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

The power of 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 selection platforms or PCR-based capsid DNA recovery methods, which can bias the selection towards 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 to existing replication competent 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

Assessment of pre-clinical liver models based on their ability to predict the liver-tropism of AAV vectors

The liver is a prime target for in vivo gene therapies using recombinant adeno-associated viral vectors (rAAV). Multiple clinical trials have been undertaken for this target in the past 15 years, however we are still to see market approval of the first liver-targeted AAV-based gene therapy. Inefficient expression of the therapeutic transgene, vector-induced liver toxicity and capsid, and/or transgene-mediated immune responses reported at high vector doses are the main challenges to date. One of the contributing factors to the insufficient clinical outcomes, despite highly encouraging preclinical data, is the lack of robust, biologically- and clinically-predictive preclinical models. To this end, this study reports findings of a functional evaluation of six AAV vectors in twelve preclinical models of the human liver, with the aim to uncover which combination of models is the most relevant for the identification of AAV capsid variant for safe and efficient transgene delivery to primary human hepatocytes. The results, generated by studies in models ranging from immortalized cells, iPSC-derived and primary hepatocytes, and primary human hepatic organoids to in vivo models, increased our understanding of the strengths and weaknesses of each system. This should allow the development of novel gene therapies targeting the human liver

New avenues of directing evolution in viral vector capsid bioengineering

Gene therapy offers the treatment of diseases caused by mutations in the patient’s genome. The delivery of a healthy copy of the mutated gene to affected cells is central to this intervention. Recombinant adeno-associated viral vectors (rAAV) are rapidly becoming the vector of choice for gene delivery applications. One of the many distinguishing features of rAAV vectors is the large diversity of capsid variants enabling a variety of cells and tissues to be targeted for gene delivery. While gene replacement and addition approaches remain the most common strategies utilising rAAVs, refinements in the use of targeted nucleases have made rAAVs a promising gene editing tool. Regardless of the intended application, the capsid variant utilised is a key determinant of clinical efficacy. This thesis presents three endeavours undertaken investigating capsid development and characterisation. The first is an investigation of high-throughput tools for identifying high-performing capsid variants and comparing non-human pre-clinical models. The second is a directed evolutionbased selection platform enabling the reliable generation of novel capsid variants that enable high levels of transgene expression. This technology was validated in two unrelated targets – the human liver and the human retina – demonstrating its broad applicability. Finally, the concept of directed evolution was applied to create a platform for generating novel capsids with increased homology-directed repair outcomes in primary T-lymphocytes and haematopoietic stem cells. This was the primary aim of the thesis and it was achieved by incorporating the lessons learned from all previous studies. In summary, over the course of this project, three key technologies were developed and validated. All these technologies are highly impactful as they enable the choice of the optimal capsids and model system as well as the most functional generation of novel capsids. Moreover, novel capsids were generated in human hepatocytes, human retina, and cells of the immune system

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. Keywords: AAV, library, directed evolution, codon optimization, DNA shuffling, capsi