Engineered Capsids for Efficient Gene Delivery to the Retina and Cornea

Abstract

Adeno-associated viral (AAV) vectors represent an ideal vehicle for human gene transfer. One advantage to the AAV vector system is the availability of multiple naturally occurring serotypes that provide selective tropisms for various target cells. Strategies to enhance the properties of the natural AAV isolates have been developed and can be divided into two approaches, rational design or directed evolution. The rational design approach utilizes knowledge of AAV capsids to make targeted changes to the capsid to alter transduction efficiency or specificity, while the directed evolution approach does not require a priori knowledge of capsid structure and includes random mutagenesis, capsid shuffling, or random peptide insertion. In this study, we describe the generation of novel variants for both AAV2 and AAV5 using a rational design approach and knowledge of AAV receptor binding, surface charge, and AAV capsid protein posttranslational modifications. The novel AAV2 and AAV5 variants demonstrate improved transduction properties in both the mouse retina and cornea. The translational fidelity of the novel AAV2 variant was confirmed in the context of the nonhuman primate (NHP) retina, whereas a NHP tissue explant model was established to allow the rapid assessment of translational fidelity between species for the AAV5 variants. The capsid-modified AAV2 and AAV5 variants described in this study have novel attributes that will add to the efficacy and specificity of their potential use in gene therapy for a range of human ocular diseases.

Optimized Reversed-Phase Liquid Chromatography/Mass Spectrometry Methods for Intact Protein Analysis and Peptide Mapping of Adeno-Associated Virus Proteins

“… 1 , 3 The most efficient viral vectors emerging from preclinical and clinical studies are adeno-associated virus (AAV), lentivirus, and adenovirus, among which AAV is the most explored owing to its lower risk in humans and efficient transduction in a variety of cells and tissues. 1 , 3 , 4 …”

Improved systemic AAV gene therapy with a neurotrophic capsid in Niemann–Pick disease type C1 mice

“…For many gene therapy studies, especially those using novel engineered capsids, nonhuman primate studies and relevant human culture models may be needed to validate and optimize a gene therapy vector for delivery to patients with NPC disease and related disorders. Advances in the identification of novel serotypes that cross the BBB in humans, and capsid engineering to derive CNS trophic variants ( Castle et al, 2016 ; Hudry et al, 2018 ; Sullivan et al, 2018 ; Hanlon et al, 2019 ; Havlik et al, 2020 ) should help improve vectors, as recent studies highlight ( Gray et al, 2013 ; Frederick et al, 2020 ; Yoon et al, 2020 ).…”

Next Step in Gene Delivery: Modern Approaches and Further Perspectives of AAV Tropism Modification

“…The influence of N-terminal acetylation on viral capsid proteins is still not clear; some suggest that the N-acetylation of VP1 and VP3 may influence viral capsid degradation and uncoating before entry into the nucleus [84]. To elucidate whether N-acetylation affects transduction properties, Frederick et al [85] yielded AAV5 mutation variants S2G, S2P, S194P, S194G, S2G/S194G and S2P/S194P. Each mutation led to the substitution from serine to either glycine or proline, which are less prone to N-terminal acetylation after the degradation of N-terminal residues by aminopeptidases [84,86], and compared their transduction properties with a wild-type strain in mice retina.…”