMD), laser photocoagulation (e.g., for diabetic retinopathy and wet AMD), and intraocular pressure reducing drugs (e.g., for glaucoma).

Also provided by the present invention are isolated nucleic acid molecules comprising the sequence of FIG. 2, vectors which contain, and/or express this sequence, and host cells which contain such vectors.

Within further aspects of the present invention gene delivery vectors are provided which direct the expression of a neurotrophic factor, or, an anti-angiogenic factor. As noted above, representative examples of neurotrophic factors include NGF, BDNF, CNTF, NT-3, NT4, FGF-2, FGF-5, FGF-18, FGF-20 and FGF-21. Representative examples of anti-angiogenic factors include soluble Flt-l, soluble Tie-2 receptor, and PEDF. Representative examples of suitable gene delivery vectors include adenovirus, retroviruses (e.g., HIV or FIV-based vectors), alphaviruses, AAV vectors, and naked DNA vectors.

Within yet other aspects of the invention non-human animal models of neovascular diseases of the eye are provided, comprising an animal having an angiogenic (i.e., pro-angiogenic) transgene in the eye. Within various embodiments, the neovascularization may be retinal or choroidal neovascularization. Within other embodiments, the animal may be a mouse or rat. As noted herein, a wide variety of angiogenic transgenes may be utilized to generate the non-human animal model, including for example, angiogenic transgenes that encode VEGF and/or an angiopoietin such as angiopoietin-1.

Also provided are methods of making such non-human animal models comprising the general steps of administering to a non-human animal a gene delivery vector which directs the expression of an angiogenic transgene. As noted above, a wide variety of gene delivery vectors (e.g., rAV and rAAV) can be utilized, as well as nucleic acid molecules which encode the angiogenic transgene (e.g., nucleic acid molecules encoding VEGF or angiopoietin). Within certain embodiments, the gene delivery vector can be administered subretinally or intravitreally. Within further embodiments the animal model can be utilized as a model for Age-related Macular Degeneration (AMD), diabetic retinopathy, or, retinopathy of prematurity (ROP).

Also provided are methods for determining the ability of an anti-angiogenic factor to inhibit neovascularization of the eye, comprising the general steps of (a) administering to an animal model as described herein an anti-angiogenic factor, and (b) determining the ability of the anti-angiogenic factor to inhibit neovascularization of the eye. As noted herein, the anti-angiogenic factor may be administered by a variety of routes, including for example, topically, subretinally, or, intravitreally. Further, the animal model may be utilized to test the efficacy of drugs, compounds, or other factors or agents for a wide variety of eye-related neovascular diseases (including AMD and ROP).

DETAILED DESCRIPTION OF THE INVENTION

A. Gene Delivery Vectors

1. Construction of Retroviral Gene Delivery Vectors

Within one aspect of the present invention, retroviral gene delivery vectors are provided which are constructed to carry or express a selected gene(s) or sequence(s) of interest. Briefly, retroviral gene delivery vectors of the present invention may be readily constructed from a wide variety of retroviruses, including for example, B, and D type retroviruses as well as spumaviruses and lentiviruses (see RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985). Such retroviruses may be readily obtained from depositories or collections such as the American Type Culture Collection ("ATCC"; Rockville, Md.), or isolated from known sources using commonly available techniques.

Any of the above retroviruses may be readily utilized in order to assemble or construct retroviral gene delivery vectors given the disclosure provided herein, and standard recombinant techniques (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Kunkel, PNAS 82:488, 1985). In addition, within certain embodiments of the invention, portions of the retroviral gene delivery vectors may be derived from different retroviruses. For example, within one embodiment of the invention, retrovirus LTRs may be derived from a Murine Sarcoma Virus, a tRNA binding site from a Rous Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.

Within one aspect of the present invention, retrovector constructs are provided comprising a 5′ LTR, a tRNA binding site, a packaging signal, one or more heterologous sequences, an origin of second strand DNA synthesis and a 3′ LTR, wherein the vector construct lacks gag/pol or env coding sequences.

Other retroviral gene delivery vectors may likewise be utilized within the context of the present invention, including for example EP 0,415,731; WO 90/07936; WO 91/0285, WO 9403622; WO 9325698; WO 9325234; U.S. Pat. No. 5,219,740; WO 9311230; WO 9310218; Vile and Hart, Cancer Res. 53:3860-3864, 1993; Vile and Hart, Cancer Res. 53:962-967, 1993; Ram et al., Cancer Res. 53:83-88, 1993; Takamniya et al., J. Neurosci. Res. 33:493-503, 1992; Baba et al., J. Neurosurg. 79:729-735, 1993 (U.S. Pat. No. 4,777,127, GB 2,200,651, EP 0,345,242 and WO91/02805).

