Creating enhanced adeno-associated viral vectors for gene delivery using directed evolution

In this thesis, I develop a method that enables the discovery and directed design of viral vectors based on the adeno-associated virus (AAV), and I employ this method to create novel variants that are able to overcome transport barriers during administration.; The transport properties of an AAV particle are determined completely by the structure of its capsid, the outer protein shell of the virus. In fact, a small number of different variants of AAV exist in nature, and each variant's capsid confers a unique set of properties. Therefore, I develop an artificial method that creates millions of AAV variants, reasoning that a few members of this library of variants will possess a set of desired properties. This method is based on the genetic modification of the AAV cap gene, the underlying blueprint for the capsid structure. The modification is carved out using a random mutagenesis and recombination process. Because the success of this technique relies upon the creation of a library of sufficient diversity, I create a computational framework that is able to predict the outcome of this procedure and aids in choosing the experimental parameters of the process that yield a desired diversity.; I have applied this technology to overcome two distinct transport barriers that can arise in gene delivery. The first has been to generate AAV vectors that have altered affinity for heparan sulfate, a proteoglycan that is distributed within a tissue in two forms: as a receptor on the cell surface and as a component of the extracellular matrix. Therefore, careful control of the penetration depth of these vectors may be possible by modulating their heparin affinity. The second barrier is the elimination of administered vectors via antibody-mediated immune processes. I demonstrate that successive rounds of mutagenesis followed by screening can yield vectors that are no longer recognized by AAV-specific antibodies, yet retain their ability for gene transfer.; In summary, this thesis tracks the development and application of a technology that allows the high-throughput generation of millions of AAV vector variants from which I isolate members that are able to overcome transport barriers to gene delivery, by using the proper screening methodology. (Abstract shortened by UMI.)...