Viral Vector Technologies and Strategies: Improving on Nature

This is the authors' abstract. We don't add key points for this paper.

Introduction Gene therapy has been known for decades but only recently has its true potential started to be realized as a growing number of therapies are reaching the clinic.1 This is in part due to the increased understanding of viral biology and advances in genetic engineering.2 In particular, the use of adeno-associated virus (AAV) is an emerging gene therapy field that holds great potential to treat a wide range of diseases because of its proven safety profile.3–5 Glybera (alipogene tiparvovec) for the treatment of lipoprotein lipase deficiency, was the first AAV gene therapy approved in the world and showed no safety concerns.6 Two AAV gene therapies, Luxturna (voretigene neparvovec-rzyl) and Zolgensma (onasemnogene abeparvovec-xioi) have been approved in the United States, for the treatment of RPE65 biallelic mutations causing retinal dystrophy and spinal muscular atrophy, respectively. These treatments utilize naturally occurring AAV serotypes and aim to express a gene that fits within the size constraints of an AAV packaging capacity.7–9 Luxturna and Zolgensma both have been successful in part due to certain low-risk aspects of treatment. For Luxturna, the subretinal route of administration is considered safe and decreases the barriers the AAV face from infecting the target cell.10 Zolgensma requires a low dose because of the target patient population and therefore the manufacturing burden, normally large for a systemic therapeutic, was decreased.11 Ophthalmic diseases are an excellent therapeutic area to employ gene therapy due to the small organ size (low manufacturing burden), accessible route of administration, clear clinical endpoints, a relatively immune-privileged compartment, and a multiplicity of ocular genetic and complex diseases with high unmet medical need.12–14 Spurred by evidence of an excellent safety profile and durable treatment effect, there are many ongoing clinical trials using viral vectors to treat various retinal diseases both monogenic conditions (choroideremia, retinitis pigmentosa, achromatopsia) and large-market complex diseases (age-related macular degeneration, Stargardt disease, and Usher syndrome).14 This, together with ever-blossoming preclinical literature supports clinical translation. The scope of this review focuses on viral gene therapy, specifically AAV gene therapy which has proven to be the main choice of delivery vehicle for several reasons including, its inability to replicate without a helper virus or integrate, the adaptability of the viral genome, and that AAV are not known to cause human disease.3,4 Other avenues of gene therapy including nonviral approaches for delivery are not covered in this review. There is, however, a clear need for improvement on currently available gene therapy technology, both in terms of capsid engineering and payload design. The main issues the viral gene therapy field is looking to address are efficiency and specific delivery and expression, and payload size limitations. In earlier times, concern had been expressed about the length of time gene therapy for ocular conditions could last. To date, reassuringly, gene therapy appears to be highly durable and long-lasting as demonstrated in both preclinical studies and clinical trials. Novel technologies and strategies are being developed to address the remaining concerns based on the continual advancement in our knowledge of viral vehicles and payload cassettes. Through ingenuity and sophisticated engineering techniques, novel approaches to improve capsid specificity to diseased cell types and to modify promoters and transgenes, are under development in many groups.8 Addressing these issues will lead to our ability to target an even broader range of diseases with viral gene therapy. This review will cover 2 main strategies to improve viral gene therapy: viral capsid engineering and payload design. Approaches to viral capsid engineering, including rational design and directed evolution are aimed at overcoming natural barriers to delivery, improved and more selective tropism, and evading preexisting immunity. Payload design will cover strategies of dual vector approaches, mini-promoter design, tissue/cell-specific promoters, minigene construction, and emerging technologies to correct underlying pathogenic mechanisms such as RNA interference (RNAi) and gene editing. This review will synthesize the progress of researchers in the academic, pharmaceutical, and biotechnology spheres and describes the many novel engineering techniques being utilized to date. Engineering the Next Generation of AAV Capsids Early AAV-based gene therapy products utilized naturally occurring [wild-type (WT)] AAV capsids, which have been extensively studied and showed efficacy in mouse models. The different protein capsids of naturally occurring AAV are typically referred to as serotypes, based on the presence of distinct antigens and a lack of antibody cross-reactivity. These serotypes have differences in their capsid protein sequences, which manifest as structural differences in key surface loop regions. Recently, the US Food and Drug Administration (FDA) approved Luxturna (voretigene an the RPE65 for the treatment of diseases by mutations in the RPE65 In in clinical trials have been for an the for the treatment of and products the and the for the treatment of retinitis and the ocular gene therapy clinical trials using AAV these have for AAV-based retinal gene therapy, the need for delivery which that will in gene therapy products for both monogenic and complex diseases of the These delivery vehicles for the development of gene therapy products for a number of as as the patient population to treat at of AAV Capsids in Gene of Administration RPE65 mutations Luxturna (voretigene neparvovec-rzyl) approved macular RPE65 mutations macular adeno-associated United Food and Drug human protein retinal protein retinitis macular retinitis for gene delivery improvement within the are of route of administration and delivery to cell types the or specific cell types in the clinical trials have to the 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