Biology subjects

Launspach, M.

Publications and source records attributed to Launspach, M..

5 recordsLinked to original sources

bioRxiv2026

CRISPR-dPCR: A unified multiplex digital PCR framework for quantitative characterization of targeted knock-in

Accurate characterization of CRISPR knock-in requires distinguishing site-specific integration from total donor abundance and donor-derived background while measuring editing at the target site. Here, we present CRISPR-dPCR, a modular multiplex digital PCR framework combining a locked nucleic acid-enhanced cut-site assay with junction-based detection and two orthogonal short-amplicon strategies: Poisson linkage and homology-arm subtraction. We evaluated assay modules across genomic loci, neuroblastoma models and primary human T cells, with selected assays compared on two dPCR platforms. In genotype-defined clones and mixtures with low residual donor, homology-arm subtraction most closely recovered expected knock-in abundance. Linkage provided highly specific evidence of target-cargo association without junction-spanning amplification, whereas junction assays supplied boundary-specific confirmation but were more sensitive to amplicon length and multiplexing. Five-channel panels integrated cut-site editing, donor abundance, copy-number normalization and site-specific knock-in in a single reaction. Residual donor DNA limited quantitative interpretation, particularly in early post-editing samples. Modular extensions quantified defined unintended outcomes, including chromosomal translocations. CRISPR-dPCR integrates complementary measurements into a scalable analytical framework for genome-editing research, cell and gene therapy product development and applications in longitudinal clinical monitoring of targeted integration and transgene persistence.

molecular biology↗

bioRxiv2026

A lipid nanoparticle platform for high yield CRISPR-targeted homology directed repair enables fully non-viral CAR T cell generation

CRISPR-mediated homology directed repair (HDR) enables targeted CAR integration with improved fitness and therapeutic potential of CAR T cells. However, current methods for generating HDR-engineered CAR T cells rely on viral transduction or electroporation, approaches that limit global implementation and constrain patient access due to their cost, toxicity, and requirement for centralized manufacturing. Through a screen of ionizable lipids, we identified LNP systems that enable CRISPR-mediated gene knock-in (KI) in primary human T cells and are amenable to hand mixing by ethanol injection as a research tool or machine formulation for larger scale manufacturing. Modifying the linear dsDNA HDR template with truncated Cas9 target sequences (tCTS) enhanced HDR rates across multiple LNP systems. We optimized two LNP formulations capable of HDR-mediated KI of a large 4kB CD19 CAR-EGFR HDR template into the TRAC locus with rates of [≥]8% and >10x improved edited cell yields compared to electroporation. We demonstrate that LNP-generated CAR T cells exhibited similar growth kinetics, activation states, differentiation states, and killing capacity compared to electroporation-generated CAR T cells. Our LNP platform components are fully disclosed, commercially sourced, and enable efficient fully non-viral CRISPR-HDR cell engineering across diverse applications.

bioengineering↗

bioRxiv2026

A Non-Viral CRISPR/Cas9 HDR Platform for Stable Engineering of Solid Tumor Models.

Virus-free genome engineering provides a flexible alternative to viral vectors for generating genetically modified cell models. Here, we establish an integrated biosafety level 1-compatible CRISPR/Cas9 homology-directed repair (HDR) workflow for stable transgene knock-in in neuroblastoma cell lines using non-viral delivery approaches. We systematically evaluated donor cassette architecture and delivery conditions across electroporation-based Cas9 ribonucleoprotein (RNP) delivery and lipid nanoparticle (LNP)-mediated co-delivery of Cas9 mRNA, sgRNA, and donor DNA. Modular AAVS1-targeting donor constructs identified a compact EF1(s)-Donor-Q8-Tag-sPA cassette that consistently yielded the strongest HDR-associated knock-in readouts, achieving up to 60% stable reporter-positive cells following electroporation without HDR enhancers. While LNP-mediated delivery enabled efficient CRISPR cargo co-delivery and generation of genetically modified tumor cell populations, knock-in efficiencies remained lower than those observed with electroporation. Subsequent enrichment approaches enabled generation of highly pure edited cell populations following both delivery strategies. Functional validation demonstrated stable transgene expression in vitro, including in three-dimensional bioprinted tumor models, and in vivo in xenograft mice without impairing tumor growth or viability. Together, these findings establish a practical non-viral HDR platform for stable engineering of solid tumor models and provide a framework for further optimization of genome editing workflows across distinct delivery modalities. Key findings- We establish a complete, virus-free CRISPR/Cas9 HDR workflow that reliably enables stable knock-in in solid tumor cell lines, demonstrated here in two neuroblastoma models under biosafety level 1 conditions. - We establish and evaluate LNP-mediated co-delivery of Cas9 mRNA, gRNA, and donor DNA for non-viral HDR knock-in in solid tumor models, revealing delivery modality-specific differences in editing efficiency, toxicity, and expression dynamics. - By systematically varying donor architectures, we identify a compact HDR template - combining a shortened custom EF1 promoter, the minimal Q8 surface reporter, and a synthetic polyadenylation signal (sPA) - that markedly improves knock-in efficiency in solid tumor cell lines, outperforming conventional cassettes. - Virus-free edited tumor cells generated using this workflow retain stable transgene expression and functional fitness in 3D bioprinted tumor constructs and xenograft mouse models, directly linking in vitro knock-in optimization to in vivo relevance. - The resulting biosafety level 1 compatible, end-to-end pipeline - integrating donor design, digital PCR-based quantification of precise integration, and enrichment strategies-offers a practical and transferable platform for engineering transgenic solid tumor models without viral vectors.

