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H - Clinical Translation of Gene and Cell Therapies -> Vector Product Engineering, Development or Manufacturing

864: Optimized Human Regulatory Sequences Achieve Targeted Expression in CNS and Decreased Liver Expression in Mice

Type: Oral Abstract Session

Presentation Details
Session Title: Vector Manufacturing and Engineering 3: Improving Vector Design and System Performance
Location: Room 201
Start Time: 5/18/2022 16:30
End Time: 5/18/2022 16:45

Adeno-associated virus (AAV)-mediated gene therapy (GT) has demonstrated transformative potential in treating genetic disorders, however targeted transgene expression is essential to its safety and efficacy. Achieving appropriate cell-specific expression in target tissues while minimizing off-target transgene expression in the liver is key due to high hepatocyte tropism of clinically validated AAV serotypes. Human genomic regulatory elements (REs) can produce exquisite control of gene expression, but efficient identification of sequence elements that confer cell-selective expression remains a challenge. We have developed a RE discovery platform that leverages expression-based functional screening and computational modeling to rapidly and iteratively discover optimized sequence elements to regulate transgene expression that can be applied to various cell and tissue types. Here, we have applied our high-throughput next generation sequencing (NGS)-based screening method to simultaneously test thousands of genomic elements in vivo to uncover 3’UTR sequence elements that selectively decrease liver expression while maintaining central nervous system (CNS) expression. In a first-generation (Gen1) screen, over 10,000 3’UTR variants comprised of human-derived sequence elements were tested in a multiplexed AAV library concomitantly delivered systemically and via intraparenchymal injection to the CNS in mice. The top 10 3’UTR sequence candidates maintained robust expression in target brain tissue and showed an up to 100-fold reduction in transgene expression in the liver compared with control 3’UTR element vectors (Figure 1). We then generated a follow-up AAV library using multiple combinations as well as sequence variants of these top 3’UTR sequences, with lead variants showing dramatically improved liver-selective de-targeting activity. In addition, using the functional data from the Gen1 screen, we built predictive sequence-based models, which were used to inform sequence selection for a second-generation (Gen2) screen. Compared with the Gen1 screen, the distribution of activity profiles of the 10,000 REs measured in the Gen2 screen showed significant further reduction in liver expression in mice, with unchanged expression in the brain. The specificity of expression achieved demonstrates that the RE prediction model of our discovery platform is able to discover genomic sequences that modulate gene expression selectively in targeted cell types. We have applied this approach to the discovery of various sequence-encoded genetic elements - enhancers, promoters and UTR elements - to achieve desired transgene expression profiles to improve the safety and efficacy of GT for a broad range of monogenic and non-monogenic diseases.

Mitchell Lopez, Serena X. Liu, Steven A. Tan, Tulasi I. Solanki, Ben Zhao, Puja Dhanota, Andrew Kim, Simon Levine, James S. Griffin, John McLaughlin, Gregory Lucey, Kenneth McCullough, Saki Shimizu, Chi Zhang, Maria Candidia Vila, Kartik Ramamoorthi, Stephanie Tagliatela, Raghavendra Hosur, Annie Tanenhaus, Martin Moorhead

Encoded Therapeutics - South San Francisco, CA, South San Francisco, CA
  A. Tanenhaus: 1; Commercial Interest i.e. Company X; Encoded Therapeutics. 1; What was received? i.e. Honorarium; Employee. 1; For what role? i.e. Speaker; Employee.

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