Packaging cell lines suitable for use with the above described retrovector constructs may be readily prepared (see U.S. Ser. No. 08/240,030, filed May 9, 1994; see also U.S. Ser. No. 07/800,921, filed Nov. 27, 1991), and utilized to create producer cell lines (also termed vector cell lines or "VCLs") for the production of recombinant vector particles.

2. Recombinant Adeno-Associated Virus Vectors

As noted above, a variety of rAAV vectors may be utilized to direct the expression of one or more desired neurotrophic factors. Briefly, the rAAV should be comprised of, in order, a 5′ adeno-associated virus inverted terminal repeat, a transgene or gene of interest operatively linked to a sequence which regulates its expression in a target cell, and a 3′ adeno-associated virus inverted terminal repeat. In addition, the rAAV vector may preferably have a polyadenylation sequence.

Generally, rAAV vectors should have one copy of the AAV ITR at each end of the transgene or gene of interest, in order to allow replication, packaging, and efficient integration into cell chromosomes. The ITR consists of nucleotides 1 to 145 at the 5′ end of the AAV DNA genome, and nucleotides 4681 to 4536 (i.e., the same sequence) at the 3′ end of the AAV DNA genome. Preferably, the rAAV vector may also include at least 10 nucleotides following the end of the ITR (ie., a portion of the "D region").

Within preferred embodiments of the invention, the transgene sequence will be of about 2 to 5 kb in length (or alternatively, the transgene may additionally contain a "stuffer" or "filler" sequence to bring the total size of the nucleic acid sequence between the two ITRs to between 2 and 5 kb). Alternatively, the transgene may be composed of same heterologous sequence several times (e.g., two nucleic acid molecules which encode FGF-2 separated by a ribosome readthrough, or alternatively, by an Internal Ribosome Entry Site or "IRES"), or several different heterologous sequences (e.g., FGF-2 and FGF-5, separated by a ribosome readthrough or an IRES).

Recombinant AVV vectors of the present invention may be generated from a variety of adeno-associated viruses, including for example, serotypes 1 through 6. For example, ITRs from any AAV serotype are expected to have similar structures and functions with regard to replication, integration, excision and transcriptional mechanisms.

Within certain embodiments of the invention, expression of the transgene may be accomplished by a separate promoter (e.g., a viral promoter). Representative examples of suitable promoters in this regard include a CMV promoter, RSV promoter, SV40 promoter, or MoMLV promoter. Other promoters that may similarly be utilized within the context of the present invention include cell or tissue specific promoters (e.g., a rod, cone, or ganglia derived promoter), or inducible promoters. Representative examples of suitable inducible promoters include tetracycline-response promoters ("Tet", see, e.g., Gossen and Bujard, Proc. Natl. Acad. Sci. USA. 89:5547-5551, 1992; Gossen et al., Science 268, 1766-1769, 1995; Baron et al., Nucl. Acids Res. 25:2723-2729, 1997; Blau and Rossi, Proc. Natl. Acad. Sci. USA. 96:797-799, 1999; Bohl et al., Blood 92:1512-1517, 1998; and Haberman et al., Gene Therapy 5:1604-1611, 1998), the ecdysone system (see e.g., No et al., Proc. Natl. Acad. Sci. USA. 93:3346-3351, 1996), and other regulated promoters or promoter systems (see, e.g., Rivera et al., Nat. Med. 2:1028-1032, 1996;).

The rAAV vector may also contain additional sequences, for example from an adenovirus, which assist in effecting a desired function for the vector. Such sequences include, for example, those which assist in packaging the rAAV vector in adenovirus-associated virus particles.

Packaging cell lines suitable for producing adeno-associated viral vectors may be readily accomplished given readily available techniques (see e.g., U.S. Pat. No. 5,872,005).

Particularly preferred methods for constructing and packaging rAAV vectors are described in more detail below in Examples 1, 2, 3, and 4.

