Engineering Vectors for Non-Invasive Gene Delivery to the Central Nervous System using Multiplexed-CREATE

Abstract

Viruses are widely modified and used as gene delivery vectors for various applications in science and therapeutics. To this end, my thesis focuses on modifying the recombinant adeno-associated viral (rAAV) vectors that are identified as a safer choice for cargo delivery compared to other known viral vectors. They are widely used in the scientific communities, have seen promising outcomes in gene therapy clinical trials, and as of today have three products approved to use in humans. However, the natural repertoire of rAAVs have broad tropism when delivered systemically, and there is room for further improvement on the efficiency and specificity, especially for gene delivery in the central nervous system (CNS). The prior work done in Dr. Gradinaru lab addresses the issue by using a directed evolution approach called CREATE, Cre recombination-based AAV targeted evolution, to identify AAV-PHP.B and AAV-PHP.eB capsids, which broadly transduce the CNS (Deverman et al, 2016; Chan et al, 2017). CREATE selects for functional lox-flipped viral DNA that crosses the blood-brain barrier (BBB) and successfully transduces a specific nerve cell-type expressing Cre, thereby applying a strong selection pressure. However, the method is limited by its ability to identify a handful of enriched variants, and may also be prone to false positives resulting from experimental biases. The effort to fully understand the selection landscape, and to select for capsids that are not just efficient towards a cell-type but also specific towards it, led to the development of Multiplexed-CREATE (M-CREATE). M-CREATE allows parallel positive selections across different cell-types of interest, enables post-hoc negative selections across off-targets using a next-generation sequencing (NGS) based capsid recovery, and retains the principles of Cre-dependent functional recovery from CREATE. The method has a synthetic library generation approach to minimize biases within selection rounds, a variant replicate feature to identify the signal versus noise within a biological system, and an analysis pipeline to group families of enriched variants based on amino acid motifs, all of which together increases the confidence in the outcome and the throughput from a single experiment. Selections across brain endothelial cells, neurons, and astrocytes yielded several AAV-PHP.B-like variants that broadly transduce the CNS, AAV-PHP.V variants that can efficiently transduce the vascular cells forming the BBB, a AAV-PHP.N variant that transduces neurons with greater specificity, and AAV-PHP.C variants that cross the BBB without murine strain specificity across tested strains. The AAV-PHP.C variants have different amino acid motifs compared to the AAV-PHP.Bs that have been previously shown to have limited CNS transduction across some mouse strains due to its interaction with the strain specific host cell surface receptor, ly6a, a homolog of which is not found in humans. (Hordeaux et al, 2018, Hordeaux et al, 2019; Huang et al, 2019; Batista et al, 2019) Therefore AAV-PHP.Cs offer some hope towards translation across other species. In summary, the M-CREATE methodology turns out to be a high-confidence, robust selection platform to yield several novel viral capsids for use in neuroscience and potential gene therapy related applications.