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Introduction: Ex vivo manufactured chimeric antigen receptor (CAR) T cell therapies are highly effective for treating B cell malignancies. However, the complexity, cost and time required to manufacture CAR T cells limits access. To overcome conventional ex vivo CAR T limitations, a novel gene therapy platform has been developed that can deliver CAR transgenes directly to T cells through systemic administration of a fusosome, an engineered, target-directed novel paramyxovirus-based integrating vector that binds specific cell surface receptors for gene delivery through membrane fusion. Here, we demonstrate that systemic administration of a CD8a-targeted, integrating vector envelope (i.e., fusogen) encoding an anti-CD20 CAR into Southern pig-tail macaques (M. nemestrina), which is a species permissive to the integrating vector-mediated transduction, results in T cell transduction and B cell depletion with no treatment-related toxicities. Methods: CD8a-specific single chain variable fragments (scFvs) were generated and measured for target specificity versus non-CD8-expressing cells in vitro. Cross-reactivity of the CD8a-specific fusogen for human and nemestrina T cells was confirmed in vitro. Targeted fusogens were then used to pseudotype integrating vector expressing an anti-CD20 CAR containing the 4-1BB and CD3zeta signaling domains (CD8a-anti-CD20CAR). Transduction and B cell killing was confirmed on human and nemestrina PBMCs. To evaluate in vivo activity, normal, healthy nemestrina macaques were treated with a single dose of CD8a-targeted anti-CD20 CAR fusosome (n=6) or saline (n=2) via intravenous infusion at 10mL/kg/hr for 1-hour and evaluated for up to 52 days for evidence of adverse effects, B cell depletion, CAR-mediated cytokine production, CAR T cell persistence and vector biodistribution using ddPCR and anti-CD20CAR transgene by RT-ddPCR to detect transgene levels. Histopathology of several organs and immunohistochemistry for CD3 and CD20 on lymph nodes, spleen, and bone marrow were performed at termination (days 35 and 52). Tolerability of the treatment was assessed by body weight, body temperature, neurological exams, serum chemistry panel, and complete blood counts pre-dose and post-dose up to 52 days. Results: The CD8a-targeted fusogen demonstrated CD8a-specificity versus human CD8 negative cell lines, and cross-reactivity and transduction efficiency in nemestrina PBMCs in vitro. Compared to a control vector (GFP), anti-CD20CAR-modified T cells showed a dose-dependent depletion of B cells using in vitro assays. Following infusion of CD8a-anti-CD20CAR fusosomes into macaques, pharmacological activity in peripheral blood was detected by a reduction of B cells in 4 of 6 animals after 7 to 10 days. Two animals showed persistent B cell depletion until study termination, with two others showing a temporary response. The presence of vector copy could be detected in the peripheral blood of all treated animals between days 3 and 10, and in isolated spleen cells in 5 of 6 animals. All control animals (saline) were negative for vector. RT-ddPCR mRNA expression similarly revealed the presence of anti-CD20CAR transcripts in isolated spleen cells from treated animals; no expression was detected in tissues from control animals. Elevations in inflammatory cytokines could be detected in the serum of treated animals between days 3 and 14. Fusosome treatment was well-tolerated in all animals with no evidence of adverse effects. Moreover, T cell transduction and B cell depletion was not associated with cytokine-related toxicities, and blood chemistry and histopathology were within normal limits. Conclusion: These data obtained in an immunologically competent animal demonstrate the proof-of-concept that systemic administration of a CD8a-anti-CD20CAR fusosome can specifically transduce T cells in vivo without pre-conditioning or T cell activation, resulting in B cell depletion in the absence of vector- or CAR T-related toxicities. Therefore, fusosome technology represents a novel therapeutic opportunity to treat patients with B cell malignancies and potentially overcome some of the treatment barriers that exist with conventional CAR T therapies. Disclosures Cunningham: Sana Biotechnology: Current Employment. Chandra: Sana Biotechnology: Current Employment. Emmanuel: Sana Biotechnology: Current Employment. Mazzarelli: Sana Biotechnology: Current Employment. Passaro: Sana Biotechnology: Current Employment. Baldwin: Sana Biotechnology: Current Employment. Nguyen-McCarty: Sana Biotechnology: Current Employment. Rocca: Sana Biotechnology: Current Employment. Joyce: Sana Biotechnology: Current Employment. Kim: Sana Biotechnology: Current Employment. Vagin: Sana Biotechnology: Current Employment. Kaczmarek: Sana Biotechnology: Current Employment. Chavan: Sana Biotechnology: Current Employment. Jewell: Sana Biotechnology: Current Employment. Lipsitz: Sana Biotechnology: Current Employment. Shamashkin: Sana Biotechnology: Current Employment. Hlavaty: Sana Biotechnology: Current Employment. Rodriguez: Sana Biotechnology: Current Employment. Co: Sana Biotechnology: Current Employment. Cruite: Sana Biotechnology: Current Employment. Ennajdaoui: Sana Biotechnology: Current Employment. Duback: Sana Biotechnology: Current Employment. Elman: Sana Biotechnology: Current Employment. Amatya: Sana Biotechnology: Current Employment. Harding: Sana Biotechnology: Current Employment. Lyubinetsky: Sana Biotechnology: Current Employment. Patel: Sana Biotechnology: Current Employment. Pepper: Sana Biotechnology: Current Employment. Ruzo: Sana Biotechnology: Current Employment. Iovino: Sana Biotechnology: Current Employment. Varghese: Sana Biotechnology: Current Employment. Foster: Sana Biotechnology: Current Employment. Gorovits: Sana Biotechnology: Current Employment. Elpek: Sana Biotechnology: Current Employment. Laska: Sana Biotechnology: Current Employment. McGill: Sana Biotechnology: Current Employment. Shah: Sana Biotechnology: Current Employment. Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Dambach: Sana Biotechnology: Current Employment.