3. Alphavirus Delivery Vectors

The present invention also provides a variety of Alphavirus vectors which may function as gene delivery vectors. For example, the Sindbis virus is the prototype member of the alphavirus genus of the togavirus family. The unsegmented genomic RNA (49S RNA) of Sindbis virus is approximately 11,703 nucleotides in length, contains a 5′ cap and a 3′ poly-adenylated tail, and displays positive polarity. Infectious enveloped Sindbis virus is produced by assembly of the viral nucleocapsid proteins onto the viral genomic RNA in the cytoplasm and budding through the cell membrane embedded with viral encoded glycoproteins. Entry of virus into cells is by endocytosis through clatharin coated pits, fusion of the viral membrane with the endosome, release of the nucleocapsid, and uncoating of the viral genome. During viral replication the genomic 49S RNA serves as template for synthesis of the complementary negative strand. This negative strand in turn serves as template for genomic RNA and an internally initiated 26S subgenomic RNA. The Sindbis viral nonstructural proteins are translated from the genomic RNA while structural proteins are translated from the subgenomic 26S RNA. All viral genes are expressed as a polyprotein and processed into individual proteins by post translational proteolytic cleavage. The packaging sequence resides within the nonstructural coding region, therefore only the genomic 49S RNA is packaged into virions.

Several different Sindbis vector systems may be constructed and utilized within the present invention. Representative examples of such systems include those described within U.S. Pat. Nos. 5,091,309 and 5,217,879, and PCT Publication No. WO 95/07994.

4. Other Viral Gene Delivery Vectors

In addition to retroviral vectors and alphavirus vectors, numerous other viral vectors systems may also be utilized as a gene delivery vector. Representative examples of such gene delivery vectors include viruses such as pox viruses, such as canary pox virus or vaccinia virus (Fisher-Hoch et al., PNAS 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad. Sci. 569:86-103. 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330 and 5,017,487; WO 89/01973); SV40 (Mulligan et al., Nature 277:108-114, 1979); influenza virus (Luytjes et al., Cell 59:1107-1113, 1989; McMicheal et al., N. Eng. J. Med. 309:13-17, 1983; and Yap et al., Nature 273:238-239, 1978); herpes (Kit, Adv. Exp. Med. Biol. 215:219-236, 1989; U.S. Pat. No. 5,288,641); HIV (Poznansky, J. Virol. 65:532-536, 1991); measles (EP 0 440,219); Semliki Forest Virus, and coronavirus, as well as other viral systems (e.g., EP 0,440,219; WO 92/06693; U.S. Pat. No. 5,166,057). In addition, viral carriers may be homologous, non-pathogenic(defective), replication competent virus (e.g., Overbaugh et al., Science 239:906-910,1988), and nevertheless induce cellular immune responses, including CTL.

5. Non-viral Gene Delivery Vectors

In addition to the above viral-based vectors, numerous non-viral gene delivery vectors may likewise be utilized within the context of the present invention. Representative examples of such gene delivery vectors include direct delivery of nucleic acid expression vectors, naked DNA alone (WO 90/11092), polycation condensed DNA linked or unlinked to killed adenovirus (Curiel et al., Hum. Gene Ther. 3:147-154, 1992), DNA ligand linked to a ligand with or without one of the high affinity pairs described above (Wu et al., J. of Biol. Chem 264:16985-16987, 1989), nucleic acid containing liposomes (e.g., WO 95/24929 and WO 95/12387) and certain eukaryotic cells (e.g., producer cells—see U.S. Ser. No. 08/240,030, filed May 9, 1994, and U.S. Ser. No. 07/800,921).

B. Neurotrophic Factors

As noted above, the term neurotrophic factor refers to proteins which are responsible for the development and maintenance of the nervous system. Representative examples of neurotrophic factors include NGF, BDNF, CNTF, NT-3, NT4, and Fibroblast Growth Factors.

Fibroblast Growth Factor refers to a family of related proteins, the first of which was isolated from the pituitary gland (see Gospodarowicz, D., Nature, 249:123-127, 1974). From this original FGF (designated basic FGF) a family of related proteins, protein muteins, and protein analogs have been identified (see, e.g., U.S. Pat. Nos. 4,444,760, 5,155,214, 5,371,206, 5,464,774, 5,464,943, 5,604,293, 5,731,170, 5,750,365, 5,851,990, 5,852,177, 5,859,208, and 5,872,226; see generally Baird and Gospodarowicz, D. Ann N.Y. Acad. Sci. 638:1, 1991. The first two members of the family to be identified were acidic fibroblast growth factor (aFGF/FGF-1) and basic fibroblast growth factor (bFGF/FGF-2). Additional members of the FGF family include: i-nt-2/FGF-3, (Smith et al., EMBO J. 7: 1013, 1988); FGF-4 (Delli-Bovi et al., Cell 50: 729, 1987); FGF-6 (Marics et al., Oncogene 4: 335 (1989); keratinocyte growth factor/FGF-7, (Finch et al., Science 245: 752, 1989); FGF-8 (Tanaka et al., Proc. Natl. Acad Sci. USA 89: 8928, 1992); and FGF-9 (Miyamoto et al., Mol. Cell Biol. 13: 4251, 1993).

