Biology subjects

Slipek, N. J.

Publications and source records attributed to Slipek, N. J..

6 recordsLinked to original sources

bioRxiv2025

A Singular Base Editing Platform for Polyfunctional Multiplex Engineering of Immune Cells

Current methods to engineer antigen-specific receptors rely on randomly integrating vectors or double-strand break induced targeted integration, both of which pose safety risks. To implement an all-in-one tool for multiplex knockout (KO) and knock in (KI), we expand the use of cytosine and adenine base editor (ABE) nickase activity to stimulate homology-directed repair (HDR) and insert clinically relevant chimeric antigen receptors (CARs) into specific loci. Through a novel sgRNA design strategy and a recombinant adeno-associated virus (rAAV) delivered DNA template, we enhanced the efficiency of ABE8e-stimulated HDR in human T cells. By combining KI of CD19-, CD33-, or mesothelin-targeting CARs with >95% quadplex gene KO (B2M/CD3{varepsilon}/PDCD1/CISH), we achieve single-step generation of highly functional off-the-shelf CAR T cell products with enhanced function. Importantly, we found no detectable translocations or significant off-target edits and demonstrated efficacy against multiple cancer lines, and a suppressive 3D spheroid culture model. This efficient engineering process of Iterative Nicking for Synchronous Engineered Reprogramming of T cells (INSERT) establishes a safe, simplified platform for advanced therapeutic CAR T engineering.

bioengineering↗

bioRxiv2024

Engineering Memory T Cells as a platform for Long-Term Enzyme Replacement Therapy in Lysosomal Storage Disorders

Enzymopathy disorders are the result of missing or defective enzymes. Amongst these enzymopathies, mucopolysaccharidosis type I, is a rare genetic lysosomal storage disorder caused by mutations in the gene encoding alpha-L-iduronidase (IDUA), ultimately causes toxic build-up of glycosaminoglycans (GAGs). There is currently no cure and standard treatments provide insufficient relief to the skeletal structure and central nervous system (CNS). Human memory T cells (Tm) migrate throughout the bodys tissues and can persist for years, making them an attractive approach for cellular-based, systemic enzyme replacement therapy. Here, we tested genetically engineered, IDUA-expressing Tm as a cellular therapy in an immunodeficient mouse model of MPS I. Our results demonstrate that a single dose of engineered Tm leads to detectable IDUA enzyme levels in the blood for up to 22 weeks and reduced urinary GAG excretion. Furthermore, engineered Tm take up residence in nearly all tested tissues, producing IDUA and leading to metabolic correction of GAG levels in the heart, lung, liver, spleen, kidney, bone marrow, and the CNS. Our study indicates that genetically engineered Tm holds great promise as a platform for cellular-based enzyme replacement therapy for the treatment of mucopolysaccharidosis type I and potentially many other enzymopathies and protein deficiencies.

bioengineering↗

bioRxiv2024

Precision Enhancement of CAR-NK Cells through Non-Viral Engineering and Highly Multiplexed Base Editing

Natural killer (NK) cells unique ability to kill transformed cells expressing stress ligands or lacking major histocompatibility complexes (MHC) has prompted their development for immunotherapy. However, NK cells have demonstrated only moderate responses against cancer in clinical trials and likely require advanced genome engineering to reach their full potential as a cancer therapeutic. Multiplex genome editing with CRISPR/Cas9 base editors (BE) has been used to enhance T cell function and has already entered clinical trials but has not been reported in human NK cells. Here, we report the first application of BE in primary NK cells to achieve both loss-of-function and gain-of-function mutations. We observed highly efficient single and multiplex base editing, resulting in significantly enhanced NK cell function. Next, we combined multiplex BE with non-viral TcBuster transposon-based integration to generate IL-15 armored CD19 CAR-NK cells with significantly improved functionality in a highly suppressive model of Burkitts lymphoma both in vitro and in vivo. The use of concomitant non-viral transposon engineering with multiplex base editing thus represents a highly versatile and efficient platform to generate CAR-NK products for cell-based immunotherapy and affords the flexibility to tailor multiple gene edits to maximize the effectiveness of the therapy for the cancer type being treated.

bioengineering↗

bioRxiv2021

Non-Viral Engineering of CAR-NK and CAR-T cells using the Tc Buster Transposon System™

