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AAV vectors for treatment of dominant retinitis pigmentosa

A vector and sequence technology, applied in the field of AAV vector for the treatment of dominant retinitis pigmentosa, can solve problems such as blindness and lack of approval

Active Publication Date: 2019-01-04
UNIV OF FLORIDA RES FOUNDATION INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

More than 100 different mutations identified in rho cause blindness
There are currently no approved drug or gene therapy treatments for adRP

Method used

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  • AAV vectors for treatment of dominant retinitis pigmentosa
  • AAV vectors for treatment of dominant retinitis pigmentosa
  • AAV vectors for treatment of dominant retinitis pigmentosa

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Example 1: Identification of short interfering RNA (siRNA) knocking down RHO

[0104] Using short interfering RNA (siRNA) design principles described by Khvorova and colleagues 4，5 , 14 siRNAs were designed to specifically cleave dog siRNAs, 12 of which also targeted human rhodopsin mRNA. Prior to proceeding, positions 2 to 19 of siRNAs were screened against the NCBI Human RefSeq database using the NCBI Blast utility (blast.ncbi.nlm.nih.gov / Blast.cgi). Two potential siRNAs were excluded because they closely matched other genes likely to be expressed in the retina. RNA versions of the 10 siRNAs were ordered from GE Healthcare Dharmacon together with non-targeting siRNAs used as controls. These were expressed in conjunction with green fluorescent protein (GFP, Figure 11The exemplary plasmid profile depicted in ) was tested in cells fused to human RHO and the reduction in green fluorescent cells was measured by fluorescence activated cell sorting (FACS). The rationa...

Embodiment 2

[0111] Example 2: RHO + / + Analysis of RHO KD in dogs

[0112] Dog 2190 (rcd1 carrier) received subretinal injections of AAV2 / 5-sc-H1-shRNA-Rho131 at the concentrations listed in Table 6. Dogs were terminated 8 weeks after injection. Several 3 mm neuroretinal biopsy punches were collected from both the bleb and non-bleb areas of each eye.

[0113] Table 6: Dog 2190

[0114]

[0115] Some non-limiting examples of the AAV2 / 5-sc-H1-shRNA constructs described herein may also be referred to as AAV2 / 5-sc-MOP500rGFP-shRNA constructs. AAV2 / 5 means that the shRNA-encoding nucleic acid is flanked by the AAV2 ITRs and is provided in rAAV particles containing the AAV5 capsid protein. In some embodiments, any interfering RNA described herein and any recombinant RHO gene described herein can be present on the same AAV nucleic acid (e.g., flanked by AAV2 ITRs) in an rAAV particle (e.g., comprising an AAV5 capsid protein). ) available on . In some embodiments, any of the interfering...

Embodiment 3

[0121] Example 3: RHO + / + Further analysis of RHO KD in dogs

[0122] Dog 2194 (rcd1 carrier) received subretinal injections of AAV2 / 5-sc-H1-shRNA-Rho820 at the concentrations listed in Table 7. Dogs were terminated 8 weeks after injection. Several 3 mm neuroretinal biopsy punches were collected from both the bleb and non-bleb areas of each eye.

[0123] Table 7: Dog 2194

[0124]

[0125] Western blot analysis:

[0126] Two biopsy punches (representing bleb or non-bleb areas) from each eye were incubated in 50 [mu]l of Lewin's buffer solution A (containing protease inhibitors) for 15 minutes on ice. Samples were sonicated with 40% amplitude, 15secON / 10secOFF X8 pulses. Then, the samples were centrifuged and the pellet was discarded. The protein concentration in the supernatant was measured by the Bradford method. Immunoblot was performed on 1 μg of total protein to visualize rhodopsin (antibody used: Millipore MAB5356, in 1:1000 dilution in blocking buffer). An...

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Abstract

Aspects of the disclosure relate to methods and compositions for treating retinitis pigmentosa. In some aspects, the disclosure provides compositions and methods for delivering an interfering nucleicacid (for example an interfering RNA) to a subject in order to reduce expression of one or both alleles of an endogenous rho gene (for example a mutant rho allele associated with retinitis pigmentosa)in the subject. In some embodiments, a replacement rho gene that is resistant to the interfering nucleic acid also is delivered to the subject.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 302,122, filed March 1, 2016, and U.S. Provisional Patent Application No. 62 / 398,451, filed September 22, 2016, the contents of each of which are incorporated by reference in their entirety Incorporated into this article. [0003] governmental support [0004] This invention was made with government support under Grant No. R24-EY022012 awarded by the National Institutes of Health. The Government has certain rights in this invention. Background technique [0005] Autosomal dominant retinitis pigmentosa (adRP) is a blinding disease that affects 1 in 12,000 people. A substantial number of these individuals carry mutations in the gene for the light harvesting pigment protein rhodopsin (rho) in photoreceptor cells in the retina. The disease is dominant because inheritance of the mutated gene from either parent results in retinal degeneration...

Claims

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Application Information

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IPC IPC(8): C12N15/79C12N15/86C12N15/85C12N15/10C12N15/11C12N15/113C07H21/02
CPCC12N15/113C12N15/86C12N2310/14C12N2310/141C12N2330/51C12N2750/14143A61P27/02C12N2310/531C12N15/1138C12N2310/122C12N7/00
Inventor 阿尔弗雷德·S·莱温威廉·W·豪斯维特迈克尔·T·马森吉尔威廉·贝尔特兰古斯塔沃·D·阿吉雷阿图尔·西代西扬塞缪尔·雅各布森
Owner UNIV OF FLORIDA RES FOUNDATION INC
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