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

• Published in: International journal of molecular sciences Vol. 21; no. 12; p. 4197
• Main Authors: Buck, Thilo M, Wijnholds, Jan
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 12-06-2020; MDPI
• Subjects: adeno-associated virus (AAV); Apoptosis; Binding sites; Biological activity; Capsid Proteins - genetics; Cassettes; Clinical trials; Clinical Trials as Topic; Deoxyribonucleic acid; Dependovirus - genetics; Disease; DNA; enhancer; Enhancers; Explants; Expression vectors; Gene expression; Gene therapy; Genetic Therapy - methods; Genetic Vectors - genetics; Medical research; Optic Nerve Diseases - genetics; Optic Nerve Diseases - therapy; Organoids; Polyadenylation; promoter; Promoter Regions, Genetic; Promoters; Quality of life; Researchers; Retina; Retinal Dystrophies - genetics; Retinal Dystrophies - therapy; retinal pigment epithelium (RPE); Review; Transgenes; Treatment Outcome; Vectors (Biology); Visual perception; Workflow; United Kingdom > UK; United States > US; polyadenylation; adeno-associated virus (AAV); enhancer; biological activity assay (BAA); Keywords. retina; retinal pigment epithelium (RPE); promoter; Cas9; transgene expression assay (TEA); pro-viral plasmid
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms21124197

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.