GNB1 encodes a beta subunit (Gβ) of heterotrimeric G proteins, which mediate signals downstream of G protein coupled receptors (GPCRs). We isolated a somatic mutant of GNB1 (K89E) by functional screening of a cDNA library derived from a blastic plasmacytoid dendritic cell neoplasm (BPDCN). A search of cancer genome databases identified recurrent mutations in GNB1 and the highly related protein GNB2. GNB1/2 K89E/T were found in B cell acute lymphoblastic leukemia (B-ALL) (1 case), follicular lymphoma (1) and myelodysplastic syndrome (MDS) (1) as well as BPDCN (1). Interestingly GNB1 K57E/T mutations were found only in myeloid diseases: [acute myeloid leukemia (2), atypical CML (2), polycythemia vera (1) and MDS (6)], while GNB1 I80N/T were found predominantly in B cell diseases [CLL (2), FL (2), DLBCL (1) and MDS (1)]. These mutated codons are all located on the GNB1 protein surface that is critical for interactions between Gβ and alpha subunits (Gα) or downstream effectors. Immunoprecipitation followed by mass spectrometry demonstrated that GNB1 K57E, I80T and K89E mutants failed to bind Gα, including GNAI2/3, GNA11/Q and GNA13 that are normally bound by wild-type (WT) GNB1. All mutations affecting these codons promoted cytokine-independent growth of human TF1 myeloid cells or mouse BaF3 lymphoid cells with activation of MEK/ERK and mTOR/PI3K pathways. Pertussis toxin treatment did not affect GNB1-dependent ERK activation or cell growth, implying a Gα-independent pathway. To investigate the function of GNB1 mutations in vivo, we performed a mouse bone marrow transplantation (BMT) experiment using wild-type and Cdkn2a-deficient donors. Loss of the cell cycle regulator CDKN2A is common in BPDCN, B-ALL, and several other hematologic malignancies. Bone marrow cells were isolated from 5-FU treated donor mice and infected with retrovirus expressing GNB1 WT, K57E, I80T or K89E. Transplantation of GNB1 mutant-expressing Cdkn2a-deficient bone marrow resulted in myeloid dendritic cell neoplasms that were CD11b+, CD11c+, CD19-, B220-, and CD3-. GNB1 mutants did not induce tumors in WT bone marrow after 12 months of observation suggesting that GNB1 requires additional cooperating mutations such as Cdkn2a loss. We performed the same BMT experiment using Cdkn2a-deficient bone morrow cells without 5-FU pretreatment. We found thatGNB1 I80T and K89E mutants induced a progenitor B cell ALL (CD11b-, CD11c-, CD19+, CD3-, TdT+). These data suggest that GNB1 mutations can promote tumorigenesis in more than one cell lineage, as observed in patients. In vivo treatment of the myeloid neoplasm with the dual PI3K/mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, we noted that GNB1 mutations co-occurred with oncogenic kinase alterations, including BCR/ABL, JAK2 V617F and BRAF V600K. Co-expression of patient-derived GNB1 alleles with the mutant kinases resulted in relative resistance to treatment with the corresponding kinase inhibitor in each context. Thus, GNB1 and GNB2 mutations confer transformation and targeted therapy resistance across a range of human tumors and may be targetable with inhibitors of PI3K/mTOR signaling. Disclosures Gotlib: Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel Support Other. Deininger:BMS, Novartis, Celgene, Genzyme, Gilead: Research Funding; BMS, ARIAD, Novartis, Incyte, Pfizer: Advisory Board, Advisory Board Other; BMS, ARIAD, Novartis, Incyte, Pfizer: Consultancy. Tyner:Constellation Pharmaceuticals: Research Funding.