FGF-5 was originally isolated as an oncogene. See Goldfarb et al. U.S. Pat. Nos. 5,155,217 and 5,238,916, Zhan et al. "Human Oncogene Detected by a Defined Medium Culture Assay" (Oncogene 1:369-376, 1987), Zhan et al. "The Human FGF-5 Oncogene Encodes a Novel Protein Related to Fibroblastic Growth Factors" (Molecular and Cellular Biology 8:3487-3495, 1988), and Bates et al. "Biosynthesis of Human Fibroblast Growth Factor 5": (Molecular and Cellular Biology 11:1840-1845, 1991).

Other FGFs include those disclosed in U.S. Pat. Nos. 4,444,760, 5,155,214, 5,371,206, 5,464,774, 5,464,943, 5,604,293, 5,731,170, 5,750,365, 5,851,990, 5 5,852,177, 5,859,208, and 5,872,226. 5,852,177, and 5,872,226, as well as FGF-20 (U.S. Provisional Application No. 60/161,162) and FGF-21 (U.S. Provisional Application No. 60/166,540).

C. Anti-Angiogenic Factors

A wide variety of anti-angiogenic factors may also be expressed from the gene delivery vectors of the present invention, including for example, Angiostatin (O'Reilly et al., Cell 79:315-328, 1994; O'Reilly et al., Nat. Med. 2:689-92, 1996; Sim et al., Cancer Res. 57:1329-34, 1997), 1,25-Di-hydroxy-vitamn D₃ (Shibuya et al., Oncogene 5:519-24, 1990; Oikawa et al., Eur. J. Pharmacol. 178:247-50, 1990; and 182:616, 1990), Endostatin (O'Reilly et al., Cell 88:277-85, 1997), Interferons alpha and beta (Sidky et al., Cancer Res. 47:5155-61, 1987; Singh et al., Proc. Natl. Acad. Sci. USA 92:4562-6, 1995), Interferon gamma (Friesel et al., J. Cell. Biol. 104:689-96, 1987), IGF-1 receptor antagonists, Interferon gamma-inducible protein IP-10 (Arenberg et al., J. Exp. Med. 1996;184:981-92; Strieter et al., J. Leukoc. Biol. 1995;57:752-62; Angiolillo et al., J. Exp. Med. 182:155-62, 1995), Interleukin 1alpha and beta (Cozzolino et al., Proc. Natl. Acad. Sci. USA 87:6487-91, 1990), Interleukin 12 (Kerbel and Hawley, J. Natl Cancer Inst. 87:557-9, 1995; Majewski et al., J. Invest. Dermatol 106:1114-8, 1996; Voest et al., J. Natl Cancer Inst. 87:581-6, 1995), 2-Methoxyestradiol (Fotsis et al., Nature 368:237-9, 1994), Platelet factor 4 (Taylor and Folkman, Nature 297:307-12, 1982; Gengrinovitch et al., J. Biol. Chem 270:15059-65, 1995), Prolactin (16 kd fragment) (Clapp et al., Endocrinology 133:1292-9, 1993; Ferrara, Endocrinology 129:896-900, 1991), Protamin, Retinoic acid (Lingen et al., Lab. Invest 74:476-83, 1996), Thrombospondin-1 and 2 (Lawler, Blood, 67:1197-209, 1986; Raugi and Lovett, Am. J. Pathol 129:364-72, 1987; Volpert et al., Biochem. Biophys. Res. Commun 217:326-32, 1995), Tissue inhibitor of metalloproteinase-1 and -2 (Moses and Langer, J. Cell Biochem 47:230-5, 1991; Ray and Stetler-Stevenson, Eur. Respir. J. 7:2062-72, 1994), Transforming growth factor beta (RayChaudhury, J. Cell. Biochem 47:224-9, 1991; Roberts et al., Proc. Natl Acad. Sci USA 83:4167-71, 1986), and Tumor necrosis factor—alpha (Frater-Schroeder et al., Proc. Natl. Acad. Sci. USA 84:5277-81, 1987; Leibovich et al., Nature 329:630-2, 1987).

Other anti-angiogenic factors that can be utilized within the context of the present invention include VEGF antagonists such as soluble Fit-1 (Kendall and Thomas, PNAS 90: 10705, 1993), pigment epithelium-derived factor or "PEDF" (Dawson et al., Science 285:245, 1999), and Ang-1 antagonists such as soluble Tie-2 receptor (Thurston et al., Science 286:2511, 1999; see also, generally Aiello et al., PNAS 92:10457, 1995; Robinson et al., PNAS 93:4851, 1996; Seo et al., Am. J. Pathol. 154:1743, 1999).

