A Novel Cell Selective Lentiviral Platform for in vivo CAR-T Engineering

Selective gene delivery to defined cell populations using targeted lentiviral vectors (LVV) represents a promising strategy for next‑generation immunotherapies such as chimeric antigen receptor (CAR) T‑cell therapy. While conventional CAR-T cell manufacturing requires multiple ex vivo manipulation steps up to two weeks, targeted LVVs can enable a short ex vivo manufacturing process or direct in vivo generation of functional CAR-T cells with substantial lower complexity and cost, improving accessibility to the therapy.

We have developed a novel paramyxovirus (PV)‑pseudotyped LVV platform designed for efficient gene transfer into non‑activated T cells ex vivo and in vivo. In this system, viral binding and fusion are mediated by modified hemagglutinin (H) and fusion (F) glycoproteins respectively. Mutation of H abolishes natural tropism, while retargeting is achieved by fusing cell‑specific single‑chain variable fragments (scFvs) or heavy‑chain variable domains (VHHs), enabling targeted transduction.

Using our high titer packaging cell line and an optimized transfection process, we produced CD4‑, CD8‑, and pan‑T‑cell–specific LVV, which efficiently generated CAR-T cells in vitro and in vivo in a PBMC-transplanted NSG mouse model. Importantly, these CAR-T cells led to robust tumor lysis in vitro and rapid tumor B-cell depletion in vivo, highlighting the clinical feasibility of this approach.

In summary, our optimized targeted LVV platform enables selective and rapid generation of defined CAR-T cell subsets, providing a foundation for safe, cost-effective and accessible CAR-T cell therapies.