Biology subjects

Snyder, G.

Publications and source records attributed to Snyder, G..

7 recordsLinked to original sources

bioRxiv2025

Allogeneic Polyclonal CD38KO/CD38-CAR γδT Cells For The Treatment Of T Cell Malignancies

Relapsed and refractory T cell malignancies are associated with poor clinical outcomes. Autologous sources of {beta}T cells have been employed for chimeric antigen receptor (CAR) therapies to eliminate the potential for graft-vs-host disease (GvHD). However, the application of CAR-T therapy for T-ALL has been hindered by an inability to obtain sufficient healthy {beta}T cells from patients combined with fratricide due to concurrent antigen expression on normal T cells. Here, we genetically engineered polyclonal {gamma}{delta}T cells, which do not cause GvHD, as an allogeneic source for cancer immunotherapy targeting the pancancer antigen CD38. Utilizing a novel expansion protocol in combination with CRISPR/AAV gene editing, we developed CD38KO/CD38-CAR polyclonal {gamma}{delta}T cells that target T-ALL. Our editing strategy enabled site-directed, on-target insertion of the CD38-CAR transgene into the CD38 locus, with no evidence of significant random CAR DNA integration (as commonly seen with lentiviral CAR transduction) or chromatin abnormalities resulting from CRISPR editing. This enhanced targeting effectively mitigated fratricide through simultaneous CD38 disruption and CAR expression. We demonstrated the efficacy of the CD38KO/CD38-CAR {gamma}{delta}T cells in vitro across multiple patient-derived T-ALL samples collected at baseline and relapse. In vivo, a single injection of CD38KO/CD38-CAR {gamma}{delta}T cells without exogenous cytokine support resulted in potent anti-leukemic efficacy. Fratricide-resistant CD38KO/CD38-CAR polyclonal {gamma}{delta}T cells thus represent a promising off-the-shelf therapeutic platform for T cell malignancies and other CD38-expressing cancers. Key PointsO_LIHybrid pan-{gamma}{delta}TCR antibody/mbIL21-41BBL feeder expansion yields high-purity, polyclonal {gamma}{delta}T cells suitable for CRISPR/AAV editing. C_LIO_LIOn-target CD38-CAR knock-in with simultaneous CD38 knockout prevents fratricide and enables potent T-ALL killing in vitro and in vivo. C_LI

immunology↗

bioRxiv2025

REMEDY: REpair of heterozygous Mutations independent of Exogenous Donor template with high efficiency using allele specific CRISPR targeting and HDR enhancers

We report the development of REMEDY (REpair of heterozygous Mutations independent of Exogenous Donor template with high efficiencY), a genome editing strategy that allows efficient repair of heterozygous mutations in human and mouse cells without necessitating an exogenous donor DNA template. Here, we used in-PAM or near-PAM CRISPR strategies to induce a double-strand break (DSB) in mutant alleles. Following the DSB, the wild-type homologous chromosome itself serves as an endogenous DNA donor template and initiates the correction of the mutant allele. Concurrently treating the cells with HDR enhancers, such as AZD7648, further improved the efficiency of the correction. We demonstrated the utility of REMEDY in the context of six different diseases with heterozygous mutations such as IBMPFD, cystic fibrosis, progeria, ITPR3-associated combined immunodeficiency, ACTA1, and TBCD in human patient derived primary cells and complementary mouse model cell lines. O_FIG O_LINKSMALLFIG WIDTH=91 HEIGHT=200 SRC="FIGDIR/small/641201v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@5f6c41org.highwire.dtl.DTLVardef@bc50d5org.highwire.dtl.DTLVardef@15edca2org.highwire.dtl.DTLVardef@e74ada_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

genetics↗

bioRxiv2024

A modified dual preparatory method for improved isolation of nucleic acids from laser microdissected fresh-frozen human cancer tissue specimens

A central theme in cancer research is to increase our understanding of the cancer tissue microenvironment (TME), which is comprised of a complex and spatially heterogeneous ecosystem of malignant and non-malignant cells, both of which actively contribute to an intervening extracellular matrix. Laser microdissection (LMD) enables histology selective harvest of cellular subpopulations from the tissue microenvironment for their independent molecular investigation, such as by high-throughput DNA and RNA sequencing. Although enabling, LMD often requires a labor-intensive investment to harvest enough cells to achieve the necessary DNA and/or RNA input requirements for conventional next generation sequencing workflows. To increase efficiencies, we sought to use a commonplace dual preparatory (DP) procedure to isolate DNA and RNA from the same LMD harvested tissue samples. While the yield of DNA from the DP protocol was satisfactory, the RNA yield from the LMD harvested tissue samples was significantly poorer compared to a dedicated RNA preparation procedure. We identified that this low yield of RNA was due to incomplete partitioning of RNA in this widely used DP protocol. Here we describe a modified DP protocol that effectively partitions nucleic acids and results in significantly improved RNA yields from LMD harvested cells.

