10 results on '"Murad R. Mamedov"'
Search Results
2. High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails
- Author
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Brian R. Shy, Vivasvan S. Vykunta, Alvin Ha, Alexis Talbot, Theodore L. Roth, David N. Nguyen, Wolfgang G. Pfeifer, Yan Yi Chen, Franziska Blaeschke, Eric Shifrut, Shane Vedova, Murad R. Mamedov, Jing-Yi Jing Chung, Hong Li, Ruby Yu, David Wu, Jeffrey Wolf, Thomas G. Martin, Carlos E. Castro, Lumeng Ye, Jonathan H. Esensten, Justin Eyquem, and Alexander Marson
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Gene Editing ,Genome ,DNA End-Joining Repair ,5.2 Cellular and gene therapies ,Biomedical Engineering ,Recombinational DNA Repair ,Bioengineering ,DNA ,Applied Microbiology and Biotechnology ,Article ,Single-Stranded ,Mutation ,Genetics ,Humans ,Molecular Medicine ,CRISPR-Cas Systems ,Development of treatments and therapeutic interventions ,Biotechnology - Abstract
Enhancing CRISPR-mediated site-specific transgene insertion efficiency by homology-directed repair (HDR) using high concentrations of double-stranded DNA (dsDNA) with Cas9 target sequences (CTSs) can be toxic to primary cells. Here, we develop single-stranded DNA (ssDNA) HDR templates (HDRTs) incorporating CTSs with reduced toxicity that boost knock-in efficiency and yield by an average of around two- to threefold relative to dsDNA CTSs. Using small-molecule combinations that enhance HDR, we could further increase knock-in efficiencies by an additional roughly two- to threefold on average. Our method works across a variety of target loci, knock-in constructs and primary human cell types, reaching HDR efficiencies of >80-90%. We demonstrate application of this approach for both pathogenic gene variant modeling and gene-replacement strategies for IL2RA and CTLA4 mutations associated with Mendelian disorders. Finally, we develop a good manufacturing practice (GMP)-compatible process for nonviral chimeric antigen receptor-T cell manufacturing, with knock-in efficiencies (46-62%) and yields (>1.5 × 109 modified cells) exceeding those of conventional approaches.
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- 2022
3. Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency
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Linda T. Vo, P. Jonathan Li, Theodore L. Roth, Murad R. Mamedov, David N. Nguyen, Peixin Amy Chen, Eric Shifrut, Jeffrey A. Bluestone, Ryan Apathy, Alexander Marson, Francis C. Szoka, Jennifer M. Puck, Daniel B. Goodman, and Victoria Tobin
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Adult ,Polymers ,CD3 ,Biomedical Engineering ,Bioengineering ,Regenerative Medicine ,Applied Microbiology and Biotechnology ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Genome editing ,Stem Cell Research - Nonembryonic - Human ,CRISPR-Associated Protein 9 ,Genetics ,Humans ,Kinetoplastida ,Progenitor cell ,Induced pluripotent stem cell ,030304 developmental biology ,Gene Editing ,0303 health sciences ,5.2 Cellular and gene therapies ,biology ,Protein Stability ,Chemistry ,Human Genome ,Polyglutamic acid ,Gene targeting ,Stem Cell Research ,Cell biology ,Haematopoiesis ,biology.protein ,RNA ,Nanoparticles ,Molecular Medicine ,Development of treatments and therapeutic interventions ,Guide ,030217 neurology & neurosurgery ,CD8 ,RNA, Guide, Kinetoplastida ,Biotechnology - Abstract
Versatile and precise genome modifications are needed to create a wider range of adoptive cellular therapies1-5. Here we report two improvements that increase the efficiency of CRISPR-Cas9-based genome editing in clinically relevant primary cell types. Truncated Cas9 target sequences (tCTSs) added at the ends of the homology-directed repair (HDR) template interact with Cas9 ribonucleoproteins (RNPs) to shuttle the template to the nucleus, enhancing HDR efficiency approximately two- to fourfold. Furthermore, stabilizing Cas9 RNPs into nanoparticles with polyglutamic acid further improves editing efficiency by approximately twofold, reduces toxicity, and enables lyophilized storage without loss of activity. Combining the two improvements increases gene targeting efficiency even at reduced HDR template doses, yielding approximately two to six times as many viable edited cells across multiple genomic loci in diverse cell types, such as bulk (CD3+) T cells, CD8+ T cells, CD4+ T cells, regulatory T cells (Tregs), γδ T cells, B cells, natural killer cells, and primary and induced pluripotent stem cell-derived6 hematopoietic stem progenitor cells (HSPCs).
