Characterisation of a novel AAV vector with enhanced neurotropism

Gene therapy is an experimental technique doctors can use to introduce healthy copies of genes in patients that are carriers of faulty genes. Popular vectors to deliver the healthy copies, also called therapeutic transgenes, are non-pathogenic viruses such as adeno-associated virus. This experimental field of medicine is currently undergoing a renaissance thanks to a number of successful clinical trials for the treatment of an inherited form of blindness, bleeding disorders and motor neuron diseases. However, gene therapies that are directed at the brain in general have been less successful. One of the issues is that viral vectors administered to the brain do not efficiently get into brain cells and do not travel far from the injection site, leading to insufficient insertion of the therapeutic gene. To overcome this hurdle, a number of technologies can be used to alter the viral capsid and make it more suitable for entry into brain cells. We have developed a new viral capsid by introducing changes to make it look more like adeno-associated viruses that are currently circulating in the human population. Viral vectors made with this capsid have an increased ability to deliver therapeutic transgenes throughout the brain and have demonstrated therapeutic potential for the treatment of a devastating disease that affects the brains of young children. Here we propose to decipher the mechanisms that contribute to the improved gene delivery abilities of this capsid. The results from this study will further assist the development of treatments for a group of diseases that affect the brain.

Adeno-associated virus (AAV) gene therapy is currently experiencing a renaissance thanks to a number of successful clinical trials. However, vector doses required to achieve therapeutic effect are high and some tissues remain difficult targets for efficient transduction. In general, gene therapy strategies directed at the brain of patients with inherited neurological or neurodegenerative disorders have shown limited efficacy. One of the main challenges for gene therapy for central nervous system (CNS) diseases is the limited distribution of AAV vectors from the injection site to the target cells. Our preliminary studies have shown that mutation of a select number of amino acids (AA) in the AAV2 capsid results in a vector with enhanced transduction and significant spread in the CNS of rodent models. Comparative biodistribution studies demonstrated that our new vector, AAV-TT, has better distribution abilities than benchmark neurotropic serotypes AAV9 and AAVrh10. Importantly, AAV-TT was the only vector able to correct a mouse model of mucopolyssacharidosis IIIC (MPSIIIC), a CNS disease caused by a lysosomal transmembrane enzyme and therefore reliant on efficient transduction throughout the brain. Here we propose a study that is aimed at delineating which mechanisms contribute to the unparalleled transduction and spread of AAV-TT in the CNS. This will be achieved by comparing the abilities of AAV2 and AAV-TT in biological assays that are designed to measure (1) cellular determinants of transduction i.e. viral attachment, cellular uptake, cytoplasmic escape, nuclear entry and uncoating; (2) interstitial transport within the brain parenchyma; (3) retro- and anterograde axonal transport within the CNS. The experiments proposed here will provide insight into the mechanisms that contribute to the increased bioactivity of AAV-TT in the CNS and will further assist the development of treatments for a group of neurological diseases that are defined by global neuropathy.

One of the main challenges for gene therapy for central nervous system (CNS) diseases is the limited distribution of AAV vectors from the injection site to the target cells. Our preliminary studies have shown that mutation of a select number of amino acids (AA) in the AAV2 capsid results in a vector with enhanced transduction and significant spread in the CNS of rodent models. Comparative biodistribution studies demonstrated that our new vector, designated AAV-True Type (AAV-TT), has better distribution abilities than benchmark neurotropic serotypes AAV9 and AAVrh10. Importantly, AAV-TT was the only vector able to correct a mouse model of mucopolyssacharidosis IIIC (MPSIIIC), a CNS disease caused by a lysosomal transmembrane enzyme and therefore reliant on efficient transduction throughout the brain. Here we propose a study that is aimed at delineating which mechanisms contribute to the unparalleled transduction and spread of AAV-TT in the CNS. We will combine state-of-the art infectivity assays with new imaging platforms to understand the biology of infection and exploit novel model systems to evaluate AAV-TT's ability to undergo interstitial and axonal transport. Together, these experiments will give critical insight into how host factors contribute to the behaviour of AAV vectors in the brain. In addition, we will use these assays to study which capsid features determine the efficacy of transduction in the CNS. Specifically, we are currently funded to investigate how the mutations in AAV-TT contribute to the observed enhanced tropism. Jointly, these studies will inform capsid design for a new generation of potent gene therapy vectors that have the potential to efficiently treat a number of devastating diseases.

The beneficiaries of this research potentially include: 1) pharmaceutical industry/biotech companies who have an interest in applying our research for the development of new therapies, 2) patients with diseases that could be treated with gene therapy, 3) pre-university and university students who have an interest in pursuing a career in science, 4) Medical students/clinicians who have an interest in developing/applying gene therapy treatments, 5) the postdoctoral research associate who will receive further training and profit from interactions with industrial partners.