June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Advances towards a dual AAV CRISPR-Cas9-based ‘Knockout and Replace’ strategy to treat rhodopsin-associated autosomal dominant Retinitis Pigmentosa
Author Affiliations & Notes
  • Mariacarmela Allocca
    Editas Medicine, Cambridge, Massachusetts, United States
  • Deepak Reyon
    Editas Medicine, Cambridge, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Mariacarmela Allocca, Editas Medicine (E); Deepak Reyon, Editas Medicine (E)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 1474. doi:
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      Mariacarmela Allocca, Deepak Reyon; Advances towards a dual AAV CRISPR-Cas9-based ‘Knockout and Replace’ strategy to treat rhodopsin-associated autosomal dominant Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2021;62(8):1474.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Mutations in the rhodopsin (RHO) gene are the most common cause of autosomal dominant Retinitis Pigmentosa (adRP), a retinal disease that results in blindness due to photoreceptor degeneration. Over 150 autosomal dominant mutations have been identified in the RHO gene. Therefore, we are developing a CRISPR-Cas9-based mutation-independent therapeutic strategy that simultaneously ‘knocks out’ endogenous RHO, and ‘replaces’ it with exogenous functional RHO using a dual AAV system.

Potent, highly specific guide RNAs and an optimized RHO expression vector have been identified and used in studies to address the following questions:

1.Could the novel alleles generated as a consequence of on-target editing be dominant negative?
2.What is the optimal ratio of the two AAV vectors?

Methods : An in vitro overexpression system was developed to quantify the effect of novel, editing-induced, alleles on cell viability. Three novel alleles were tested, one in each open-reading-frame as an indicator of potential novel alleles generated by on-target editing. The system was benchmarked using RHO-P23H as the dominant negative allele and WT RHO as the normal allele. To confirm in vitro results, and to identify the optimal ratio of the two AAVs, the dual AAV system was tested at multiple ratios in humanized RHO mice. The optimal ratio of vectors and percentage of potential novel deleterious alleles was determined by quantifying on-target editing by NGS at 6 and 13-weeks post subretinal injection.

Results : Examination of the on-target editing site in humanized mice by NGS revealed low percentage of potential deleterious allele and a consistent INDEL profile at 6 and 13-weeks post injection, demonstrating that the risk of generating novel dominant negative alleles is minimal. Different ratios of dual AAV vectors showed corresponding change in the level of RHO KO and replacement. The configuration that provides the highest level of RHO replacement and sufficient on-target editing has been identified as the ‘optimal ratio’ for future studies.

Conclusions : We have demonstrated minimal risk of generating novel dominant negative alleles and the feasibility of achieving therapeutically relevant levels of editing and replacement using an optimized ratio of the two AAV vectors. We are moving forward to preclinical studies for further characterization of dual AAV system in vivo.

This is a 2021 ARVO Annual Meeting abstract.

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