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- 2019
4. Hybrid ssDNA repair templates enable high yield genome engineering in primary cells for disease modeling and cell therapy manufacturing
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Hong Li, Alvin Ha, Lumeng Ye, Franziska Blaeschke, Jonathan H. Esensten, Jeffrey L. Wolf, Alexis Talbot, Vivasvan Vykunta, Brian R. Shy, Alexander Marson, Yan Yi Chen, Justin Eyquem, Murad R. Mamedov, Thomas G. Martin, David N. Nguyen, Jing-Yi Chung, Shane Vedova, and Theodore L. Roth
- Subjects
Homology directed repair ,Cell therapy ,chemistry.chemical_compound ,chemistry ,Cas9 ,Transgene ,CRISPR ,Computational biology ,Biology ,Genome ,DNA ,Genome engineering - Abstract
CRISPR-Cas9 offers unprecedented opportunities to modify genome sequences in primary human cells to study disease variants and reprogram cell functions for next-generation cellular therapies. CRISPR has several potential advantages over widely used retroviral vectors including: 1) site-specific transgene insertion via homology directed repair (HDR), and 2) reductions in the cost and complexity of genome modification. Despite rapid progress with ex vivo CRISPR genome engineering, many novel research and clinical applications would be enabled by methods to further improve knock-in efficiency and the absolute yield of live knock-in cells, especially with large HDR templates (HDRT). We recently reported that Cas9 target sequences (CTS) could be introduced into double-stranded DNA (dsDNA) HDRTs to improve knock-in, but yields and efficiencies were limited by toxicity at high HDRT concentrations. Here we developed a novel system that takes advantage of lower toxicity with single-stranded DNA (ssDNA). We designed hybrid ssDNA HDRTs that incorporate CTS sites and were able to boost knock-in percentages by >5-fold and live cell yields by >7-fold relative to dsDNA HDRTs with CTS. Knock-in efficiency and yield with ssCTS HDRTs were increased further with small molecule inhibitor combinations to improve HDR. We demonstrate application of these methods across a variety of target loci, knock-in constructs, and primary human cell types to reach ultra-high HDR efficiencies (>80-90%) which we use for pathogenic gene variant modeling and universal gene replacement strategies for IL2RA and CTLA4 mutations associated with mendelian immune disorders. Finally, we develop a GMP-compatible method for fully non-viral CAR-T cell manufacturing, demonstrating knock-in efficiencies of 46-62% and generating yields of >1.5 x 109 CAR+ T cells, well above current doses for adoptive cellular therapies. Taken together, we present a comprehensive non-viral approach to model disease associated mutations and re-write targeted genome sequences to program immune cell therapies at a scale compatible with future clinical application.
