A3 - AAV Vectors - Capsid Engineering
311: AAV Capsid Selection at Spatial and Single-Cell Resolution in Non-Human Primate Retina
Type: Oral Abstract Session
Presentation Details
Session Title: AAV Capsid Engineering: Multilevel Approaches for Enhanced AAV Delivery
The development of novel adeno-associated viruses (AAVs) with tissue and cell-type-specific tropism is crucial to meet the need for efficient delivery of gene therapies. AAV selection methodologies have given rise to a new class of engineered AAV vectors capable of improved gene delivery at lower doses and with greater specificity than wild-type AAVs. However, a challenge remains in accurately resolving transduction status within heterogeneous cell populations and across multiple tissues. Existing approaches for identifying cell-type-specific tropism of engineered AAVs through single-cell RNA sequencing are acknowledged to be either low-throughput and/or cost-prohibitive. This limitation is, in part, due to their reliance on droplet-based single-cell or nuclei partitioning. Moreover, the use of fluorescent transgenes for detection raises concerns about potential toxicity, particularly in neuronal populations. Our lab has overcome these constraints by establishing an efficient approach for extensively multiplexed profiling of AAV tropism in specific cell types and regions. Our approach utilizes a modified version of the combinatorial indexing technique for sequencing single-cell transcriptomes (SPLiT-seq), first described by Rosenberg et al. in 2018, to simultaneously capture transcriptome and AAV transduction information at single-cell resolution. This strategy offers numerous benefits in terms of scalability. A single experiment can profile ~150,000 cells or nuclei derived from hundreds of biological samples and enables the simultaneous assessment of tens to hundreds of AAV capsid variants. Importantly, our approach doesn't necessitate specialized equipment and is applicable across various tissues and species. To illustrate the utility of AAV SPLiTseq in defining the cell-type-specific tropism of clinically translatable capsids we performed a mixed pool capsid screen with 90 PM-AAVs from two parental serotypes delivered intravitreal to the retina of non-human primates (NHP). The cellular diversity of the NHP and human retina significantly varies spatially, and the degree of damage or sparing of the foveal cones differs across human retinal diseases. To ensure adequate coverage of therapeutically relevant retinal cell types, we isolated nuclei from both peripheral and foveal regions for single-cell sequencing. Capsid transduction was assessed at single-cell resolution using AAV SPLiTseq and more broadly using bulk RNA and DNA sequencing. This allowed for integrated analysis across these complementary screening modalities. Top-performing capsid variants were identified by comparative analysis of bulk and single-cell data and further evaluated for performance and cell-type tropism. In summary, we demonstrate the successful implementation of AAV SPLiT-seq for regional and cell-type-specific tropism to identify clinically relevant PM-AAVs for gene therapy delivery.
Ashley Robbins1,2, Yonghong Chen2, Brian Lewandowski2, Dominika Houserova2, Paul T. Ranum2, Luis Tecedor2, Beverly L. Davidson2,3
1Neuroscience Graduate Group, University of Pennsylvania, Philadelphia, PA,2Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA,3Department of Pathology & Laboratory Medicine, University of Pennsylvania, Philadelphia, PA"
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