Engineering adeno-associated viral vectors with novel structure-function relationships for improved gene delivery.

摘要

Viral gene delivery vectors, in particular adeno-associated viral (AAV) vectors, have demonstrated strong potential in both animal models and human clinical trials to treat various diseases, such as cystic fibrosis and hemophilia. However, several gene delivery barriers, such as pre-existing immunity and poor transduction of some cells, reduce the therapeutic efficacy of the viral vectors; likely because nature never intended for viruses to function as gene therapy vectors. Therefore, I sought to develop innovative high-throughput directed evolution platforms to both engineer and better elucidate the structure-function relationships of the AAV capsid to generate enhanced AAV gene delivery vectors.;Initially, I employed a novel transposon-based mutagenesis strategy to explore the optimal capsid location of a given peptide and develop several AAV vectors that can be purified in a single chromatography step. I generated highly diverse (>107), novel AAV libraries through random mutagenesis of the viral cap gene, the genetic template for the viral capsid. Selections of an AAV2 library generated AAV2 vectors with altered receptor binding properties and significantly enhanced resistance to pre-existing neutralizing antibodies. Furthermore, I extended this random mutagenesis strategy to develop an innovative forward genetics platform to better elucidate the structure-function relationships of the AAV capsid. Application of this platform to two highly divergent AAV serotypes (5 and 6) identified the key capsid regions that confer sialic acid and protein receptor binding.;In parallel, I employed a complementary capsid engineering strategy involving in vitro recombination to construct a highly chimeric AAV library with cap genes from AAV1, 2, 4-6, 8, and 9 and elucidate the diverse array of gene delivery properties possessed by these chimeras. Finally, we applied our directed evolution strategies towards two unmet clinical needs. We generated an AAV vector that mediated high levels of gene delivery to primary human airway epithelia and fully corrected the cystic fibrosis epithelial Cl - transport defect. In addition, I evolved several novel AAV vectors capable of efficient gene delivery to astrocytes, a key support cell of the central nervous system, and thus, these vectors hold tremendous promise for treatment of genetic disorders, such as Alzheimer's and amyotrophic lateral sclerosis.