Biology subjects

Naeimi Kararoudi, M.

Publications and source records attributed to Naeimi Kararoudi, M..

5 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↗

bioRxiv2021

CRISPR-Targeted CAR Gene Insertion Using Cas9/RNP and AAV6 Enhances Anti-AML Activity of Primary NK Cells

Human peripheral blood natural killer (NK) cells have intense antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity. Recently, we described an efficient method for gene targeting in NK cells using Cas9/ribonucleoprotein (RNP) complexes. Here we combined this approach with single-stranded (ss) or self-complementary (sc) Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a safe-harbor locus using a wide variety of homology arms for homology repair (HR) and non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. For proof-of-concept, we generated mCherry-expressing primary NK cells and determined that sc vectors with 300bp homology arms had optimal transduction efficiency. Then, we generated CD33-targeting CAR NK cells with differing transmembrane and signaling domains (CD4/4-1BB+CD3{zeta} and NKG2D/2B4+CD3{zeta}) and expanded them on CSTX002 feeder cells. Expansion kinetics were unaltered and the expanded NK cells maintained high CAR expression (mean 68% CAR+). The CD33-CAR-NK cells showed increased activation markers and enhanced antileukemic activity with improved killing kinetics against CD33-positive acute myeloid leukemia (AML) cell lines and primary samples. Using targeted sequencing we demonstrated the accuracy of CAR gene insertion in human primary NK cells genome. Site-directed insertion using RNP and scAAV6 is an efficient method for stable genetic transfer into primary NK cells that has broad potential for fundamental discovery and therapeutic applications.

immunology↗

bioRxiv2021

Synchronization Temperature Determines the Location of RSV Fusion During Entry in Cultured Cells

Respiratory syncytial virus (RSV) is the most frequent cause of bronchiolitis in children under five years of age. No vaccines against this virus are currently available. RSV infection of a cell is initiated by fusion between the virion membrane and a cellular membrane, but it is not clear if the fusion process takes place at the plasma membrane or within an endosome. Most such experiments have been initiated at the traditional synchronization temperature of 4{degrees}C, an abnormal temperature for animal cells and one at which cellular homeostasis may be negatively affected. We have compared two synchronization temperatures (4{degrees}C and 22{degrees}C) to determine the kinetics of RSV entry into human bronchial epithelial cells. Following inoculation, virus entry was halted at different times by the addition of neutralizing antibody or temperature reduction to 4{degrees}C. We engineered a virus that encodes an extra viral gene, beta-lactamase fused to the viral phosphoprotein (P), to enable rapid detection after infection initiation. We found that the synchronization temperature used during inoculation determines the site of fusion. Transition from 4{degrees}C to 37{degrees}C resulted in RSV entry via the endosomal pathway but also induced F-actin disruption and plasma membrane blebbing, whether the cells were inoculated with RSV or not. Transition from 22{degrees}C to 37{degrees}C resulted in RSV entry by fusion at the plasma membrane and without the F-actin and plasma membrane disruptions. These results suggest that RSV normally enters cells by fusion at the plasma membrane and that the induction of endocytosis by infection synchronization at 4{degrees}C may be an artefact caused by distortion of the plasma membrane-supporting cytoskeleton. Author SummaryIn order to understand the overall mechanism driving infection, it is important to determine how the virus enters cells. The pathway that RSV uses to infect cells is unclear. It is a common practice to attach the virions at 4{degrees}C to synchronize the viral infection. In this report, we found that warming up primary cultures of undifferentiated normal human bronchial epithelial cells to 37{degrees}C from 4{degrees}C triggered dramatic changes in their cell membrane and cytoskeleton totally unrelated to the presence of the virions. The assessment of viral content delivery to the cytoplasm using RSV engineered to express BlaM allowed us to find that the virions attached at 4{degrees}C or 22{degrees}C fused their envelope with endosome or plasma membrane, respectively. Consequently, the entry via endosome after attachment at 4{degrees}C is an experimental artefact and RSV infects by fusing its envelope with the plasma membrane. The implications go beyond RSV since the entry of several virus species have been explored by synchronizing the infection after attachment at 4{degrees}C.

microbiology↗

bioRxiv2019

Highly efficient site-directed gene insertion in primary human natural killer cells using homologous recombination and CRISPaint delivered by AAV

Human peripheral blood natural killer (NK) cells have strong antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity. However, genetic modification of NK cells has been challenging due to the high expression of innate sensing mechanisms for viral nucleic acids. Recently, we described an efficient vector-free method using Cas9/ribonucleoprotein complexes for gene deletion in NK cells. Here, we combined this approach with single-stranded (ss) or self-complementary (sc) Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a user-defined locus using homology repair (HR) and non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. Using these approaches, we identified scAAV6 as the superior serotype for successful generation of stable mCherry-expressing primary NK cells (up to 89%). To maximize transgene packaging in HR-directed gene insertion, we identified minimum optimal homology arm lengths of 300bp for the flanking region of the Cas9-targeting site. Lastly, we demonstrate that mCherry positive NK cells can be expanded to large numbers using feeder cells expressing membrane-bound IL-21. This efficient method for site-directed insertion of genetic material into NK cells has broad potential for basic discovery and therapeutic applications for primary NK cells.

immunology↗