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Abstract Autologous chimeric antigen receptor (CAR) T cell therapies have revolutionized the treatment of B cell malignancies and multiple myeloma, with emerging potential in autoimmune disorders. However, current approaches face limitations, including complex manufacturing, high costs, variable product quality, and risks associated with lymphodepletion. Allogeneic CAR-T cells present additional challenges, such as graft-versus-host disease (GVHD), donor selection constraints, and limited cell persistence, leading to less clinical efficacy. In vivo CAR T generation is an alternative strategy to the ex vivo cell engineering utilized in all currently approved CAR T cell therapies. Regardless of the delivery vehicle, all in vivo genetic engineering approaches share a key challenge—the precision delivery of the genetic medicine to the intended target cell type. To address these limitations, we developed a novel platform for in vivo CAR-T cell generation using an engineered third-generation lentiviral vector. This platform features rationally designed fusogen and binder moieties for precise T cell targeting. Utilizing mutated cocal glycoprotein, its native receptor LDL-R was disrupted without affecting its fusogenic potential. This enables the retargeting of lentiviral vectors to specific T cells via a high-affinity CD7 binder molecule provided as a second envelope protein. Viral vectors encoding a green fluorescent protein (GFP) transgene were used to show the specificity of T cell transduction. This non-replicating, self-inactivating lentiviral vector was used to deliver transgene encoding CARs against BCMA with humanized VHH nanobody (IASO 206), CD19 with humanized scFv (IASO 207) and CD20 with humanized VHH nanobody (IASO 208) to T cells through CD7 mediated T cell transduction under a high yield T cell specific promotor/enhancer to ensure T cell specific expression of CAR structures. Furthermore, the reduction of macrophage phagocytosis and the T cell mediated viral vector elimination were achieved by CD47expression and the knocking out of MHC I gene, respectively, to extend the lifespan of lentiviral vector dosed in vivo. IASO 207 and 208 were found to specifically transduce T cells and generate functional CAR T cells capable of killing B cells using donor-derived PBMC. In vivo delivery of IASO 207 and 208 to HSC engrafted humanized mice via single tail vein injection resulted in depletion of both circulating and spleen B cells. Similarly, in vivo delivering of IASO 206 to human PBMC reconstituted NPG mice via single tail vein injection resulted in tumor regression of RPMI-8226 multiple myeloma xenograft without showing any mice toxicity. Taking together, these results validate our platform's ability to generate functional CAR-T cells in vivo, offering a promising off-the-shelf solution for relapsed/refractory multiple myeloma, NHL, and severe autoimmune diseases.