Next-generation human liver gene therapy

Project Summary/Abstract Pioneering clinical trials of hepatocyte-targeted liver gene therapy in hemophilias have established the principal therapeutic efficacy of adenoassociated virus (AAV) vectors. Clinical trials have also revealed limitations of current AAV vectors, mainly lower than expected therapeutic efficacy and dose-dependent toxicity undermining both safety and efficacy. These clinical findings show that animal models, even monkeys or mice engrafted with human hepatocytes, fail to accurately predict the performance of AAV vectors in humans. The limitations of current AAV vectors need to be overcome for broad application of AAV liver gene therapy because most liver diseases require more hepatocytes to be transduced to achieve a therapeutic effect than hemophilias. To maximize the therapeutic effect that can be achieved within a safe AAV vector dose limit, we will identify AAV capsids that transduce hepatocytes in the human liver with maximum efficiency but no or limited off-targeting. For this, we will harness the near-clinical conditions provided by normothermic machine perfusion (NMP) of human livers. We will compare capsids currently used in clinical trials of liver gene therapy to engineered capsids reported to efficiently target mouse liver or human hepatocytes engrafted in mice. To compare capsids side by side, we have established analysis of AAV vector-expressed barcodes by single-cell RNA sequencing of comprehensive cell populations isolated from human livers after NMP. We will also target capsids de novo in human livers maintained by NMP by screening a library of 1 million chimeric capsids generated by shuffling of the DNA sequences of naturally occurring AAV capsids. In addition to achieving unprecedented levels of efficiency and specificity of transduction of hepatocytes, we aim to establish transduction of cholangiocytes, thereby facilitating the development of gene therapies for biliary diseases. We will also target activated hepatic stellate cells (myofibroblasts), the source of excessive collagen in liver fibrosis, and reactive cholangiocytes, which form the ductular reaction characteristic for cholestatic liver diseases. Efficient and specific in vivo gene delivery to these pathogenic and abundant cells will facilitate therapeutic strategies based on inactivation or repurposing, for example, reprogramming into hepatocytes. By identifying or generating capsids that transduce hepatocytes and other therapeutically relevant liver cell types with the highest level of efficiency and specificity our results will directly inform clinical trials that are at the planning stage and provide the basis for extending the reach of AAV liver gene therapy to common liver diseases like fatty liver disease and biliary diseases, including liver fibrosis as their common end stage.