Delivery strategies for CRISPR/Cas genome editing tool for retinal dystrophies: challenges and opportunities

Abstract

CRISPR/Cas, an adaptive immune system in bacteria, has been adopted as an efficient and precise tool for site-specific gene editing with potential therapeutic opportunities. It has been explored for a variety of applications, including gene modulation, epigenome editing, diagnosis, mRNA editing, etc. It has found applications in retinal dystrophic conditions including progressive cone and cone-rod dystrophies, congenital stationary night blindness, X-linked juvenile retinoschisis, retinitis pigmentosa, age-related macular degeneration, leber's congenital amaurosis, etc. Most of the therapies for retinal dystrophic conditions work by regressing symptoms instead of reversing the gene mutations. CRISPR/Cas9 through indel could impart beneficial effects in the reversal of gene mutations in dystrophic conditions. Recent research has also consolidated on the approaches of using CRISPR systems for retinal dystrophies but their delivery to the posterior part of the eye is a major concern due to high molecular weight, negative charge, and in vivo stability of CRISPR components. Recently, non-viral vectors have gained interest due to their potential in tissue-specific nucleic acid (miRNA/siRNA/CRISPR) delivery. This review highlights the opportunities of retinal dystrophies management using CRISPR/Cas nanomedicine.

Keywords: CRISPR/Cas9; Gene editing; Non-viral nanocarriers; Retinal dystrophies.

Graphical abstract

Fig. 1

Various forms of CRISPR/Cas9 including plasmid, mRNA and RNPs that could be delivered to achieve significant gene editing to treat retinal dystrophies.

Fig. 2

Applications of CRISPR/Cas technology.

Fig. 3

VEGF A gene editing efficiency of Cas9 RNPs in retinal dystrophy in mice. (A) The overall study outline, herein, CNV model was developed in mice using laser followed by subretinal injection of VEGF A targeting Cas9 RNP. After 7 d injection of RPE complexes were analyzed for CNV area and deep sequencing was performed to evaluate gene editing in the targeted cells/tissues. Meanwhile, after 3 d injection of VEGFA ELISA was also performed. (B) Representative laser-induced CNV stained with isolectin B4 (IB4) in C57BL/6 J mice injected with the Rosa26-specific Cas9 RNP (as a control) or the VEGFA-RNP. The area of CNV is demonstrated as yellow line. (C) CNV area. (D) level of VEGF A in the CNV area. (E) Gene editing in terms of indel in RPE cells at VEGFA targeted site. Reprinted from , licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/legalcode). Copyright © 2017 the authors. Published by Cold Spring Harbor Laboratory Press.

Fig. 4

Schematic representation of nanomedicine trafficking in the treatment of retinal dystrophic conditions starting from (1) intravitreal injection, (2) diffusion through vitreous fluid, (3) cellular uptake, (4) endosomal escape and (5) interest specific gene editing.

Fig. 5

Non-viral vectors being explored for the delivery of CRISPR/Cas RNPs.

Fig. 6

In vivo gene editing efficiency of RNP loaded nanocapsule in mice.Graphicalillustration of the (a) TdTomato gene, (b) site of intravitreal injection in mice eye, (c) ATRA targeted nanocapsule. (d) post injection in vivo gene editing in RPE layer quantified in terms of TdTomato positive area (%) in different treatment groups, (e) mounted RPE layer of the mouse treated with PBS, RNP, NCs and NC-ATRA, herein black area represents TdTomato signals after 12 d treatment. Reprinted by permission from . Copyright ©2019 the authors under exclusive licence to Springer Nature Limited.

Fig. 7

Schematic representation of mRNA editing using Cas13b in retinal dystrophic conditions.