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SummaryT cells are the central effectors and regulators of the adaptive immune response. This protocol provides a step-by-step approach for isolating and enriching total T cells by negative selection from complex murine tissues, including bone marrow (BM), liver, heart, and kidneys. We describe steps for tissue harvesting, preparation of single-cell suspensions, and immunomagnetic enrichment. We then outline procedures for flow cytometric assessment of cell purity and viability. This protocol enables efficient recovery of high-quality T cells for reliable downstream analyses. Graphical abstract Highlights•Isolation of leukocytes from murine BM, liver, heart and kidneys•Non-enzymatic dissociation of kidney and heart tissue•Protocol for immunomagnetic enrichment of T cells•Flow cytometry analysis of T cell purity and viability Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. T cells are the central effectors and regulators of the adaptive immune response. This protocol provides a step-by-step approach for isolating and enriching total T cells by negative selection from complex murine tissues, including bone marrow (BM), liver, heart, and kidneys. We describe steps for tissue harvesting, preparation of single-cell suspensions, and immunomagnetic enrichment. We then outline procedures for flow cytometric assessment of cell purity and viability. This protocol enables efficient recovery of high-quality T cells for reliable downstream analyses.

Background and Objectives: Recent studies have identified variants in the kinesin family member 5A (KIF5A) gene that predispose to amyotrophic lateral sclerosis (ALS). These ALS-linked KIF5A variants lead to the exclusion of exon 27, resulting in the production of a mutated protein with an altered C-terminal region (KIF5A ΔExon27). Through whole genome sequencing, we identified a novel KIF5A intronic variant, rs1057522322 (c.2993-6C > A; chr12:57582596C > A, GRCh38.p14), in a family segregating ALS. Our goal is to investigate the effect of this variant on exon 27 splicing and to assess its functional consequences on KIF5A-mediated cargo transport. Methods: Induced pluripotent stem cells (iPSCs) were generated from siblings with and without the c.2993-6C > A variant. RT-PCR was performed on RNA extracted from iPSC-derived neurons to assess exon 27 splicing. Functional studies were conducted on iPSC-derived motor neurons (MNs). Results: RT-PCR confirmed that the c.2993-6C > A variant induced exon 27 skipping in KIF5A. Immunofluorescent staining showed that KIF5A ΔExon27 abolished the axonal interaction with splicing factor proline- and glutamine-rich, a cargo specifically transported by KIF5A. Under stress conditions, MNs carrying the c.2993-6C > A variant exhibited TDP-43 proteinopathy. Discussion: KIF5A intronic variant c.2993-6C > A could be a risk factor for ALS. KIF5A ΔExon27 impairs KIF5A-mediated cargo transport and contributes to ALS pathogenesis in a TDP-43–dependent manner.

Most cancer patients diagnosed with late-stage head and neck squamous cell carcinoma are treated with chemoradiotherapy, which can lead to toxicity. One potential alternative is tumor-limited conversion of a prodrug into its cytotoxic form. We reason this could be achieved by transient and tumor-specific expression of purine nucleoside phosphorylase (PNP), an Escherichia coli enzyme that converts fludarabine into 2-fluoroadenine, a potent cytotoxic drug. To efficiently express bacterial PNP in tumors, we evaluate 44 chemically distinct lipid nanoparticles (LNPs) using species-agnostic DNA barcoding in tumor-bearing mice. Our lead LNP, designated LNP intratumoral (LNPIT), delivers mRNA that leads to PNP expression in vivo. Additionally, in tumor cells transfected with LNPIT, we observe upregulated pathways related to RNA and protein metabolism, providing insight into the tumor cell response to LNPs in vivo. When mice are treated with LNPIT-PNP, then subsequently given fludarabine phosphate, we observe anti-tumor responses. These data are consistent with an approach in which LNP-mRNA expression of a bacterial enzyme activates a prodrug in solid tumors. Lipid nanoparticles (LNPs) delivering mRNA after intratumoral administration could be a promising cancer treatment strategy. Here this group reports the intratumoral delivery of mRNA with LNPs inducing the expression of purine nucleoside phosphorylase and inhibiting the progression of head and neck squamous cell carcinoma in vivo.