Chimeric antigen receptor (CAR) T cells have demonstrated their efficacy in treating blood-based cancers. However, the durability of responses is often hindered by challenges related to long-term T cell persistence and engraftment. The success of CAR T cell immunotherapy relies on the differentiation status and overall fitness of the CAR T cell product. Current protocols involve the activation and ex vivo expansion of patient T cells, however, activation leads to irreversible differentiation, compromising their therapeutic potency. Our recent work showed that a manufacturing protocol utilizing non-activated T cells results in superior differentiation characteristics and reduced exhaustion, with concordant benefits in long-term tumor control. Nevertheless, as quiescent T cells are highly resistant to lentiviral infection, CAR T manufacturing yield is a significant limitation with non-activated T cells. The goal of this study is to harness the intrinsic stemness qualities of non-activated CAR T cells and improve their transduction efficiency and effector function, thereby enhancing their durable efficacy following infusion. Quiescent T cells initiate a type I interferon (IFN1)-mediated innate response upon lentiviral vector transduction, which limits CAR T cell transduction efficiency. Our preliminary data indicate that pre-treatment of non-activated T cells with an IFN1-binding protein enhances CAR T cell transduction efficiency, and promotes a more naïve and central memory phenotype. This research will delve into the impact of IFN1 blockade on CAR lentivirus transduction and function of non-activated T cells, both in vitro and in xenograft models in vivo. Given that sustained type I IFN signaling facilitates tumor immune escape and resistance to therapies, we hypothesize that continuous IFN1 blockade not only enhances T cell fitness by inhibiting the innate response to the lentiviral vector but also amplifies the therapeutic efficacy of T cells in tumors reliant on IFN-mediated immune evasion. To explore this further, we will evaluate the effect of sustained IFN1 blockade by constructing lentiviral transfer plasmids encoding both a CAR and a secreted anti-IFN1 binding protein in various xenograft models of cancer. Another key aspect of our investigation is the interplay between SAMHD1 and IFN1 signaling pathways in quiescent T cells. SAMHD1 restricts nucleotide availability for reverse transcription and is upregulated by IFN1. Given that Vpx, a component of natural HIV, degrades SAMHD1, we will assess the impact of Vpx incorporation on the efficiency of reverse transcription and vector integration of CAR lentivirus in non-activated T cells. Our hypothesis is that restoring Vpx, which targets rate-limiting steps of the viral transduction pathway, will synergize with IFN1 inhibition in quiescent T cells, ultimately enhancing lentiviral transduction efficiency and bolstering the function in non-activated CAR T cells. These studies represent a signif...