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Background CAR-T cell therapies have revolutionized cancer treatment resulting in FDA approval of 7 drugs for B cell malignancies. However, their lengthy, complex and expensive manufacturing limits patient access, with over 40% of eligible patients unable to receive products due to disease progression. In contrast, AAV is a cost-effective, safe, off-the-shelf treatment option that is currently used in 7 FDA-approved gene therapies. AVO-100 is a gene therapy that uses AAV with capsids containing T cell-targeting (TCeT) moieties to produce CD19-specific CAR-T cells in patients.Methods pFastBac plasmids encoding a CD19-specific CAR, TCeT-containing AAV capsid and AAV rep proteins were generated using standard molecular biology techniques. They were transformed into DH10Bac cells to produce bacmids, which were transfected into Sf9 insect cells to create recombinant baculoviruses. The baculoviruses were used to produce AVO-100 in Sf9 cells, and AVO-100 was used to transduce human PBMCs in vitro. CAR-T cell phenotype was analyzed 72 hours later via flow cytometry. Cytotoxicity was assessed in a 48-hour-long coculture assay with CD19+EGFP+ Raji lymphoma cells. Luciferase-expressing Raji cells were administered IV to NSG mice and treated 3 days later with human PBMCs and AVO-100 (also IV) to generate CAR-T cells in vivo. Tumor growth was monitored via IVIS imaging and CAR-T cell persistence was analyzed by flow cytometry.Results Analysis of CAR surface expression showed that the transduction efficiency ranged between 42% and 54% ( figure 1A). Reduction in%CD19+ B cells 72 hours post-transduction, compared to non-transduced controls, indicated B cell depletion by functional CAR-T cells (figure 1B). Coculture assays confirmed this, demonstrating 55%–85% specific killing of Raji lymphoma cells (figure 1C). In vivo studies in NSG mice, infused with Raji cells, human PBMCs, and AVO-100 IV, showed significantly reduced tumor burden in treated mice compared to untreated controls, as measured by IVIS imaging over 24 days (figure 2A). Untreated mice died by day 16, while all treated mice survived until day 24 (figure 2B). At the experiment’s end, 0.3%–69.9% of human T cells in mouse blood expressed the CAR (figure 2C), confirming in vivo CAR-T cell generation and persistence, with effective anti-tumor activity against B cell lymphoma.Conclusions The preclinical data so far suggest that AVO-100 can be used to generate CD19-specific T cells from non-activated T cells in human PBMCs both in vitro and in vivo and the CAR-T cells can control the growth of Raji lymphoma in vitro and in NSG mice.Abstract 1001 Figure 1Flow cytometry analysis of CAR-T cell transduction efficiency (A), B cell depletion from PBMCs transduced with AVO-100 (B) and CAR-T cell-mediated killing of Raji cells in vitro (C)Abstract 1001 Figure 2CD19-specific CAR-T cells, generated in vivo from human PBMCs using AVO-100, control Raji tumor growth (A), improve mouse survival (B) and persist for at least 24 days (C)