CRISPR/Cas9 genome editing to create nonhuman primate models for studying stem cell therapies for HIV infection
This review examines opportunities provided by recent advances in CRISPR/Cas9 gene editing technologies to refine nonhuman primate (NHP) HIV models. These modifications can assist in investigating genetic factors affecting HIV replication and designing cellular therapies that exploit genetic barriers to HIV infections, including engineering mutations into CCR5 that confer resistance to HIV/simian immunodeficiency virus (SIV) infections. Thus, we provide an overview of recent advances and challenges in gene editing NHP embryos. We also discuss the value of genetically engineered animal models for developing novel stem cell-based therapies for curing HIV.
Allogeneic MHC-matched T-cell receptor α/β-depleted bone marrow transplants in SHIV-infected, ART-suppressed Mauritian cynomolgus macaques
Sci Rep. 2022
In this study, we modeled allogeneic hematopoietic stem cell transplants (allo-HSCTs) in four antiretroviral (ART)-suppressed simian-human immunodeficiency virus (SHIV)-infected Mauritian cynomolgus macaques (MCMs) to illuminate factors contributing to transplant-induced viral reservoir decay. The transplants produced ~ 85% whole blood donor chimerism without causing high-grade graft-versus-host disease (GvHD). Consequently, three MCMs had undetectable SHIV DNA in their blood post-transplant. However, SHIV-harboring cells persisted in various tissues, with detectable viral DNA in lymph nodes and tissues between 38 and 62 days post-transplant. Further, removing one MCM from ART at 63 days post-transplant resulted in SHIV rapidly rebounding within 7 days of treatment withdrawal. Thus, this study concluded that transplanting SHIV-infected MCMs with allogeneic MHC-matched α/β T cell-depleted bone marrow cells prevented high-grade GvHD and decreased SHIV-harboring cells in the blood post-transplant but did not eliminate viral reservoirs in tissues.
§co-senior authors Pubmed Link
Generation of SIV-resistant T cells and macrophages from nonhuman primate induced pluripotent stem cells with edited CCR5 locus
Stem Cell Reports 2022
In this study, we edited the CCR5 locus of iPSCs generated from T cells (T-iPSCs) or fibroblasts (fib-iPSCs) from Mauritian cynomolgus macaques (MCM) using CRISPR-Cas9 technology. We found that CCR5 editing does not affect hematopoietic and T cell differentiation potentials of fib-iPSCs. However, T-iPSCs with edited CCR5 lost their capacity to differentiate into CD4+CD8+ T cells while maintaining myeloid differentiation potential. T cells and macrophages produced from CCR5-edited MCM iPSCs did not support replication of the CCR5-tropic simian immunodeficiency viruses SIVmac239 (T cell tropic) and SIVmac316 (macrophage-tropic). Overall, these results provide a platform for exploring gene-edited iPSC-based AIDS therapies in nonhuman primates.
§co-senior authors PubMed Link
Transplantation of T-cell receptor α/β-depleted allogeneic bone marrow in nonhuman primates
Here we evaluated the outcomes of major histocompatibility complex (MHC)-matched T-cell receptor α/β-depleted allogeneic hematopoietic stem cell transplants (alloHSCTs) in Mauritian cynomolgus macaques (MCMs). Following T-cell receptor α/β depletion, bone marrow cells were transplanted into MHC-identical MCMs conditioned with total body irradiation. Graft-versus-host disease (GvHD) prophylaxis included sirolimus alone in two animals or tacrolimus with cyclophosphamide in another two animals. Macaques treated with sirolimus developed nearly complete donor chimerism with acute GvHD. Conversely, in the cyclophosphamide and tacrolimus-treated group, macaques developed mixed chimerism without GvHD, with long-term engraftment observed in one animal. These results demonstrate the feasibility of alloHSCT engraftment without GvHD in an MHC-identical MCM model following complete myeloablative conditioning and anti-GvHD prophylaxis with posttransplant cyclophosphamide and tacrolimus. They also suggest that this alloHSCT model can be used to elucidate the mechanisms of GvH-mediated elimination of HIV-infected cells.
§co-senior authors PubMed Link
Identifying a Minor Histocompatibility Antigen in Mauritian Cynomolgus Macaques Encoded by APOBEC3C
Front. Immunol. 2020
In this study, we used a combination of alloimmunization, whole exome sequencing, and bioinformatics to identify a minor histocompatibility antigen (mHAg) in Mauritian cynomolgus macaques (MCMs). We mapped the minimal optimal epitope to a 10-mer peptide (SW10) in apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3C (APOBEC3C) and determined the major histocompatibility complex class I restriction element as Mafa-A1∗063, which is expressed in almost 90% of MCMs. APOBEC3C SW10-specific CD8+ T cells recognized immortalized B cells but not fibroblasts from a mHAg-positive MCM. These results provide a framework for identifying mHAgs in a non-transplant setting and suggest that APOBEC3C SW10 could be used as a model antigen to test mHAg-targeted therapies in NHPs.
Isolation of a monoclonal antibody from a phage display library binding the rhesus macaque MHC class I allomorph Mamu-A1*001
This study isolated an antibody that recognizes the common Indian rhesus macaque MHC class I molecule, Mamu-A1*001. We induced Mamu-A1*001-binding antibodies by alloimmunizing a female Mamu-A1*001-negative rhesus macaque with peripheral blood mononuclear cells (PBMC) from a male Mamu-A1*001-positive donor. A Fab phage display library was constructed with PBMC from the alloimmunized macaque and panned to isolate an antibody that binds to Mamu-A1*001 but not to other common rhesus macaque MHC class I molecules. The isolated antibody distinguishes PBMC from Mamu-A1*001-positive and -negative macaques. Additionally, the Mamu-A1*001-specific antibody binds the cynomolgus macaque MHC class I ortholog Mafa-A1*001:01 but not variants Mafa-A1*001:02/03, indicating a high degree of binding specificity. We anticipate that the Mamu-A1*001-specific antibody will be useful for identifying Mamu-A1*001-positive rhesus macaques, detecting Mamu-A1*001-positive cells in populations of Mamu-A1*001-negative cells, and examining disease processes that alter the cell surface expression of Mamu-A1*001. Moreover, the alloimmunization strategy will be useful for isolating additional MHC allomorph-specific monoclonal antibodies or antibodies against other polymorphic host proteins, which are difficult to isolate with traditional technologies.