AAV mediated delivery of Exon-specific U1 snRNA corrects ELP1 splicing and prevents retinal degeneration in familial dysautonomia

Abstract

Purpose : Familial dysautonomia (FD) is an autosomal recessive neurodegenerative disease caused by a splicing mutation in the gene encoding Elongator complex protein 1 (ELP1, also known as IKBKAP). A T-to-C base change in the 5’ splice site of ELP1 exon 20 results in exon 20 skipping with tissue specific reduction of ELP1 protein predominantly in the central and peripheral nervous system. In addition to a complex neurological phenotype, FD patients exhibit progressive retinal degeneration that severely affects their quality of life. To rescue retinal degeneration in FD, we have developed a novel splicing-targeted therapeutic approach based on Exon specific U1 small nuclear RNA (ExspeU1 snRNA)). Using a phenotypic mouse model, TgFD9; Ikbkap^D20/flox, we have tested the therapeutic efficacy of ExspeU1 to correct ELP1 mis-splicing and rescue of retinal degeneration.

Methods : Adeno Associated Vector expressing ExspeU1 was delivered to the retina (AAV2- ExspeU1) by Intravitreal injection. RT-PCR analysis was performed to analyze ELP1 splicing in the retina. Optical Coherence Tomography (OCT) analysis was used to evaluate the thickness of the retinal layers. Retinal whole-mount staining was performed to count the number of retinal ganglion cells (RGCs), and immunohistochemical staining was performed to detect the transduction of AAV2- ExspeU1 in the retina.

Results : To restore ELP1 splicing defect and prevent RGC loss in FD, we have designed a novel splice targeted therapy using ExSpeU1s that permits targeted binding to intronic sequences downstream of the mutant 5' splice site enhancing recruitment of the spliceosomal machinery. In vivo delivery of AAV2- ExspeU1 to the retina showed broader transduction throughout the retina, predominantly in RGCs. Our findings indicated a significant improvement in ELP1 splicing correction in the retina in AAV2- ExspeU1 injected mice (n=6) compared to sham injected mice (p<0.001) and support the therapeutic potential of an AAV2- ExspeU1 based treatment to rescue RGC loss in FD.

Conclusions : Our findings demonstrate that delivery of AAV2- ExspeU1 to the retina in FD mice by intravitreal injection corrected the ELP1 splicing defect and therfore it has the potential to rescue the retinal degeneration observed in FD patients. These findings highlight the therapeutic value of AAV2- ExspeU1 delivery to treat retinal degeneration in FD.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.