Mechanisms that Enhance and Suppress HIV-1 Resistance in Gene Edited Primary Human Cells

增强和抑制基因编辑原代人类细胞中 HIV-1 耐药性的机制

• 批准号: 10700726
• 负责人: Amanda M Dudek
• 金额: $ 13.12万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-12 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10700726
• 关键词: Alleles; Allogenic; Alternative Splicing; Anti-Retroviral Agents; Autologous; Biological Assay; Biology; Bone Marrow; CCR5 gene; CD34 gene; CD4 Positive T Lymphocytes; CXCR4 gene; Cell Line; Cell Transplantation; Cells; Characteristics; Clinical Research; Clustered Regularly Interspaced Short Palindromic Repeats; Cyclophilin A; Data; Dendritic Cells; Disease; Disparity; Dominant-Negative Mutation; Engineering; Genes; Genetic Transcription; Goals; HIV; HIV Infections; HIV therapy; HIV-1; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Human; Individual; Infection; Intrinsic factor; Investigation; Ionomycin; Knock-in; Knock-out; Low Prevalence; Macrophage; Membrane; Memory; Modeling; Modification; Molecular; Morbidity - disease rate; Null Lymphocytes; Patients; Pattern; Peptides; Peripheral Blood Mononuclear Cell; Pharmacotherapy; Protein Isoforms; Proteins; Regimen; Resistance; Rhesus; Stem cell transplant; T-Lymphocyte; T-Lymphocyte Subsets; TRIM Gene; TRIM5 gene; Techniques; Technology; Testing; Therapeutic; Toxic effect; Transplantation; Treatment Efficacy; Umbilical Cord Blood; Viral; Viral Proteins; Virus; Virus Diseases; Virus Replication; adeno-associated viral vector; antiretroviral therapy; base editing; cell immortalization; cell type; conditioning; cytokine; improved; innovation; monocyte; novel; prevent; repaired; resistance mutation; response; stem cell therapy; stem cells; therapeutic genome editing; viral rebound

While there have been great advances in HIV therapies over the past decades, the only true cure so far has been through hematopoietic stem cell transplant (HSCT) of cells naturally lacking the HIV co-receptor CCR5. Due to the toxicity of conditioning regimens prior to HSCT and significant morbidity due to graft-verses-host disease (GVHD), allogeneic HSCT is not a viable option for most patients. With the advent of CRISPR, modification of a patient’s own cells to prevent GVHD is now possible, but thus far the efficiency at which cells have been modified was not enough to prevent viral rebound in the absence of anti-retroviral therapy. In line with the rationale for combination anti-retroviral drug therapy to target multiple steps in replication, we have developed a multi-factor knock-out/knock-in strategy for editing CD34+ hematopoietic stem and progenitor cells which allows greater than 90% deletion of CCR5, as well as up to 50% allelic knock-in of two inhibitory peptides targeting either fusion (C46V2o) or uncoating (a human-rhesus chimeric TRIM5a). When using this strategy to edit primary human CD4+ T cells, the efficiency of knock-out/knock-in is sufficient for complete inhibition of CCR5-tropic virus (BaL), and an average of more than 700-fold inhibition of CXCR4-tropic virus (NL4-3) when tested across 5 different primary human T cell donors. While these data are extremely promising, there was a large disparity in the efficiency of inhibition of CXCR4-tropic replication by the human- rhesus TRIM5a, with some T cell donors showing greater than 1,000-fold inhibition in the hRhTRIM5a knock- in condition, and some showing little or no significant inhibition of replication. This observation was in spite of sustained levels of allelic knock-in and hRhTRIM5a RNA expression throughout the course of the infection, and no resistance mutations observed in the infectious virus at endpoint. We therefor propose here to study the underlying T-cell/TRIM/virus interactions which may be uniquely donor specific and influence viral replication including T cell cytokine expression, viral reactivation, and expression of endogenous cellular factors that may act as a dominant negative to our inhibitory hRhTRIM5a in addition to investigation of other TRIM-related factors for inhibition such as TRIMCyp. Although the final goal is to develop a therapy through transplantation of edited hematopoietic stem and progenitor cells, this proposal is entirely based on editing and investigating the mechanisms of restriction in differentiated primary human target cells such as CD4+ T cells, monocytes, and macrophages in order to better understand the biology within our editing platform and allow eventual improvement of the platform. Successful completion of this proposal will allow a better understanding of mechanisms of restriction in primary cells where often these mechanisms have been predominantly studied in immortalized cell lines. In addition, completion of this proposal to improve our therapeutic platform will widen the pool of patients potentially able to be treated using genetically modified autologous HSCT such as patients later in infection that may have predominantly CXCR4-tropic virus.

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