Engineering hematopoietic stem cells to generate therapeutic antibody secreting B cells - PROJECT SUMMARY Hypercholesterolemia is a major public health crisis, underscored by the fact that cardiovascular disease is the leading cause of death globally. The standard treatment for hypercholesterolemia is statins, but more recently effective biologics have emerged, including antibodies targeting proprotein convertase subtilisin/kexin type 9 (PCSK9). Inhibition of PCSK9 results in greater liver low density lipoprotein receptor (LDLR) expression and lower cholesterol levels in the bloodstream, making it an effective treatment for hypercholesterolemia. While anti-PSCK9 antibodies are very effective, they require repeated dosing for a patient’s lifetime to maintain efficacy – highlighting the need for of a singular, lifelong therapeutic solution for hypercholesterolemia. Our group has pioneered use of precision CRISPR/Cas9 genome-editing of hematopoietic stem and progenitor cells (HSPCs) to not only correct underlying genetic defects, but to engineer cells with new biologic function. In this proposal, I will use this technology to test my hypothesis that engineering HSPCs to produce B cells that secrete anti-PCSK9 antibodies upon engraftment would be an effective and durable treatment for hypercholesterolemia. I will accomplish this by targeting an engineered antibody transgene delivered by a recombinant adeno-associated virus 6 (AAV6) vector into a genomic safe-harbor site to minimally disrupt normal B cell function and allow for antibody expression from a synthetic B cell promoter. I have demonstrated feasibility of this gene-targeted antibody (GT-Ab) strategy through efficient targeted integration of antibody cassettes in human HSPCs and subsequent engraftment and multi-lineage reconstitution in immunodeficient mice. In addition, I have shown that B cells engineered with GT-Abs secrete functional antibodies in vitro. In order to further evaluate the effects and therapeutic potential of engineering GT-Ab HSPCs, I propose to (i) characterize the effect of GT-Ab engineering of HSPCs on B cell development (ii) study GT-Ab B cell expression and reconstitution in vivo by engraftment of human GT-Ab HSPCs, and (iii) evaluate therapeutic efficacy of GT-Ab HSPCs in a disease model of hypercholesterolemia. Overall, the completion of this proposal will provide important safety and efficacy data for engineering a hematopoietic system capable of generating antibody-secreting B cells to inhibit PCSK9 for the lifelong treatment of hypercholesterolemia. This fellowship will take place at Stanford University School of Medicine in the Institute of Stem Cell Biology and Regenerative Medicine, providing robust scientific and training infrastructure for the proposed work. Dr. Matthew Porteus is the ideal sponsor for this project due to his expertise in genome-engineering HSPCs and translating therapies to the clinic as well as his extensive track record of mentoring independent investigators. I will also be supported by an advisory team with the diverse expertise necessary to carry out this proposal, comprised of Drs. Peter Kim (antibody engineering), Hiromitsu Nakauchi (hematopoietic stem cell transplant), Kara Davis (B cell development, single-cell immune profiling), and Joshua Knowles (hypercholesterolemia).