Cancer immunotherapy using T cells and NK cells modified with viral vectors to express a chimeric antigen receptor (CAR) has shown remarkable efficacy in treating hematological malignancies in clinical trials. However, viral vectors are limited in their cargo size capacity, and large-scale manufacturing for clinical use remains complex and cost prohibitive. As an alternative, CAR delivery via DNA transposon engineering is a superior and cost-effective production method. Engineering via transposition is accomplished using a two-component system: a plasmid containing a gene expression cassette flanked by transposon inverted terminal repeats (ITRs) paired with a transposase enzyme that binds to the ITRs, excises the transposon from the plasmid, and stably integrates the transposon into the genome. Here, we used the newly developed hyperactive Tc Buster (Bio-Techne) transposon system to deliver a transposon containing a multicistronic expression cassette (CD19-CAR, mutant DHFR, and EGFP) to primary human peripheral blood (PB) NK cells and T cells. We optimized methods to avoid DNA toxicity and maximize efficiency. Our cargo contained a mutant dihydrofolate reductase (DHFR) which allowed us to enrich for stable transposon integration using methotrexate (MTX) selection. We then tested CAR-NK and CAR-T cells in functional assays against CD19-expressing Raji cells. CAR-expressing NK and T cells produced significantly more cytokines than CAR-negative controls and efficiently killed target cells. We recognize that cryopreservation manufactured CAR-expressing cells will be necessary for clinical translation. We observed reduced cytotoxicity of CAR-NK cells immediately after thaw, but increasing the NK dose overcame this loss of function. Our work provides a platform for robust delivery of multicistronic, large cargo via transposition to primary human NK and T cells. We demonstrate that CAR-expressing cells can be enriched using MTX selection, while maintaining high viability and function. This non-viral approach represents a versatile, safe, and cost-effective option for the manufacture of CAR-NK and CAR-T cells compared to viral delivery.

bioengineering↗

bioRxiv2020

Internal checkpoint regulates T cell neoantigen reactivity and susceptibility to PD1 blockade

While neoantigen-specific tumor infiltrating lymphocytes (TIL) can be derived from in antigen-expressing tumors, their adoptive transfer fails to consistently elicit durable tumor regression. There has been much focus on the role of activation/exhaustion markers such as PD1, CD39 and TOX in TIL senescence. We found these markers were inversely expressed to Cytokine-Induced SH2 protein (CISH), a negative regulator of TCR signaling and tumor immunity in mice. To evaluate the physiological role of CISH in human TIL we developed a high-efficiency CRIPSR-based method to knock out CISH in fully mature TIL. CISH KO resulted in increased T cell receptor (TCR) avidity, tumor cytolysis and neoantigen recognition. CISH expression in the tumor resections correlated with TIL inactivity against p53 hotspot mutations and CISH KO in TIL unmasked reactivity against these universal neoantigens. While CISH KO resulted in T cell hyperactivation and expansion it did not alter maturation, perhaps by preferential PLC{gamma}-1 and not AKT inhibition. Lastly, CISH KO in T cells increased PD1 expression and the adoptive transfer of Cish KO T cells synergistically combines with PD1 antibody blockade resulting in durable tumor regression and survival in a preclinical animal model. These data offer new insights into the regulation of neoantigen recognition, expression of activation/exhaustion markers, and functional/maturation signals in tumor-specific T cells.

immunology↗

bioRxiv2018

Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors

Chimeric antigen receptor engineered T cell (CAR-T) immunotherapy has shown efficacy against a subset of hematological malignancies1,2, yet its autologous nature and ineffectiveness against epithelial and solid cancers limit widespread application. To overcome these limitations, targeted nucleases have been used to disrupt checkpoint inhibitors and genes involved in alloreactivity3-6. However, the production of allogeneic, \"off-the-shelf\" T cells with enhanced function requires multiplex genome editing strategies that risk off-target effects, chromosomal rearrangements, and genotoxicity due to simultaneous double-strand break (DSB) induction at multiple loci7-10. Moreover, it has been well documented that DSBs are toxic lesions that can drive genetic instability11,12. Alternatively, CRISPR/Cas9 base editors afford programmable enzymatic nucleotide conversion at targeted loci without induction of DSBs13,14. We reasoned this technology could be used to knockout gene function in human T cells while minimizing safety concerns associated with current nuclease platforms. Through systematic reagent and dose optimization, we demonstrate highly efficient multiplex base editing and consequent protein knockout in primary human T cells at loci relevant to the generation of allogeneic CAR-T cells including the T cell receptor constant (TRAC) locus, {beta}-2 microglobulin (B2M), and programmed cell death 1 (PDCD1). Multiplex base edited T cells equipped with a CD19 CAR killed target cells more efficiently; and importantly, both DSB induction and translocation frequency were greatly reduced compared to cells engineered with Cas9 nuclease. Collectively, our results establish a novel multiplex gene editing platform to enhance both the safety and efficacy of engineered T cell-based immunotherapies.

genetics↗