bioengineering↗

bioRxiv2025

Personalized CRISPR Knock-In Cytokine Gene Therapy to Remodel the Tumor Microenvironment and Enhance CAR T Cell Therapy in Solid Tumors

The immunosuppressive tumor microenvironment (TME) remains a central barrier to effective immunotherapy in solid tumors. To address this, we developed a novel gene therapeutic strategy that enables localized remodeling of the TME via tumor-intrinsic cytokine expression. Central to this approach is CancerPAM, a multi-omics bioinformatics pipeline that identifies and ranks patient-specific, tumor-exclusive CRISPR-Cas9 knock-in sites with high specificity and integration efficiency. Using neuroblastoma--a pediatric solid tumor with a suppressive TME--as a model, we applied CancerPAM to sequencing data from cell lines and patients to identify optimal integration sites for pro-inflammatory cytokines (CXCL10, CXCL11, IFNG). CRISPR-mediated CXCL10 knock-in into tumor cells significantly enhanced CAR T cell infiltration and antitumor efficacy both in vitro and in vivo. In vivo, CXCL10-expressing tumors showed significantly increased early CAR T cell infiltration and prolonged survival compared to controls. CancerPAM rankings correlated strongly with target-site specificity and knock-in efficiency, validating its predictive performance. Our findings establish CancerPAM as a powerful tool for safe and effective CRISPR-based interventions and provide a conceptual framework for integrating cytokine-driven TME remodeling with cellular immunotherapies. This personalized strategy holds promise for enhancing CAR T cells and other immunotherapies across immune-refractory solid tumors.

bioengineering↗

bioRxiv2025

Revisiting the need for mRNA nucleoside modification in CAR T cell engineering

mRNA-based chimeric antigen receptor (CAR)-T cells offer the promise of enhanced safety and simplified manufacturing. However, in vitro-transcribed (IVT) mRNA is known to trigger antiviral immune responses, inflammatory signaling, and apoptosis in transfected cells. To address these challenges and enable efficient IVT-mRNA expression, modified nucleosides, such as N1-methyl-pseudouridine (m1{Psi}), have become the gold standard for CAR-T cell production, albeit at increased cost. In this study, immune responses to IVT-mRNA were evaluated across five primary human cell types, including T-cells. Unexpectedly, T-cells, unlike other immune and non-immune cell types tested, exhibited no immune activation in response to unmodified mRNA. T-cell viability and cytokine secretion patterns remained unaffected, regardless of whether unmodified mRNA was delivered via lipid nanoparticles (LNPs) or electroporation. Furthermore, CAR expression levels in T-cells were not influenced by mRNA modification with m1{Psi} or 5-methoxy-uridine (5moU) nucleosides. The absence of nucleoside modifications did not compromise CAR-T cell cytotoxic potency, demonstrating that such modifications are not required for producing functional CAR-T cells. These findings eliminate the need for nucleoside modification in T-cell mRNA, simplifying and reducing the cost of CAR-T cell manufacturing while positioning IVT-mRNA as a highly efficient and minimally invasive tool for CAR-T cell engineering.

bioengineering↗