Session: 801. Gene Therapies: Poster I
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Autoimmune disorders, Translational Research, Diseases, Immune Disorders, Lymphoid Malignancies, Technology and Procedures, Gene editing
Here, we evaluated the role of key restriction factors in resting CD8+ T cells by performing CRISPR/Cas9 knockout studies. We identified IFITM1 and SAMHD1 as critical factors that act through distinct mechanisms to block efficient transduction of resting CD8+ T cells by CD8-targeted fusosome. As part of an effort to increase transduction efficiency, we identified temsirolimus and IL-7 as clinically relevant pharmaceutical agents that lift restriction by IFITM1 and SAMHD1, respectively, in resting CD8+ T cells. Pre-treatment of resting CD8+ T cells with temsirolimus and IL-7 alone or in combination prior to transduction by CD8-targeted CD19CAR fusosome resulted in significantly increased gene transfer and surface expression of CD19CAR (At 10 SupT1 IU/CD8, the increase in percent transduction was on average 2.4-fold with IL-7, 10.3-fold with temsirolimus, or 25.0-fold with the combination, using cells from 6 donors).
While IL-7 supports T cell expansion and function apart from regulating SAMHD1 activity, temsirolimus is an immunosuppressive molecule that inhibits mTOR and therefore can interfere with cytotoxic function, expansion, and survival of T cells. We tested the functional effect of temsirolimus on resting CD8+ T cells prior to transduction and exposure to NALM6 tumor cells. Pre-treatment with temsirolimus reduced CAR-driven cytotoxic ability and expansion of transduced resting CD8+ T cells compared to untreated cells from two donors.
To identify a novel pharmaceutical agent that could relieve IFITM1-mediated restriction in CD8+ T cells without negatively affecting cytotoxic function, we performed a high-throughput screen of clinically-approved small molecules for their ability to increase transduction in resting T cells. Amphotericin B was identified as an agent that led to IFITM1 degradation in resting CD8+ T cells and increased their transduction by fusosome (At 5 SupT1 IU/T cell, percent transduction was increased by an average of 5.7-fold using cells from 4 donors). In addition, pre-treatment of resting CD8+ T cells with amphotericin B prior to transduction by CD8-targeted CD19CAR fusosome led to an increased ability to control NALM6 tumor cell growth over a serial challenge assay without a delay in cytotoxicity or expansion.
Avoiding lymphodepletion and the complexities of autologous CAR T manufacturing makes in vivo delivery of a CD8-targeted CD19CAR fusosome an attractive option for the treatment of B cell malignancies and other B cell-mediated diseases. The findings reported here further our understanding of resting CD8+ T cell transduction, informing the potential use of pharmaceutical interventions or vector modifications to overcome restriction and increase the potency of a CD8-targeted fusosome.
Disclosures: Ott: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Aamer: Sana Biotechnology: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Humes: Sana Biotechnology: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Burch: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Proano: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Dolinski: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Vedenova: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Benson: Sana Biotechnology: Current equity holder in publicly-traded company, Ended employment in the past 24 months. White: Sana Biotechnology: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Tareen: Sana Biotechnology: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Green: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Elpek: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. van Hoeven: Sana Biotechnology: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Fry: United States Patent and Trademark Office: Patents & Royalties: WO2015084513A1; United States Patent and Trademark Office: Patents & Royalties: WO2019178382A1; Sana Biotechnology: Consultancy, Current equity holder in publicly-traded company, Ended employment in the past 24 months; United States Patent and Trademark Office: Patents & Royalties: WO2017205747A1.