Recombinant Adeno-Associated Viral Vectors (rAAV)-Vector Elements in Ocular Gene Therapy Clinical Trials and Transgene Expression and Bioactivity Assays

Abstract

Inherited retinal dystrophies and optic neuropathies cause chronic disabling loss of visual function. The development of recombinant adeno-associated viral vectors (rAAV) gene therapies in all disease fields have been promising, but the translation to the clinic has been slow. The safety and efficacy profiles of rAAV are linked to the dose of applied vectors. DNA changes in the rAAV gene cassette affect potency, the expression pattern (cell-specificity), and the production yield. Here, we present a library of rAAV vectors and elements that provide a workflow to design novel vectors. We first performed a meta-analysis on recombinant rAAV elements in clinical trials (2007–2020) for ocular gene therapies. We analyzed 33 unique rAAV gene cassettes used in 57 ocular clinical trials. The rAAV gene therapy vectors used six unique capsid variants, 16 different promoters, and six unique polyadenylation sequences. Further, we compiled a list of promoters, enhancers, and other sequences used in current rAAV gene cassettes in preclinical studies. Then, we give an update on pro-viral plasmid backbones used to produce the gene therapy vectors, inverted terminal repeats, production yield, and rAAV safety considerations. Finally, we assess rAAV transgene and bioactivity assays applied to cells or organoids in vitro, explants ex vivo, and clinical studies.

Human stem cell-based retina on chip as new translational model for validation of AAV retinal gene therapy vectors

“…Here, we further extended this concept by an in-depth analysis of various capsids linked to retinal gene therapy. The naturally occurring serotypes AAV2, AAV8, and the developed AAV2.7m8 were chosen, as they are currently applied in clinical trials or approved retinal therapy ( Buck and Wijnholds, 2020 ; Shahryari et al., 2019 ). Furthermore, AAV2.7m8 is a second-generation AAV developed via in vivo directed evolution, created and selected for more efficient delivery in ocular gene therapies ( Dalkara et al., 2013 ).…”

Inflammation in Viral Vector-Mediated Ocular Gene Therapy: A Review and Report From a Workshop Hosted by the Foundation Fighting Blindness, 9/2020

“…Although gene therapies based on lentiviral and nonviral vectors delivery are in development, vectors derived from adeno-associated virus (AAV) are currently the most commonly used platform for delivery of ocular gene therapy. 4 , 5 Wild-type AAV is a small DNA virus with many naturally occurring capsid serotypes, which can be used directly for gene therapy or first intentionally modified. The virus is primarily non-integrating but persists as episomal DNA.…”

The Ocular Gene Delivery Landscape

“…One of the first approaches to show gene transfer in mouse retina used rAAV vectors to deliver a reporter gene by subretinal injection into an adult mouse retina [62]. Since then, AAV-mediated gene delivery into the retina has been used in several proof-of-concept studies to target photoreceptors or the RPE in mice and larger models (such as dogs) [63][64][65]. Notably, one of the studies involving the delivery of the RPE65 gene into mutant dogs showed therapeutic potential, which subsequently led to the approval of the first gene therapy drug (Luxturna TM ) by the Food and Drug Administration in 2018 [66].…”