Project summary Adoptive cell therapy has revolutionized cancer treatment and has immense potential to cure a variety of cancer types. Multiple critical barriers exist for current CAR-T therapy particularly in solid tumor, including insufficient T cells trafficking to the cancer site, lack of effective cancer cell killing, severe toxicity, strong immunosuppression in the tumor microenvironment, and lack of persistence. Overcoming these barriers is critical to achieve the full potential of CAR-T. To overcome these challenges, it is important to identify gene targets that can boost multiple features of T cells function, especially from fully unbiased genetic screens performed in primary T cells. We have recently developed such T cell CRISPR-based screening approaches. These genetic screens, coupled with stringent selection of T cells for properties – including enhanced potency, persistence and in vivo infiltration/activity – have identified gene modifications that maximally enhance desired T cell properties. We conducted knock-out and gain of function in vitro and in vivo CRISPR screening in primary T cells and identified multiple novel negative/positive regulators of T cell function, in animal tumor models using different CAR-Ts (e.g. CD19, CD22 and HER-2 CARs). We identified two especially potent gene modifications that augment CART-Ts: 1) PRODH2 overexpression resulting in metabolic boost and 2) PRDM1 (exon 3) deletion resulting in epigenetic modulation. This initial research and methods pave the way for further enhancements. Our central hypothesis is that engineering CAR-T cells with a metabolic boost from PRODH2 overexpression as well as epigenetic regulation by PRDM exon3 deletion can enhance HER2 CAR-T cell performance and enhance anti-tumor efficacy in vivo. To do this, we will utilize our knock-in/knock-out (KIKO) protocol to introduce CAR and gene modification sequences precisely at the TRAC locus to generate four HER2 CAR-Ts: PRODH2- OE, PRDM1-KO, PRODH2-OE+PRDM1-KO and control CAR-T. The in vitro characterization (level of CAR expression, proliferation, immune phenotyping, serial killing ability) as well as in vivo evaluations (efficacy, CAR- T infiltration and persistence) of each of the 4 CAR-Ts will be performed using at least two cancer cell lines/mouse model. Our vector and manufacturing processes in this proposal are identical to the planned GMP clinical approach, allowing smooth transition to clinical development. If successful, this study will identify our best performing HER2 CART and provide sufficient data to support a Phase II SBIR submission, and allow a path towards an IND and first-in-human Phase 1 clinical trial, thus delivering a superior CAR-T candidate for patients with solid tumors. In broad terms, the major goal of this study is to evaluate if metabolic boost and epigenetic reprograming when combined result in more effective CAR-Ts relative to either modification alone.