- Published
- 2021
5. Genome-wide CRISPR screens reveal metabolic and transcriptional regulation of BTN3A and cancer susceptibility to Vγ9Vδ2 T cell targeting
- Author
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Murad R Mamedov, Shane Vedova, Jacob W. Freimer, Avinash Das Sahu, Peixin Amy Chen, Amrita Ramesh, Kristina Hanspers, Vinh Q. Nguyen, Erin J Adams, and Alexander Marson
- Subjects
Immunology ,Immunology and Allergy - Abstract
γδ T cells are potent anti-cancer effectors with potential to target tumors broadly, independent of neoantigens or HLA-background. γδ T cells can sense conserved cell stress signals prevalent in transformed cells, although the mechanisms governing how γδ T cells sense and kill stressed target cells remain poorly characterized. Vγ9Vδ2 T cells – the most abundant subset of human γδ T cells – recognize a protein complex containing butyrophilin 3A1 (BTN3A1), a ubiquitously expressed cell surface protein that is activated by phosphoantigens abundantly produced by tumor cells. Here we performed genome-wide CRISPR screens in target cancer cells to identify pathways that regulate: (1) γδ T cell activity with a functional co-culture killing screen, and (2) BTN3A1 cell surface expression in a phenotypic screen. Multilayered regulation of BTN3A1 expression was uncovered: transcriptional regulation (IRF1, CTBP1, ZNF217, RUNX1), intracellular trafficking, sialylation, oxidative phosphorylation (OXPHOS), purine metabolism, and other metabolic pathways. Consistent with these results, we found upregulated BTN3A1 on cells undergoing an energy crisis due to glucose deprivation, glycolysis inhibition, or OXPHOS inhibition. Furthermore, we discovered that this BTN3A1 upregulation was induced by activation of the AMP-activated protein kinase (AMPK). Also, CRISPR screen-derived gene expression signatures correlated with higher survival in low-grade glioma patients whose tumors had high Vγ9Vδ2 T cell infiltration. Uncovering this AMPK-dependent mechanism of metabolic stress-induced ligand activation deepens our understanding of γδ T cell stress surveillance and suggests new avenues to enhance γδ T cell anti-cancer activity. M.R.M. is a Cancer Research Institute (CRI) Irvington Fellow supported by CRI and was funded by the Human Vaccines Project Michelson Prize for Human Immunology.
- Published
- 2022
6. A Cas9 nanoparticle system with truncated Cas9 target sequences on DNA repair templates enhances genome targeting in diverse human immune cell types
- Author
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Theodore L. Roth, Francis C. Szoka, Eric Shifrut, Jeffrey A. Bluestone, Puck Jm, Murad R. Mamedov, Peixin Amy Chen, Jia Jie Li, David N. Nguyen, Ryan Apathy, Alexander Marson, Tobin, Daniel B. Goodman, and Linda T. Vo
- Subjects
0303 health sciences ,Cas9 ,DNA repair ,medicine.medical_treatment ,Gene targeting ,Biology ,Genome ,Cell biology ,Homology directed repair ,03 medical and health sciences ,0302 clinical medicine ,Cancer immunotherapy ,030220 oncology & carcinogenesis ,medicine ,Progenitor cell ,Reprogramming ,030304 developmental biology - Abstract
Virus-modified T cells are approved for cancer immunotherapy, but more versatile and precise genome modifications are needed for a wider range of adoptive cellular therapies1–4. We recently developed a non-viral CRISPR–Cas9 system for genomic site-specific integration of large DNA sequences in primary human T cells5. Here, we report two key improvements for efficiency and viability in an expanded variety of clinically-relevant primary cell types. We discovered that addition of truncated Cas9 target sequences (tCTS) at the ends of the homology directed repair (HDR) templates can interact with Cas9 ribonucleoproteins (RNPs) to ‘shuttle’ the template and enhance targeting efficiency. Further, stabilizing the Cas9 RNPs into nanoparticles with poly(glutamic acid) improved editing, reduced toxicity, and enabled lyophilized storage without loss of activity. Combining the tCTS HDR template modifications with polymer-stabilized nanoparticles increased gene targeting efficiency and viable cell yield across multiple genomic loci in diverse cell types. This system is an inexpensive, user-friendly delivery platform for non-viral genome reprogramming that we successfully applied in regulatory T cells (Tregs), γδ-T cells, B cells, NK cells, and primary and iPS-derived6 hematopoietic stem progenitor cells (HSPCs).