The ability of a given molecule to be "anti-angiogenic" can be readily assessed utilizing a variety of assays, including for example, the HMVEC assay provided in

Example 15.

D. Method for Treating and/or Preventing Diseases of the Eye and Pharmaceutical Compositions

As noted above, the present invention provides methods which generally comprise the step of intraocularly administering a gene delivery vector which directs the expression of one or more neurotrophic factor to the eye, or an anti-angiogenic factor to the eye in order to treat, prevent, or inhibit the progression of an eye disease. As utilized herein, it should be understood that the terms "treated, prevented, or, inhibited" refers to the alteration of a disease course or progress in a statistically significant manner. Determination of whether a disease course has been altered may be readily assessed in a variety of model systems, discussed in more detail below, which analyze the ability of a gene delivery vector to delay, prevent or rescue photoreceptors, as well as other retinal cells, from cell death.

1. Diseases of the Eye

A wide variety of diseases of the eye may be treated given the teachings provided herein. For example, within one embodiment of the invention gene delivery vectors are administered to a patient intraocularly in order to treat or prevent macular degeneration. Briefly, the leading cause of visual loss in the elderly is macular degeneration (MD), which has an increasingly important social and economic impact in the United States. As the size of the elderly population increases in this country, age related macular degeneration (AMD) will become a more prevalent cause of blindness than both diabetic retinopathy and glaucoma combined. Although laser treatment has been shown to reduce the risk of extensive macular scarring from the "wet" or neovascular form of the disease, there are currently no effective treatments for the vast majority of patients with MD.

Within another embodiment, gene delivery vectors can be administered to a patient intraocularly in order to treat or prevent an inherited retinal degeneration. One of the most common inherited retinal degenerations is retinitis pigmentosa (RP), which results in the destruction of photoreceptor cells, and the RPE. Other inherited conditions include Bardet-Biedl syndrome (autosomal recessive); Congenital arnaurosis (autosomal recessive); Cone or cone-rod dystrophy (autosomal dominant and X-linked forms); Congenital stationary night blindness (autosomal dominant, autosomal recessive and X-linked forms); Macular degeneration (autosomal dominant and autosomal recessive forms); Optic atrophy, autosomal dominant and X-linked forms); Retinitis pigmentosa (autosomal dominant, autosomal recessive and X-linked forms); Syndromic or systemic retinopathy (autosomal dominant, autosomal recessive and X-linked forms); and Usher syndrome (autosomal recessive). This group of debilitating conditions affects approximately 100,000 people in the United States alone.

As noted above, within other aspects of the invention, gene delivery vectors which direct the expression of a neurotrophic growth factor can be administered to a patient intraocularly in order to treat or prevent glaucoma. Briefly, glaucoma is not a uniform disease but rather a heterogeneous group of disorders that share a distinct type of optic nerve damage that leads to loss of visual function. The disease is manifest as a progressive optic neuropathy that, if left untreated leads to blindness. It is estimated that as many as 3 million Americans have glaucoma and, of these, as many as 120,000 are blind as a result. Furthermore, it is the number one cause of blindness in African-Americans. Its most prevalent form, primary open-angle glaucoma, can be insidious. This form usually begins in midlife and progresses slowly but relentlessly. If detected early, disease progression can frequently be arrested or slowed with medical and surgical treatment. Representative factors that may be expressed from the vectors of the present invention to treat glaucoma include neurotrophic growth factors such as FGF-2, 5, 18, 20, and, 21.

Within yet other embodiments gene delivery vectors can be administered to a patient intraocularly in order to treat or prevent injuries to the retina, including retinal detachment, photic retinopathies, surgery-induced retinopathies, toxic retinopathies, retinopathies due to trauma or penetrating lesions of the eye.

As noted above, the present invention also provides methods of treating, preventing, or, inhibiting neovascular disease of the eye, comprising the step of administering to a patient a gene delivery vector which directs the expression of an anti-angiogenic factor. Representative examples of neovascular diseases include diabetic retinopathy, AMD (wet form), and retinopathy of prematurity. Briefly, choroidal neovascularization is a hallmark of exudative or wet Age-related Macular Degeneration (AMD), the leading cause of blindness in the elderly population. Retinal neovascularization occurs in diseases such as diabetic retinapathy and retinopathy of prematurity (ROP), the most common cause of blindness in the young.

Particularly preferred vectors for the treatment, prevention, or, inhibition of neovascular diseases of the eye direct the expression of an anti-angiogenic factor such as, for example, soluble tie-2 receptor or soluble FIt-1.

