CD90 as a Target for In Vivo Gene and Cell Therapies

Abstract

Hematopoiesis occurs through a hierarchical process of differentiation beginning with multipotent self-renewing hematopoietic stem cells (HSCs) that persist throughout the human lifespan and ending with transient terminally differentiated unipotent cells. This progression allows for a malleable hematopoietic system capable of responding to pathological and physical assaults to the body. The hierarchical differentiation of the hematopoietic system can be exploited in the clinic for benign and malignant hematology. A patients' pathological hematopoietic system can be cured by replacing their HSCs with new HSCs from a healthy donor (i.e. allogeneic transplantation). Furthermore, the advent of gene modification therapies allows for pathological mutations in a patient's HSCs to be treated by gene therapy and re-transfused into patients (i.e autologous transplantation). The best examples of this therapeutic platform being the treatment of hemoglobinopathies. The reliance of current ex vivo HSC gene therapy approaches on prolonged in vitro culture time, toxic myeloablative conditioning regimens to remove unmodified HSCs, and highly specialized infrastructure severely impact the accessibility of these therapie. Therefore, novel HSC-specific gene therapy platforms with simplified manufacturing protocols for in vivo applications are needed. Previous work refined the target for HSC gene therapy and identified a phenotypically defined subset of cells (CD34+CD90+) that is exclusively responsible for rapid recovery onset, robust long-term multilineage engraftment, and entire reconstitution of the bone marrow (BM) stem cell compartment. My long-term goal is to target this refined HSC subset ex vivo and in vivo for gene and cellular therapies. In this dissertation I develop CD90-recognizing chimeric molecules successfully engineered them onto viral vectors. First proof-of-concept studies in vitro showed that CD90-targeted lentiviral vectors (CD90-LVs) delivered their cargo, with high specificity, into CD90+ cell lines and primary human CD34+CD90+ HSCs. Encouraged by these results, we applied and evaluated the safety of CD90-targeted viral vectors for in vivo applications to transduce and edit human HSCs. Sufficient conditioning with low-dose chemotherapeutics before transplantation of gene-edited HSC is crucial for efficient ex vivo HSC gene therapy. This pretreatment, while genotoxic, enables the engraftment of gene-therapy cell products at therapeutic chimeric thresholds. In vivo gene therapy does not require pre-conditioning. Therefore, targeted, nongenotoxic selection platforms post-vector delivery can be leveraged to increase gene marked/corrected chimerism. Targeting the CD34+CD90+ HSC pool for these enrichment strategies would significantly improve gene modified chimerism. I hypothesized that using CD90-targeted gene and cellular therapies in vivo will not only efficiently deliver gene therapeutics to human HSCs with high target specificity, but gene-marked/edited HSCs can subsequently repopulate the hematopoietic hierarchy after anti-CD90-targeted selection (i.e., chimeric antigen receptor (CAR) T cells). In summary, CD90-LVs can be easily implemented into currently existing ex vivo HSC gene therapy approaches to replace the utilization of cytotoxic chemical transduction enhancers and increase the efficiency of HSC delivery. Furthermore, the ability to target LVs to HSCs in vivo will significantly enhance the accessibility of HSC gene therapy in areas with limited biomedical infrastructure. Additionally, protecting HSCs from CD90-targeted immunotherapies and discovering CD90-dependent signaling pathways will minimize off-target toxicities of potential therapies and open a new category of anti-tumor targets in the clinic. This strategy could also enrich gene-edited HSCs to treat sickle cell disease (SCD).