- Published
- 2019
7. Individual heritable differences result in unique cell lymphocyte receptor repertoires of naïve and antigen-experienced cells
- Author
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Cornelia L. Dekker, Gary E. Swan, Daniel Gadala-Maria, Jason A. Vander Heiden, Helen M. McGuire, Christopher R. Bolen, Mark M. Davis, Ghia Euskirchen, Steven H. Kleinstein, Lindsay G. Cowell, Mikhail K. Levin, Florian Rubelt, and Murad R. Mamedov
- Subjects
CD4-Positive T-Lymphocytes ,0301 basic medicine ,Lymphocyte ,Science ,General Physics and Astronomy ,Adaptive Immunity ,CD8-Positive T-Lymphocytes ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,0302 clinical medicine ,Antigen ,medicine ,Humans ,Epigenetics ,Gene ,Genetics ,B-Lymphocytes ,Multidisciplinary ,Genes, Immunoglobulin ,Reverse Transcriptase Polymerase Chain Reaction ,Repertoire ,V(D)J recombination ,Twins, Monozygotic ,General Chemistry ,Acquired immune system ,V(D)J Recombination ,Genes, T-Cell Receptor ,030104 developmental biology ,medicine.anatomical_structure ,CD8 ,030215 immunology - Abstract
The adaptive immune system's capability to protect the body requires a highly diverse lymphocyte antigen receptor repertoire. However, the influence of individual genetic and epigenetic differences on these repertoires is not typically measured. By leveraging the unique characteristics of B, CD4+ T and CD8+ T-lymphocyte subsets from monozygotic twins, we quantify the impact of heritable factors on both the V(D)J recombination process and on thymic selection. We show that the resulting biases in both V(D)J usage and N/P addition lengths, which are found in naïve and antigen experienced cells, contribute to significant variation in the CDR3 region. Moreover, we show that the relative usage of V and J gene segments is chromosomally biased, with ∼1.5 times as many rearrangements originating from a single chromosome. These data refine our understanding of the heritable mechanisms affecting the repertoire, and show that biases are evident on a chromosome-wide level., The diversity of antigen receptor specificities is largely generated by random recombination of its segments. Here the authors show, by genetic comparison of monozygotic twin lymphocyte subsets, that individual genetic and epigenetic biases also contribute to the shape of the B and T cell repertoires.
- Published
- 2016
8. A Macrophage Colony-Stimulating-Factor-Producing γδ T Cell Subset Prevents Malarial Parasitemic Recurrence
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Florian Rubelt, Mark M. Davis, Yue Zhang, Ramesh V. Nair, Katherine Cumnock, Robert W. Sauerwein, Murad R. Mamedov, Damian L. Trujillo, Naresha Saligrama, David Schneider, Justin A. Kenkel, Anja Scholzen, Yueh-hsiu Chien, and Jose Henrique M. Oliveira
- Subjects
0301 basic medicine ,Macrophage colony-stimulating factor ,Chemokine ,Immunology ,lnfectious Diseases and Global Health Radboud Institute for Molecular Life Sciences [Radboudumc 4] ,Parasitemia ,Lymphocyte Activation ,CCL5 ,Plasmodium chabaudi ,03 medical and health sciences ,Mice ,0302 clinical medicine ,All institutes and research themes of the Radboud University Medical Center ,Immunity ,Recurrence ,T-Lymphocyte Subsets ,parasitic diseases ,medicine ,Immunology and Allergy ,Animals ,Humans ,biology ,Macrophage Colony-Stimulating Factor ,Plasmodium falciparum ,Receptors, Antigen, T-Cell, gamma-delta ,biology.organism_classification ,medicine.disease ,Colony-stimulating factor ,Malaria ,030104 developmental biology ,Infectious Diseases ,biology.protein ,Female ,030215 immunology - Abstract
Despite evidence that γδ T cells play an important role during malaria, their precise role remains unclear. During murine malaria induced by Plasmodium chabaudi infection and in human P. falciparum infection, we found that γδ T cells expanded rapidly after resolution of acute parasitemia, in contrast to αβ T cells that expanded at the acute stage and then declined. Single-cell sequencing showed that TRAV15N-1 (Vδ6.3) γδ T cells were clonally expanded in mice and had convergent complementarity-determining region 3 sequences. These γδ T cells expressed specific cytokines, M-CSF, CCL5, CCL3, which are known to act on myeloid cells, indicating that this γδ T cell subset might have distinct functions. Both γδ T cells and M-CSF were necessary for preventing parasitemic recurrence. These findings point to an M-CSF-producing γδ T cell subset that fulfills a specialized protective role in the later stage of malaria infection when αβ T cells have declined.