2. Animal Models

In order to assess the ability of a selected gene therapy vector to be effective for treating diseases of the eye which involve neovascularization, a novel model for neovascularization (either choroidal or subretinal) can be generated by subretinal injection of a recombinant virus (e.g., rAV or rAAV) containing an angiogenic transgene such as VEGF and/or angiopoietin. Within certain embodiments, an angiogenic transgene such as angiopoietin-l can be used in combination with another factor such as VEGF, in order to generate neovascularization. The extent and duration of neovascularization induced by the gene delivery vectors containing an angiogenic transgene such as VEGF can be determined using fundus photography, fluorescein angiography and histochemistry.

To assess the ability of anti-angiogenic molecules to prevent neovascularization in the model described above, a D10-sFlt-1 rAAV (or other gene delivery vector which directs the expression of an anti-angiogenic factor) is intraocularly injected, either by subretinal or intravitreal routes of injection. Generally, subretinal injection of the gene delivery vector may be utilized to achieve delivery to both the choroidal and inner retinal vasculature. Intravitreal injection can be utilized to infect Muller cells and retinal ganglion cells (RGCs), which deliver anti-angiogenic protein to the retinal vasculature. Muller cells span the retina and would secrete the therapeutic protein into the subretinal space.

Such injections may be accomplished either prior to, simultaneous with, or subsequent to administration of an angiogenic factor or gene delivery vector which expresses an angiogenic factor. After an appropriate time interval, inhibition of neovascularization can be determined using fundus photography, fluorescein angiography and/or histochemistry.

While there are many animal models of retinal neovascularization such as oxygen-induced ischemic retinopathy (Aiello et al., PNAS 93: 4881, 1996.) and the VEGF transgenic mouse (Okamoto et al., Am. J. Pathol. 151: 281, 1997), there are fewer models of choroidal neovascularization (e.g., laser photocoagulation as described by Murata et al., IOVS 39: 2474, 1998). Subretinal neovascularization from the retinal rather than choroidal blood supply is also observed in VEGF transgenic animals (Okamoto et al., Am. J. Pathol. 151: 281, 1997). Hypoxic stimulation of VEGF expression is known to correlate with neovascularization in human ocular disease.

The pathologic hallmark of glaucomatous optic neuropathy is the selective death of retinal ganglion cells (RGCs) (Nickells, R. W., J. Glaucoma 5:345-356. 1996; Levin, L. A. and Louhab, A., Arch. Ophihalmol. 114:488-491, 1996.; Kerrigan, L. A., Zack, D. J., Quigley, H. A., Smith, S. D. and Pease, M. E., Arch. Ophihalmol. 115:1031-1035, 1997). Recent studies indicate that RGCs die with characteristics of apoptosis after injury to the axons of adult RGCs such as axotomy of the optic nerve (ON), and in glaucoma and anterior ischemic optic neuropathy in humans (Nickells, 1996). Thus, damage to the optic nerve by axotomy is used by many researchers as a model for selective apoptotic cell death of adult RGCs.

The loss of-RGCs caused by ON transection in adult mammals varies from 50% to more than 90% depending on the techniques used to identify RGCs, the proximity of the lesion to the eye, and the age and species of the animal. For example, in a study in adult rats, in which retrogradely transported tracers were used to distinguish RGCs from displaced amacrine cells (Villegas-Pérez, M. P., Vidal-Sanz, M., Bray, G. M. and Aguayo, A. J., J. Neurosci. 8:265-280, 1988). ON transection near the eye (0.5-1 mm) leads to the loss of more than 90% of the RGCs by 2 weeks. In contrast, in adult animals in which the ON was cut nearly 10 mm from the eye, 54% of RGCs survived by 3 months (Richardson, P. M., Issa, V. M. K. and Shemie, S., J. Neurocytol. 11:949-966, 1982.).

Briefly, the posterior pole of the left eye and the origin of the optic nerve (ON) are exposed through a superior temporal intraorbital approach. A longitudinal excision of the ON dural sheath is performed. The ON is then gently separated from the dorsal aspect of this sheath and completely transected within the orbit, within 1 mm of the optic disc. Care is taken to avoid damage to the ophthalmic artery, which is located on the inferomedial dural sheath of the ON.

RGC survival and death following gene delivery can also be examined using two alternative models of ON injury: 1) ON crush; and (2) increased intraocular pressure. In the first model the ON is exposed, and then clamped at a distance of about one millimeter from the posterior pole using a pair of calibrated forceps as previously described (Li et al., Invest. Ophihalmol. Vis. Sci. 40:1004, 1999). In the second model, chronic moderately elevated intraocular pressure can be produced unilaterally by cauterization of three episcieral vessels as described by Neufeld et al. in PNAS 17:9944, 1999).

