255. A Method for Developing More Potent AAV Capsids by Prolonging the Vector Half-Lives in the Blood Circulation
Authors: Miyuki Kimura, Kei Adachi, Hiroyuki Nakai
Journal: Molecular Therapy (2016)

Adeno-associated virus (AAV) has been used successfully as a vector for in vivo gene therapy. Many different serotypes are under investigation, but AAV9 vectors especially show robust in vivo transduction by systemic administration via the periphery. Although the mechanism of this robustness is not yet completely understood, the superior ability to cross the endothelial cell barriers including the blood-brain barrier has been presumed to play a key role in efficient systemic gene delivery by AAV9. In this regard, our laboratory has accumulated provocative evidence that AAV permeability across the endothelium is not substantially different among many AAV strains (serotypes and mutants) per se, but that AAV9's in vivo robustness following intravascular administration is due to its distinctively delayed blood clearance. Given that many AAV strains transduce cells better than AAV9 when directly injected into the parenchyma, it should be reasonable to hypothesize that prolonging the half-life of an AAV could substantially increase in vivo transduction efficiency following systemic administration. Here, we show that substitution of a small subset of amino acids in the C-terminal half of the viral capsid can substantially prolong the half-life of AAV1 in the blood and confer a superior in vivo transduction efficiency that is better than that of AAV9 following systemic vector administration. Based on the results from our preliminary AAV1 and AAV9 capsid domain swapping study and a capsid amino acid sequence alignment study, we identified 7 capsid segments comprising 5 to 23 amino acids that might contribute to the persistent circulation of AAV9 in the blood. Each of these 7 segments can be replicated in the AAV1 capsid, which shows a rapid blood clearance rate, by substituting 3 to 11 amino acids of the AAV1 capsid with those of the AAV9 capsid. In the study, we created a panel of 27=128 AAV1-derived strains that carry these segment(s) in any possible combinations (note: one of the 128 is the same as the wild-type AAV1 capsid). We then determined blood clearance rates of all the 128 AAV1-derived strains and AAV9 following intravenous injection into mice using the AAV Barcode-Seq approach. The results show that 42 mutants that showed a >30-fold increase in persistence in the blood at 24 hours post-injection compared to the parental wild-type AAV1 all shared either of the following segment combinations, xxx9xx9 or xxxxx99 (note: each letter represents a segment and × can be either AAV1 or AAV9). This indicates that the 7th segment plays an indispensable role but requires either of the 4th and 6th segments for substantially prolonging the half-life. A preliminary tissue transduction analysis in mice revealed that these AAV1-derived mutants with a long half-life showed enhanced in vivo gene delivery compared to AAV9. In a separate study, we also identified a 16 amino acid-long segment in the N-terminal half of the AAV9 capsids that is essential for maintaining high levels of circulating viral particles in the blood at 72 hours post-injection. We are currently investigating whether this amino acid motif, when combined with the above-described xxx9xx9 or xxxxx99 motifs, could further enhance persistence of AAV1-derived mutants and augment in vivo transduction, using another set of 24 AAV1-derived composite mutant capsids in mice. In summary, the study establishes proof-of-principle of a new approach to create more portent AAV vectors by modulating the vector half-life. This approach will be particularly useful for developing novel AAV capsids that can transduce the brain tissues more efficiently by systemic vector administration.