High-throughput engineering and in situ screening of targeted gene delivery vectors
Gene delivery has become an essential strategy for neuroscience research and offers the promise of therapeutic applications. Adeno-associated viruses (AAVs) have been of particular interest as a gene delivery vehicle (vector) due to their low toxicity and high engineering potential, although their low efficiency and selectivity have required invasive surgical procedures and transgenic animals. To fulfill the pressing need for toolkits for efficient and precisely targeted non-invasive gene delivery, we have developed high-throughput engineering and screening approaches for advancing recombinant AAV vectors. Initially, we developed a high-throughput selection platform based on directed evolution, named Cre recombinase-based AAV targeted evolution (CREATE), that allows us to effectively narrow down a vast library of capsid variants to dozens of promising candidates over a couple of iterations. With this method, we identified several capsid variants that can deliver genes broadly and efficiently to the central and peripheral nervous systems of mice and non-human primates through systemic administration. Next, to further characterize the tropism of AAV vectors, we developed an ultrasensitive sequential fluorescence in situ hybridization (USeqFISH) method for the spatial transcriptomic profiling of both AAV and endogenous transcripts in intact brain tissue. This method achieves exceptional sensitivity that only requires the unique sequence of 14-nucleotide (nt) in cultured cells and 40-nt in tissue for selective RNA visualization, allowing short barcoding of AAV genomes. With an RNA-retaining tissue clearing and a two-step signal quenching method, we established USeqFISH available for quantitative detection of endogenous and virally delivered genes (up to ~50) via sequential labeling in three-dimensional, intact brain tissue. Using USeqFISH, we profiled the transduction of pooled systemic AAVs carrying unique barcodes across tens of genetically defined cell types in diverse mouse brain regions, revealing the distinct cell-subtype tropism of each variant. We also demonstrated the applicability of USeqFISH to the non-human primate (NHP) brain, showing its potential translation into in situ AAV profiling and multimodal, single-cell, intact-tissue analysis in this species. We believe these two approaches provide a powerful, high-throughput technology that will accelerate the engineering of targeted non-invasive gene delivery vectors and bring us closer to successfully translating AAV vectors into safer, more accessible gene therapeutics.
Dr. Min Jee (Min) Jang is a postdoctoral scholar and NARSAD Young investigator under the mentorship of Dr. Viviana Gradinaru at California Institute of Technology. During her postdoctoral training, she developed high-throughput approaches for engineering and screening gene delivery vectors, focusing on adeno-associated viruses (AAV). She has contributed to the development of systemic AAV vectors that can deliver genes broadly, efficiently, and selectively to the nervous systems through intravenous administration. Recently she developed a new spatial transcriptomics method for characterizing pooled systemic AAV vectors against genetically defined cell types in 3D intact tissue. Before Caltech, Min received her BS, MS and PhD in the Department of Bio and Brain Engineering at Korean Advanced Institute of Science and Technology (KAIST) and a short-term postdoctoral fellowship at Korea University College of Medicine, studying brain-on-a-chip technologies. She is a combination of a tool developer, an engineer and a neuroscientist who believes technology development can lead to scientific breakthroughs and immediate solutions for human health.