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Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). Over 30 ModPoKI screens across human TCR- and CAR-T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically stimulated CAR-T cells and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate an ∼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram T cell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions.

Precise targeting of large transgenes to T cells using homology-directed repair has been transformative for adoptive cell therapies and T cell biology. Delivery of DNA templates via adeno-associated virus (AAV) has greatly improved knockin efficiencies, but the tropism of current AAV serotypes restricts their use to human T cells employed in immunodeficient mouse models. To enable targeted knockins in murine T cells, we evolved Ark313, a synthetic AAV that exhibits high transduction efficiency in murine T cells. We performed a genome-wide knockout screen and identified QA2 as an essential factor for Ark313 infection. We demonstrate that Ark313 can be used for nucleofection-free DNA delivery, CRISPR-Cas9-mediated knockouts, and targeted integration of large transgenes. Ark313 enables preclinical modeling of Trac-targeted CAR-T and transgenic TCR-T cells in immunocompetent models. Efficient gene targeting in murine T cells holds great potential for improved cell therapies and opens avenues in experimental T cell immunology.

The efficacy of adoptive T cell therapies for cancer treatment can be limited by suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints1,2. Targeted gene editing has the potential to overcome these limitations and enhance T cell therapeutic function3-10. Here we performed multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to identify genes that can be targeted to prevent T cell dysfunction. These screens converged on RASA2, a RAS GTPase-activating protein (RasGAP) that we identify as a signalling checkpoint in human T cells, which is downregulated upon acute T cell receptor stimulation and can increase gradually with chronic antigen exposure. RASA2 ablation enhanced MAPK signalling and chimeric antigen receptor (CAR) T cell cytolytic activity in response to target antigen. Repeated tumour antigen stimulations in vitro revealed that RASA2-deficient T cells show increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent cancer cell killing. RASA2-knockout CAR T cells had a competitive fitness advantage over control cells in the bone marrow in a mouse model of leukaemia. Ablation of RASA2 in multiple preclinical models of T cell receptor and CAR T cell therapies prolonged survival in mice xenografted with either liquid or solid tumours. Together, our findings highlight RASA2 as a promising target to enhance both persistence and effector function in T cell therapies for cancer treatment.

Summary: TRIM21 is an interferon‐stimulated E3 ligase that controls the activity of pattern‐recognition signaling via ubiquitination of interferon regulatory factors and DDX41. Previous studies on the role of TRIM21 in innate immune responses have yielded contradictory results, suggesting that the role of TRIM21 is cell specific. Here, we report that bone‐marrow‐derived macrophages (BMDMs) generated from Trim21−/− mice have reduced expression of mature macrophage markers. Reflecting their reduced differentiation in response to macrophage colony‐stimulating factor (M‐CSF), Trim21−/− BMDMs had decreased expression of M‐CSF signature genes. Although Trim21−/− BMDMs responded normally to Toll‐like receptor 9 (TLR9) activation, they produced lower levels of pro‐inflammatory cytokines in response to the TLR2 agonist PAM3CSK4. In line with this, the response to infection with the Bacillus Calmette–Guérin strain of Mycobacterium bovis was also diminished in Trim21−/− BMDMs. Our results indicate that TRIM21 controls responses to TLR2 agonists. [ABSTRACT FROM AUTHOR]

Discovering the role of fibroblasts residing in the tumor microenvironment (TME) requires controlled, localized perturbations because fibroblasts play critical roles in regulating immunity and tumor biology at multiple sites. Systemic perturbations can lead to unintended, confounding secondary effects, and methods to locally genetically engineer fibroblasts are lacking. To specifically investigate murine stromal cell perturbations restricted to the TME, we developed an adeno-associated virus (AAV)-based method to target any gene-of-interest in fibroblasts at high efficiency (>80%). As proof of concept, we generated single (sKO) and double gene KOs (dKO) of Osmr , Tgfbr2 , and Il1r1 in cancer-associated fibroblasts (CAFs) and investigated how their cell states and those of other cells of the TME subsequently change in mouse models of melanoma and pancreatic ductal adenocarcinoma (PDAC). Furthermore, we developed an in vivo knockin-knockout (KIKO) strategy to achieve long-term tracking of CAFs with target gene KO via knocked-in reporter gene expression. This validated in vivo gene editing toolbox is fast, affordable, and modular, and thus holds great potential for further exploration of gene function in stromal cells residing in tumors and beyond., Competing Interests: M.F.K. is a founder and shareholder of FOUNDERY innovations. J.E. is a compensated co-founder at Mnemo Therapeutics and a compensated scientific advisor to Cytovia Therapeutics. J.E. owns stocks in Mnemo Therapeutics and Cytovia Therapeutics. J.E. has received a consulting fee from Casdin Capital, Resolution Therapeutics and Treefrog Therapeutics. The J.E. lab has received research support from Cytovia Therapeutics, Mnemo Therapeutics, and Takeda Pharmaceutical Company.

Adoptive chimeric antigen receptor T-cell (CAR-T) therapy is transformative and approved for hematologic malignancies. It is also being developed for the treatment of solid tumors, autoimmune disorders, heart disease, and aging. Despite unprecedented clinical outcomes, CAR-T and other engineered cell therapies face a variety of manufacturing and safety challenges. Traditional methods, such as lentivirus transduction and electroporation, result in random integration or cause significant cellular damage, which can limit the safety and efficacy of engineered cell therapies. We present hydroporation as a gentle and effective alternative for intracellular delivery. Hydroporation resulted in 1.7- to 2-fold higher CAR-T yields compared to electroporation with superior cell viability and recovery. Hydroporated cells exhibited rapid proliferation, robust target cell lysis, and increased pro-inflammatory and regulatory cytokine secretion in addition to improved CAR-T yield by day 5 post-transfection. We demonstrate that scaled-up hydroporation can process 5 x 10 8 cells in less than 10 s, showcasing the platform as a viable solution for high-yield CAR-T manufacturing with the potential for improved therapeutic outcomes., Competing Interests: B.J.S., S.B., F.F., L.H.W., and R.S.P. are or were employed by and have an equity interest in Indee Labs. L.M.R.F., J.B., L.L., F.L.P. and J.E. are or were consultants to Indee Labs. R.S.P. is an investor in and a venture partner at both Pioneer Fund and Axial, which have a financial interest in Indee Labs. The Ferreira Lab received support from Indee Labs as a subaward from the National Institute of Diabetes and Digestive and Kidney Diseases (Grant No. 1R43DK133029-01). Indee Labs has a commercial interest in developing patents related to Hydropore™ (WO2016109864A1 & WO2019084624A1). M.F. and R.B. are or were employed by and have an equity interest in CellChorus. CellChorus received support from the National Center for Translational Sciences (R44TR005137) and the National Institute of General Medical Sciences (R44GM149106) of the National Institutes of Health and the National Science Foundation (NSF2229323). CellChorus has a commercial interest in developing the TIMING assay. J.E. is a compensated co-founder at Mnemo Therapeutics; owns stocks in Mnemo Therapeutics and Cytovia Therapeutics; is a compensated scientific advisor for Enterome, Treefrog Therapeutics and Resolution Therapeutics; and is a holder of patents pertaining to but not resulting from this work. The Eyquem Lab received research support from Cytovia Therapeutic, Mnemo Therapeutics, Takeda and Indee Labs as a subaward from the National Cancer Institute (Contract No. 7591022C00053).