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RETGENTX Report Summary

Project ID: 282085
Funded under: FP7-IDEAS-ERC
Country: Italy

Final Report Summary - RETGENTX (Overcoming the challenge of large gene transfer for the therapy of inherited retinal diseases)

Inherited retinal diseases (IRDs) cause blindness in more than 2 million people worldwide (1). The majority are due to mutations in genes expressed in photoreceptors (PR) in the retina (2). Others and us have recently demonstrated the safety and efficacy of gene therapy for IRDs in patients with Leber congenital amaurosis (LCA)(3-8). One of the major limitations to extend this clinical success to other blinding conditions is that many are caused by mutations in genes with large (> 5 kb) coding sequences that exceed the cargo capacity of the most efficient gene transfer vector for PR, the adeno-associated virus (AAV). Conversely, vectors with larger cloning capacity like lentiviral (LV), helper-dependent adenoviral (HD-Ad) and herpesviral (HV) vectors have poor PR tropism.
This project aimed at overcoming the challenge of large gene delivery to PR for the treatment of common IRDs due to mutations in PR genes with large coding sequences such as Stargardt disease (STGD1), due to mutations in the ABCA4 gene (9) and Usher Syndrome type IB (USHIB), due to mutations in the MYO7A gene (2). To accomplish this we proposed to: i. identify large-capacity vector platforms with improved PR transduction efficiency, by screening a series of existing LV, Ad and HV vectors; ii. exploit the possibility of expanding AAV cargo capacity by generating dual AAV vectors each containing one of 2 halves of a large gene which is reconstituted upon AAV intermolecular concatemerization in the nucleus of target cells (10-12). We proposed to assess the efficiency of these vector platforms to transduce murine and porcine PR and then to use the platform with highest PR transduction efficiency to correct the retinal phenotype of murine models of common severe IRDs due to mutations in large genes.
We have evaluated the mouse retinal transduction profiles of vectors derived from 16 different Ad serotypes, 7 LV pseudotypes and from a bovine HV. Most of the vectors tested transduced efficiently the retinal pigment epithelium while their overall PR transduction efficiency appeared lower than the one of AAV8, that remains the golden standard among naturally occurring AAV serotypes for inherited PR diseases (13).
In parallel to the evaluation of these large-capacity vector platforms, we generated dual AAV vectors, which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We compared their transduction efficiency in vitro and in the retina. We found that dual AAV trans-splicing and hybrid AK approaches transduce mouse and pig PR, with a transduction efficiency significantly higher in the large cone-enriched pig retina than in mice. Notably, we found that subretinal administration of dual AAV trans-splicing and hybrid AK vectors encoding for the large ABCA4 and MYO7A genes resulted in significant improvement of the retinal phenotype of Abca4-/- and sh1 murine models, respectively (14, 15). Our initial studies, however, had also highlighted a critical issue associated with the use of dual AAV vectors: the production of truncated proteins from both the 5′- and 3’-half vectors. To solve this issue we have explored the use of various protein degradation signals, and we identified a signal that effectively mediates selective degradation of the truncated protein produced from the 5’-haf vector without affecting full-length protein production, thus improving the safety of the platform (16). Additionally, in view of a potential clinical translation of dual AAV-ABCA4 vectors for STGD1 treatment, we have worked on the identification of a more suitable promoter than the Rhodopsin promoter (RHO) we have initially used in our studies. Indeed, RHO is a rod-specific promoter, which is a desirable feature when treating the Abca4 -/- mouse retina where the majority of PR are rods. In contrast, in the human macula that is affected in STGD1 patients, both rod and cone PR are present and express ABCA4 (17), thus effective treatment would require the use of a promoter that targets both types of PR. To this aim, we compared the PR transduction properties of both the human G protein-coupled receptor kinase (GRK1) and interphotoreceptor retinoid binding protein (IRBP) promoters, which have been both described to drive high levels of combined rod and cone PR transduction in various species. We found that, although both promoters give comparable levels of PR transduction, higher levels of cone PR transduction can be achieved with the GRK1 promoter in pigs (16). In conclusion, we have developed dual AAV-based strategy for STGD1 and USH1B treatment and we are planning further clinical development of these gene therapy products.

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2. Dryja T, Retinitis Pigmentosa and stationary night blindness, in The Online Metabolic & Molecular Bases of Inherited Diseases C. Scriver, et al., Editors. 2001, McGraw-Hill: New York, NY. p. 5903-5933.
3. Bainbridge J W, Smith A J, Barker S S, et al. (2008) Effect of gene therapy on visual function in Leber's congenital amaurosis. N Engl J Med. 358(21): p. 2231-9.
4. Cideciyan A V, Hauswirth W W, Aleman T S, et al. (2009) Vision 1 year after gene therapy for Leber's congenital amaurosis. N Engl J Med. 361(7): p. 725-7.
5. Maguire A M, High K A, Auricchio A, et al. (2009) Age-dependent effects of RPE65 gene therapy for Leber's congenital amaurosis: a phase 1 dose-escalation trial. Lancet. 374(9701): p. 1597-605.
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7. Simonelli F, Maguire A M, Testa F, et al. (2010) Gene therapy for Leber's congenital amaurosis is safe and effective through 1.5 years after vector administration. Mol Ther. 18(3): p. 643-50.
8. Jacobson S G, Acland G M, Aguirre G D, et al. (2006) Safety of recombinant adeno-associated virus type 2-RPE65 vector delivered by ocular subretinal injection. Mol Ther. 13(6): p. 1074-84.
9. Allikmets R, Singh N, Sun H, et al. (1997) A photoreceptor cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy. Nat Genet. 15(3): p. 236-46.
10. Duan D, Sharma P, Yang J, et al. (1998) Circular intermediates of recombinant adeno-associated virus have defined structural characteristics responsible for long-term episomal persistence in muscle tissue. J Virol. 72(11): p. 8568-77.
11. Yan Z, Zhang Y, Duan D, et al. (2000) Trans-splicing vectors expand the utility of adeno-associated virus for gene therapy. Proc Natl Acad Sci U S A. 97(12): p. 6716-21.
12. Ghosh A, Yue Y, Lai Y, et al. (2008) A hybrid vector system expands adeno-associated viral vector packaging capacity in a transgene-independent manner. Mol Ther. 16(1): p. 124-30.
13. Puppo A, Cesi G, Marrocco E, et al. (2014) Retinal transduction profiles by high-capacity viral vectors. Gene Ther.
14. Trapani I, Colella P, Sommella A, et al. (2014) Effective delivery of large genes to the retina by dual AAV vectors. EMBO Mol Med. 6(2): p. 194-211.
15. Colella P, Trapani I, Cesi G, et al. (2014) Efficient gene delivery to the cone-enriched pig retina by dual AAV vectors. Gene Ther. 21(4): p. 450-6.
16. Trapani I, Toriello E, de Simone S, et al. (2015) Improved dual AAV vectors with reduced expression of truncated proteins are safe and effective in the retina of a mouse model of Stargardt disease. Hum Mol Genet. 24(23): p. 6811-25.
17. Molday L L, Rabin A R, and Molday R S (2000) ABCR expression in foveal cone photoreceptors and its role in Stargardt macular dystrophy. Nat Genet. 25(3): p. 257-8.

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