A variety of animal models can be utilized for photoreceptor degeneration, including the RCS rat model, P23H transgenic rat model, the rd mouse, and the S334ter transgenic rat model.

Briefly, in the S334ter transgenic rat model, a mutation occurs resulting in the truncation of the C-terminal 15 amino acid residues of rhodopsin (a seven-transmembrane protein found in photoreceptor outer segments, which acts as a photopigment). The S334ter mutation is similar to rhodopsin mutations found in a subset of patients with retinitis pigmentosa (RP). RP is a heterogeneous group of inherited retinal disorders in which individuals experience varying rates of vision loss due to photoreceptor degeneration. IN many RP patients, photoreceptor cell death progresses to blindness. Transgenic S334ter rats are born with normal number of photoreceptors. The mutant rhodopsin gene begins expression at postnatal day 5 in the rat, and photoreceptor cell death begins at postnatal day 10-15. In transgenic line S334ter-3 , approximately 70% of the outer nuclear layer has degenerated by day 60 in the absence of any therapeutic intervention. The retinal degeneration in this model is consistent from animal to animal and follows a predictable and reproducible rate. This provides an assay for therapeutic effect by morphological examination of the thickness of the photoreceptor nuclear layer and comparison of the treated eye to the untreated (contralateral) eye in the same individual animal.

S334ter rats are utilized as a model for RP as follows. Briefly, S334ter transgenic rats are euthanized by overdose of carbon dioxide inhalation and immediately perfused intracardially with a mixture of mixed aldehydes (2% formaldehyde and 2.5 % glutaraldehyde). Eyes are removed and embedded in epoxy resin, and 1 μm thick histological sections are made along the vertical meridian. Tissue sections are aligned so that the ROS and Miller cell processes crossing the inner plexiform layer are continuous throughout the plane of section to assure that the sections are not oblique, and the thickness of the ONL and lengths of RIS and ROS are measured. These retinal thickness measurements are plotted and establish the baseline retinal degeneration rates for the animal model. The assessment of retinal thickness is as follows: briefly, 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 retina. The greatest 3 contiguous values for ONL thickness in each retina is also compared in order to determine if any region of retina (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.

One particularly preferred line of transgenic rats, TgN(s334ter) linc 4 (abbreviated s334ter 4) can be utilized for in vivo experiments. Briefly, in this rat model expression of the mutated opsin transgene begins at postnatal day P5 in these rats, leading to a gradual death of photoreceptor cells. These rats develop an anatomically normal retina up to P15, with the exception of a slightly increased number of pyknotic photoreceptor nuclei in the outer nuclear layer (ONL) than in non-transgenic control rats. In this animal model , the rate of photoreceptor cell death is approximately linear until P60, resulting in loss of 40-60% 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 remaining.

3. Methods of Administration

Gene delivery vectors of the present invention may be administered intraocularly to a variety of locations depending on the type of disease to be treated, prevented, or, inhibited, and the extent of disease. Examples of suitable locations include the retina (e.g., for retinal diseases), the vitreous, or other locations in or adjacent to the eye.

Briefly, the human retina is organized in a fairly exact mosaic. In the fovea, he mosaic is a hexagonal packing of cones. Outside the fovea, the rods break up the close hexagonal packing of the cones but still allow an organized architecture with cones rather evenly spaced surrounded by rings of rods. Thus in terms of densities of the different photoreceptor populations in the human retina, it is clear that the cone density is highest in the foveal pit and falls rapidly outside the fovea to a fairly even density into the peripheral retina (see Osterberg, G. (1935) Topography of the layer of rods and cones in the human retina. Acta Ophthal. (suppl.) 6, 1-103; see also Curcio, C. A., Sloan, K. R., Packer, O., Hendrickson, A. E. and Kalina, R. E. (1987) Distribution of cones in human and monkey retina: individual variability and radial asymmetry. Science 236, 579-582).

Access to desired portions of the retina, or to other parts of the eye may be readily accomplished by one of skill in the art (see, generally Medical and Surgical Retina: Advances, Controversies, and Management, Hilet Lewis, Stephen J. Ryan, Eds., medical illustrator, Timothy C. Hengst. St. Louis: Mosby, c1994. xix, 534; see also Retina,Stephen J. Ryan, editor in chief,. 2nd ed., St. Louis, Mo.: Mosby, c1994. 3 v. (xxix. 2559 p.).

The amount of the specific viral vector applied to the retina is uniformly quite small as the eye is a relatively contained structure and the agent is injected directly into it. The amount of vector that needs to be injected is determined by the intraocular location of the chosen cells targeted for treatment. The cell type to be transduced will be determined by the particular disease entity that is to be treated.