biochemistry↗

bioRxiv2023

Flow cytometry methods for targeted isolation of ctenophore cells

Cell suspension fluidics, such as flow cytometry (FCS) and fluorescence-activated cell sorting (FACS), facilitates the identification and precise separation of individual cells based on phenotype. Since its introduction, flow cytometry has been used to analyze cell types and cellular processes in diverse non-vertebrate taxa, including cnidarians, molluscs, and arthropods. Ctenophores, which diverged very early from the metazoan stem lineage, have emerged as an informative clade for the study of metazoan cell type evolution. We present standardized methodologies for flow cytometry-mediated identification and analyses of cells from the model ctenophore Mnemiopsis leidyi that can also be applied to isolate targeted cell populations. Here we focus on the identification and isolation of ctenophore phagocytes. Implementing flow cytometry methods in ctenophores allows for fine scale analyses of fundamental cellular processes conserved broadly across animals, as well as potentially revealing novel cellular phenotypes and behaviors restricted to the ctenophore lineage.

cell biology↗

bioRxiv2022

FAVOR: Functional Annotation of Variants Online Resource and Annotator for Variation across the Human Genome

Large-scale whole genome sequencing (WGS) studies and biobanks are rapidly generating a multitude of coding and non-coding variants. They provide an unprecedented resource for illuminating the genetic basis of human diseases. Variant functional annotations play a critical role in WGS analysis, result interpretation, and prioritization of disease- or trait-associated causal variants. Existing functional annotation databases have limited scope to perform online queries or are unable to functionally annotate the genotype data of large WGS studies and biobanks for downstream analysis. We develop the Functional Annotation of Variants Online Resources (FAVOR) to meet these pressing needs. FAVOR provides a comprehensive online multi-faceted portal with summarization and visualization of all possible 9 billion single nucleotide variants (SNVs) across the genome, and allows for rapid variant-, gene-, and region-level online queries. It integrates variant functional information from multiple sources to describe the functional characteristics of variants and facilitates prioritizing plausible causal variants influencing human phenotypes. Furthermore, a scalable annotation tool, FAVORannotator, is provided for functionally annotating and efficiently storing the genotype and variant functional annotation data of a large-scale sequencing study in an annotated GDS file format to facilitate downstream analysis. FAVOR and FAVORannotator are available at https://favor.genohub.org.

genetics↗

bioRxiv2021

Powerful synergistic effects of a STING agonist and an IL-2 superkine in cancer immunotherapy against MHC I-deficient and MHC I+ tumors

Cyclic dinucleotides (CDNs) and TLR ligands mobilize antitumor responses by NK cells and T cells, potentially serving as complementary therapies to immune checkpoint therapy. In the clinic thus far, however, CDN therapy has yielded mixed results, perhaps because it initiates responses potently, but does not provide signals to sustain activation and proliferation of activated cytotoxic lymphocytes. To improve efficacy, we combined CDNs with a half-life extended IL-2 superkine, H9-MSA. CDN/H9-MSA therapy induced dramatic long-term remissions of the most difficult-to-treat MHC I-deficient and MHC I+ tumor transplant models. H9-MSA combined with CpG oligonucleotide also induced potent responses. Mechanistically, tumor elimination required CD8 T cells and not NK cells in the case of MHC I+ tumors and NK cells but not CD8 T cells in the case of MHC-deficient tumors. Furthermore, combination therapy resulted in more prolonged and more intense NK cell activation, cytotoxicity and expression of cytotoxic effector molecules in comparison to monotherapy. Remarkably, in a primary autochthonous sarcoma model that is refractory to PD-1 checkpoint therapy, the combination of CDN/H9-MSA combined with checkpoint therapy yielded long-term remissions in the majority of animals, mediated by T cells and NK cells. This novel combination therapy has potential to activate responses in tumors resistant to current therapies and prevent MHC I-loss accompanying acquired resistance of tumors to checkpoint therapy. One sentence summaryPowerful immunotherapy effects mediated by the combination of innate agonists and superkine.

immunology↗

bioRxiv2021

Genomics analysis of hexanoic acid exposure in Drosophila species

Drosophila sechellia is a dietary specialist endemic to the Seychelles islands that has evolved to consume the fruit of Morinda citrifolia. When ripe, the fruit of M. citrifolia contains octanoic acid and hexanoic acid, two medium chain fatty acid volatiles that deter and are toxic to generalist insects. D. sechellia has evolved resistance to these volatiles allowing it to feed almost exclusively on this host plant. The genetic basis of octanoic acid resistance has been the focus of multiple recent studies, but the mechanisms that govern hexanoic acid resistance in D. sechellia remain unknown. To understand how D. sechellia has evolved to specialize on M. citrifolia fruit and avoid the toxic effects of hexanoic acid, we exposed adult D. sechellia, D. melanogaster and D. simulans to hexanoic acid and performed RNA sequencing comparing their transcriptional responses to identify D. sechellia specific responses. Our analysis identified many more genes responding transcriptionally to hexanoic acid in the susceptible generalist species than in the specialist D. sechellia. Interrogation of the sets of differentially expressed genes showed that generalists regulated the expression of many genes involved in metabolism and detoxification whereas the specialist primarily downregulated genes involved in the innate immunity. Using these data we have identified interesting candidate genes that may be critically important in aspects of adaptation to their food source that contains high concentrations of HA. Understanding how gene expression evolves during dietary specialization is crucial for our understanding of how ecological communities are built and how evolution shapes trophic interactions.

genomics↗