Base editing approaches to enable an HIV cure

The HIV reservoir is restricted to cells of the immuno-hematopoietic lineage. Rare but well-studied instances of HIV cures in patients who underwent allogeneic hematopoietic stem cell transplantation (alloSCT) from HIV- resistant donors and who experienced acute graft-versus-host disease indicate that the recipient’s immuno- hematopoietic compartment must be quickly replaced, leaving no opportunity for re-seeding of viral reservoirs as would occur with slow or incomplete immune reconstitution. The goal of this project is to phenocopy these cases of HIV cure in humans by devising a genetically engineered cellular system to achieve simultaneous HIV resistance and efficient T cell-mediated depletion of the viral reservoir without the risk of uncontrollable alloreactivity. This will be tested in non-human primates that are infected with a SHIV and treated with autologous stem cells and chimeric antigen receptor T cells engineered for HIV resistance. We recently developed a platform to eradicate the hematopoietic compartment using chimeric antigen receptor (CAR) T cells directed against the pan-leukocyte antigen CD45. Since T cells also express CD45, to prevent fratricide (self-killing by T cells) we mapped the target epitope of the anti-CD45 CAR and used CRISPR base editing to mutate a single amino acid (Y232C) in the CD45 extracellular domain to generate anti-CD45 redirected CAR T cells that were themselves resistant to CAR45. To allow hematopoietic reconstitution in the face of anti- CD45 immune attack, we also mutated Y232C in CD34+ hematopoietic stem/progenitor cells (HSPC) and demonstrated normal hematopoietic engraftment and development. This platform allowed us to develop a universal blood cancer immune therapy and has paved the way to the immuno-hematopoietic ablation and replacement that we postulate is required for erasing the HIV reservoir. We have now also shown that we can create dual CD45-resistant and HIV-resistant cells, using multiplexed CRISPR base editing to introduce the CD45Y232C mutation (installing resistance to anti-CD45 CART) and a CCR5start codon off mutation (CCR5BE) that installs HIV resistance. These genetic modifications led to HIV-resistant and anti-CD45 resistant CART cells that could effectively eliminate HIV-infected CD4 T cells in mouse xenografts while protecting the engrafted cells from infection. In this project, we will test our therapeutic strategy in SHIV-infected animals, by testing the hypothesis that an autologous cellular immunotherapy platform can clear the viral reservoir. In Aim 1 we will replace endogenous hematopoiesis with multiplex base edited HSC in Rhesus macaques (RM). In Aim 2 we will test the ability of autologous anti-CD45 CART cells to eliminate the immune-hematopoietic compartment in RM. In Aim 3 we will treat SHIV-infected RM engrafted with engineered CAR45-resistant, HIV-resistant HSC using autologous anti- CD45 CART cells.