For example, a single 20-microliter volume (of 10¹³ physical particle titer/ml rAAV) may be used in a subretinal injection to treat the macula and fovea. A larger injection of 50 to 100 microliters may be used to deliver the rAAV to a substantial fraction of the retinal area, perhaps to the entire retina depending upon the extent of lateral spread of the particles.

A 100-ul injection will provide several million active rAAV particles into the subretinal space. This calculation is based upon a titer of 10¹³ physical particles per milliliter. Of this titer, it is estimated that 1/1000 to 1/10,000 of the AAV particles are infectious. The retinal anatomy constrains the injection volume possible in the subretinal space (SRS). Assuming an injection maximum of 100 microliters, this would provide an infectious titer of 10⁸ to 10⁹ rAAV in the SRS. This would have the potential of infecting all of the ˜150×10⁶ photoreceptors in the entire human retina with a single injection.

Smaller injection volumes focally applied to the fovea or macula may adequately transfect the entire region affected by the disease in the case of macular degeneration or other regional retinopathies.

Gene delivery vectors can alternately be delivered to the eye by intraocular injection into the vitreous. In this application, the primary target cells to be transduced are the retinal ganglion cells, which are the retinal cells primarily affected in glaucoma. In this application, the injection volume of the gene delivery vector could be substantially larger, as the volume is not constrained by the anatomy of the subretinal space. Acceptable dosages in this instance can range from 25 ul to 1000 ul.

4. Assays

A wide variety of assays may be utilized in order to determine appropriate dosages for administration, or to assess the ability of a gene delivery vector to treat or prevent a particular disease. Certain of these assays are discussed in more detail below.

a. Electroretinographic Analysis

Electroretinographic analysis can be utilized to assess the effect of gene delivery administration into the retina. Briefly, rats are dark adapted overnight and then in dim red light, then anesthetized with intramuscular injections of xylazine (13 mg/kg) and ketamine (87 mg/kg). Full-field scotopic ERGs are elicited with 10-μsec flashes of white light and responses were recorded using a UTAS-E 2000 Visual Electrodiagnostic System (LKC Technologies, Inc., Gaithersburg, Md.). The corneas of the rats are the anesthetized with a drop of 0.5% proparacaine hydrochloride, and the pupils dilated with 1% atropine and 2.5% phenylephrine hydrochloride. Small contact lenses with gold wire loops are placed on both corneas with a drop of 2.5% methylcellulose to maintain corneal hydration. A silver wire reference electrode is placed subcutaneously between the eyes and a ground electrode is placed subcutaneously in the hind leg. Stimuli are presented at intensities of -1.1, 0.9 and 1.9 log cd m^-2 at 10-second, 30-second and 1-minute intervals, respectively. Responses are amplified at a gain of 4,000, filtered between 0.3 to 500 Hz and digitized at a rate of 2,000 Hz on 2 channels. Three responses are averaged at each intensity. The a-waves are measured from the baseline to the peak in the cornea-negative direction, and b-waves are 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 are averaged for a given animal.

b. Retinal Tissue Analysis

As described in more detail above and below, retinal tissue analysis can also be utilized to assess the effect of gene delivery administration into the retina.

5. Pharmaceutical Compositions

Gene delivery vectors may be prepared as a pharmaceutical product suitable for direct administration. Within preferred embodiments, the vector should be admixed with a pharmaceutically acceptable carrier for intraocular administration. Examples of suitable carriers are saline or phosphate buffered saline.

Deposit Information

The following material was deposited with the American Type Culture Collection:

The above material was deposited by Chiron Corporation with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209, telephone 703-365-2700. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for purposes of Patent Procedure. The deposit will be maintained for a period of 30 years following issuance of this patent, or for the enforceable life of the patent, whichever is greater. Upon issuance of the patent, the deposits will be available to the public from the ATCC without restriction.

This deposit is provided merely as a convenience to those of skill in the art, and is not an admission that a deposit is required under 35 U.S.C. §112. The nucleic acid sequence of this deposit, as well as the amino acid sequence of the polypeptide encoded thereby, are incorporated herein by reference and should be referred to in the event of an error in the sequence described herein. A license may be required to make, use, or sell the deposited materials, and no such license is granted hereby.
 

Claim 1 of 3 Claims

1. A method of inhibiting angiogenesis in a diseased eye of a subject, comprising, administering intraocularly a recombinant adeno-associated virus (rAAV) gene delivery vector which directs the expression of an anti angiogenic factor, such that administration of said vector inhibits neovascularization of the diseased eye.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.