AAV-p40 Bioengineering Platform for Variant Selection Based on Transgene Expression.

The power of adeno-associated viral (AAV)-directed evolution for identifying novel vector variants with improved properties is well established, as evidenced by numerous publications reporting novel AAV variants. However, most capsid variants reported to date have been identified using either replication-competent (RC) selection platforms or polymerase chain reaction-based capsid DNA recovery methods, which can bias the selection toward efficient replication or unproductive intracellular trafficking, respectively. A central objective of this study was to validate a functional transduction (FT)-based method for rapid identification of novel AAV variants based on AAV capsid mRNA expression in target cells. We performed a comparison of the FT platform with existing RC strategies. Based on the selection kinetics and function of novel capsids identified in an in vivo screen in a xenograft model of human hepatocytes, we identified the mRNA-based FT selection as the most optimal AAV selection method. Lastly, to gain insight into the mRNA-based selection mechanism driven by the native AAV-p40 promoter, we studied its activity in a range of in vitro and in vivo targets. We found AAV-p40 to be a ubiquitously active promoter that can be modified for cell-type-specific expression by incorporating binding sites for silencing transcription factors, allowing for cell-type-specific library selection.

A User's Guide to the Inverted Terminal Repeats of Adeno-Associated Virus.

Ongoing development of recombinant vectors based on adeno-associated virus (rAAV) is providing an increasingly powerful and widely used toolkit for gene transfer and genome editing applications. While conceptually simple, the system harbors considerable complexity that presents many potential pitfalls for the inexperienced user. The short inverted terminal repeats (ITRs) can prove to be particularly problematic during vector engineering due to inherent instability necessitating diligent quality control measures during vector manufacture. This is especially important from a clinical standpoint when consistent purity and potency are paramount, and all components of the system are rigorously scrutinized by regulatory agencies. Despite the discovery over 30 years ago that the AAV ITRs are the only cis-acting elements of the virus required for vector production, there is a scarcity of reviews specifically focused on these complex elements. This review provides an overview of the ITR with the dual purpose of acting as a user's guide in the application of AAV vector technology and as a roadmap for ongoing vector development and optimization.

Codon-Optimization of Wild-Type Adeno-Associated Virus Capsid Sequences Enhances DNA Family Shuffling while Conserving Functionality.

Adeno-associated virus (AAV) vectors have become one of the most widely used gene transfer tools in human gene therapy. Considerable effort is currently being focused on AAV capsid engineering strategies with the aim of developing novel variants with enhanced tropism for specific human cell types, decreased human seroreactivity, and increased manufacturability. Selection strategies based on directed evolution rely on the generation of highly variable AAV capsid libraries using methods such as DNA-family shuffling, a technique reliant on stretches of high DNA sequence identity between input parental capsid sequences. This identity dependence for reassembly of shuffled capsids is inherently limiting and results in decreased shuffling efficiency as the phylogenetic distance between parental AAV capsids increases. To overcome this limitation, we have developed a novel codon-optimization algorithm that exploits evolutionarily defined codon usage at each amino acid residue in the parental sequences. This method increases average sequence identity between capsids, while enhancing the probability of retaining capsid functionality, and facilitates incorporation of phylogenetically distant serotypes into the DNA-shuffled libraries. This technology will help accelerate the discovery of an increasingly powerful repertoire of AAV capsid variants for cell-type and disease-specific applications.