UCLA

Hematopoietic stem cell (HSC) transplant with gene therapy has recently emerged as a successful clinical treatment of a number of previously incurable genetic blood diseases. This approach aims to permanently fix genetic defects in HSCs, a rare and specialized type of cell with the unique ability to regenerate the entire blood system throughout a patient’s lifetime. In this approach, bone marrow (BM) or mobilized peripheral blood (mPB) is collected from a patient, enriched for HSCs, transduced with an engineered lentiviral vector (LV) encoding the correct genetic sequence, and transplanted back into the patient. After transplant, modified HSCs engraft in the BM and produce healthy blood cells throughout the patient’s lifetime.

While the last decade of research has yielded major advances including successful Phase I/II gene therapy clinical trials, clinical and commercial scaling of this technology to a broader range of patients and diseases has revealed a number of hurdles. One major limitation is the great expense and difficulty of producing clinical-grade LV, which I address in Chapters 2 and 3 by presenting two methods that improve the efficiency of LV transduction of HSC. In Chapter 4, I demonstrate the successful application of a new LV gene therapy for an autoimmune blood disease.

Chapter 2 presents a method to enhance the enrichment of HSCs from the heterogeneous cell population obtained from the collection of bone marrow cells, addressing a critical limitation in creating cost-effective, clinical-grade LV vector. This method utilizes immunomagnetic beads to purify CD34+CD38- cells, a population highly enriched for HSCs beyond standard CD34+ selection. Using immune-deficient xenograft models, we demonstrate that enrichment of CD34+CD38- cells reduces gene therapy culture scale and lentiviral vector requirements by ~10-fold while still maintaining the long-term gene-marked engraftment required for clinical benefit. Therefore, this strategy represents an easily translatable method which can conserve valuable clinical grade LV preparations and could lower the cost per patient, or allow for the treatment of a greater number of patients.

Chapter 3 presents a method to further improve HSC transduction efficiency with the use of two compounds: Prostaglandin E2 (PGE2) and poloxamer synperonic F108 (PS-F108). While transduction enhancement with each individual compound has previously been reported, the combination of these compounds leads to a synergistic and marked improvement in LV transduction of HSCs using a globin LV. Remarkably, this synergistic combination achieved a 6-fold improvement in gene transfer to long-term engrafting HSCs while using a LV dose 10-fold lower than the dose in our current clinical protocol. Thus this strategy has two major advantages: it reduces the amount of viral particles required to transduce HSCs, and also allows for better gene transfer and ultimate globin transgene expression, which is critical to improving clinical efficacy.

Finally, chapter 4 demonstrates the effectiveness of a newly engineered LV for the treatment of a severe form of genetic autoimmunity called IPEX syndrome. IPEX is caused by mutations in FoxP3, the key lineage-determining transcription factor required for the development and function of regulatory T cells (Treg cells). We developed a new LV using endogenous human FOXP3 regulatory elements to restore FoxP3 expression in a developmentally appropriate manner. We use this LV to transduce HSCs and restore functional Treg development in a mouse model of FoxP3 deficiency and successfully rescue autoimmune defects associated with this phenotype. These findings demonstrate preclinical efficacy for the treatment of IPEX patients by autologous HSC transplant and may provide further insight into new cell therapies for autoimmunity. Collectively, the work described here advances the field of gene therapy by improving the efficiency of the manufacturing process and expanding the range of diseases which can be treated by this method.