- Published
- 2017
9. Arginine-Rich Motifs Are Not Required for Hepatitis Delta Virus RNA Binding Activity of the Hepatitis Delta Antigen
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John L. Casey, Brittany L. Griffin, Ali Soroush, Leighton H. Daigh, and Murad R. Mamedov
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viruses ,Amino Acid Motifs ,DNA Mutational Analysis ,Immunology ,RNA-dependent RNA polymerase ,RNA-binding protein ,Plasma protein binding ,Biology ,Arginine ,Microbiology ,Virus ,Cell Line ,Virology ,Humans ,Sequence Deletion ,Hepatitis delta Antigens ,Structure and Assembly ,RNA-Binding Proteins ,RNA ,Non-coding RNA ,Molecular biology ,RNA editing ,Insect Science ,Mutagenesis, Site-Directed ,Nucleic acid ,RNA, Viral ,Hepatitis Delta Virus ,Protein Binding - Abstract
Hepatitis delta virus (HDV) replication and packaging require interactions between the unbranched rodlike structure of HDV RNA and hepatitis delta antigen (HDAg), a basic, disordered, oligomeric protein. The tendency of the protein to bind nonspecifically to nucleic acids has impeded analysis of HDV RNA protein complexes and conclusive determination of the regions of HDAg involved in RNA binding. The most widely cited model suggests that RNA binding involves two proposed arginine-rich motifs (ARMs I and II) in the middle of HDAg. However, other studies have questioned the roles of the ARMs. Here, binding activity was analyzed in vitro using HDAg-160, a C-terminal truncation that binds with high affinity and specificity to HDV RNA segments in vitro . Mutation of the core arginines of ARM I or ARM II in HDAg-160 did not diminish binding to HDV unbranched rodlike RNA. These same mutations did not abolish the ability of full-length HDAg to inhibit HDV RNA editing in cells, an activity that involves RNA binding. Moreover, only the N-terminal region of the protein, which does not contain the ARMs, was cross-linked to a bound HDV RNA segment in vitro . These results indicate that the amino-terminal region of HDAg is in close contact with the RNA and that the proposed ARMs are not required for binding HDV RNA. Binding was not reduced by mutation of additional clusters of basic amino acids. This result is consistent with an RNA-protein complex that is formed via numerous contacts between the RNA and each HDAg monomer.
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- 2013
10. Novel M-CSF-producing γδ T cells protect against recurrent malaria
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Murad R. Mamedov, Anja Scholzen, Ramesh V. Nair, Katherine Cumnock, Justin A. Kenkel, Jose H. M. Oliveira, Damian L. Trujillo, Naresha Saligrama, Yue Zhang, Florian Rubelt, David S. Schneider, Yueh-hsiu Chien, Robert Sauerwein, and Mark M. Davis
- Subjects
Immunology ,Immunology and Allergy - Abstract
In 2016, there were 216 million malaria cases – 445,000 of which resulted in deaths. Despite overwhelming evidence that γδ T cells strongly respond during malaria infection and vaccination, their functional and phenotypic characteristics remain the least understood facets of the adaptive immune response. Therefore, we studied the role of these cells in human and mouse malaria. In both Plasmodium falciparum-infected subjects and in P. chabaudi-infected mice, we found γδ T cells expanding rapidly after resolution of acute parasitemia, in contrast to αβ T cells that expanded at the acute stage and then declined. Silencing the murine γδ T cells led to recurrent rounds of Plasmodium parasitemia. Single-cell T cell receptor sequencing of the expanded mouse cells revealed oligoclonal γδ T cells restricted to the TRAV15N-1 (Vδ6.3) V-region and converging complementarity-determining region 3 (CDR3) motifs. Also, RNA-seq of the expanded γδ T cells showed an unexpected transcriptional profile characterized by myeloid-modulating factors, previously unseen in γδ T cells. The expanded TRAV15N-1 γδ T cells abundantly produced M-CSF, which was necessary for preventing parasitemic recurrence. Interestingly, αβ T cells were the major source of M-CSF during acute infection, while γδ T cells filled that role during the post-acute stage. We have uncovered a novel γδ T cell subset that fills a protective role in the late stage of malaria. These cells could provide the mechanism for other observed correlations between γδ T cell and myeloid activity in cancer and infectious disease.
- Published
- 2018
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