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

Towards the Treatment of Human Genetic Liver Disease by AAV-Mediated Genome Editing and Selective Expansion of Repaired Hepatocytes

Gene repair involves the correction of the genetic mutation directly at the defective locus with retention of physiological expression. The biggest challenge of this approach, however, is that gene repair by homologous recombination occurs at levels that are unlikely to be sufficient to confer therapeutic benefit in the majority of cell-autonomous liver disease phenotypes, such as OTC deficiency, the most common urea cycle disorder. To overcome this challenge, gene correction can be complemented by selective expansion strategies designed to expand repaired hepatocytes to frequencies required for therapeutic benefit. In vivo expansion can be achieved, for instance, by conferring a selective advantage to gene-corrected cells. In this study, human-specific genetic inhibitors were designed to exploit a selective expansion strategy based on the modulation of the tyrosine catabolism pathway and were successfully validated in humanised (Fah-/-, Rag2-/-, IL2rg-/-) FRG mice. Another way to increase the frequency of gene repair is to use nucleases to create DNA breaks at the target site to promote homology-directed repair (HDR). Recombinant AAV vectors carrying human-specific reagents for CRISPR/Cas9-mediated genome editing were developed in order to correct a single nucleotide mutation in exon 9 of the OTC gene. Initially, the editing reagents were evaluated in OTC-deficient mice with a transposed engineered “minigene” version of the OTC gene. Editing reagents functionally validated in this model were then evaluated in vivo on the native OTC locus in primary human hepatocytes, including patient-derived hepatocytes, xenografted into FRG mice. Availability of novel synthetic AAV capsids, such as NP59, facilitated high targeting efficiency of human hepatocytes which in turn resulted in up to 29% OTC alleles being corrected by HDR. The studies described in this thesis show for the first time precise gene repair of a disease-causing mutation in primary human hepatocytes in vivo