Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

Abstract

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (cγ(-/-))-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

Lentiviral Vector-Based Dendritic Cell Vaccine Suppresses HIV Replication in Humanized Mice

“…To provide a source of naive MHC class I-restricted anti-SL9 CD8 T cells, a fraction of the CD34 + hematopoietic stem cells (HSCs) used in the reconstitution was transduced with a vector encoding an SL9-specific human T cell receptor (TCR). [44][45][46] Analysis of the PBMCs by flow cytometry showed that >60% of the lymphocytes in the BLT mice were of human origin ( Figure S1).…”

“…The other half were injected retro-orbitally after transplant. 46 For all humanized mice, the engraftment frequency was determined by staining the blood lymphocytes and splenocytes for human CD45 and found to be 60%-90%, resulting in 0.5-2 Â 10 6 human lymphocytes/mL in the blood and 1-3 Â 10 7 human lymphocytes in the spleen. For the SL9 TCR BLT mice, engraftment of the SL9 TCR-transduced CD34 + cells resulted in the expression of SL9 TCR in 0.1%-1.4% of the human CD8 T cells.…”

Novel AIDS therapies based on gene editing

“…Rhesus macaques inoculated with an AAV vector expressing this antibody were protected from several infectious challenges with SIV and thus functioned like an effective HIV-1 vaccine [97]. The data from these studies and others [98–100] suggest that gene therapy with CAR may be a potentially effective therapy for chronic HIV infection.…”

Engineering HIV-Specific Immunity with Chimeric Antigen Receptors

“…[30][31][32] The use of this CD4fCAR has important advantages, in that it has been found to be a safe reagent in multiple long-term clinical trials with over 500 patient years of clinical safety data 33,34 and this is strong evidence that it does not induce cytokine storms that have been an unwanted element with other CAR-based approaches in treating malignancies. 35,36 It is also unlikely to generate escape variants of HIV envelope as the loss of CD4 binding of an escape variant will likely have a dramatic effect on viral fitness.…”

“…We and others have done extensive testing of the antiviral function of CD8 + T cells transduced with CD4f CAR, finding that transduced cells are capable of killing HIV-infected cells and suppressing viral replication [30][31][32]34,37 and (unlike the HIV-specific TCR) that this activity is independent of HLA-I molecules. 38 CARs can also function in CD4 + T cells to act as HIV-1-specific helper cells.…”