UCLA

Autologous hematopoietic stem cell gene therapy with lentiviral vectors has recentlyemerged as a successful treatment option for a number of previously incurable genetic blood diseases. However, a limitation of a majority of lentiviral vectors to date is the inability to physiologically regulate the expressed transgene. Many vectors are driven by constitutively active promoter elements in an effort to drive therapeutic levels of expression in the target cell lineage(s). Although this approach has been efficacious for some gene targets, this lack of regulation is highly unfavorable for other gene targets that require strict physiological regulation and expression. To address these limitations, we present the development of a novel bioinformatics-guided approach for the design of endogenously regulated lentiviral vectors. In Chapter 2, we implement our bioinformatics approach to elucidate the regulatory elements of the CYBB gene to design a highly regulated vector for the treatment of X-Linked Chronic Granulomatous Disease (X-CGD), able to recapitulate the physiological expression and regulation of the native gene. We also demonstrate the capability to delineate the minimal functional boundaries of the key enhancer within the vector cassette, leading to an improvement in titer, infectivity and expression for an optimized vector design. In Chapter 3, we expand our developmental pipeline to generate a lentiviral vector for the treatment of Wiskott-Aldrich Syndrome (WAS). Using a similar approach to identify the endogenous regulatory elements of the WAS gene, we were able to pinpoint critical lineage specific enhancers for high-level expression in all affected lineages, especially in megakaryocytes. This high-level expression addresses an unmet medical need posed by the current clinic gene therapy vector for WAS which under expresses in the megakaryocyte lineage, consequently leaving patients thrombocytopenic. In Chapter 4, we combine our bioinformatics-guided approach with an in-vivo multiplexed screen and identified key lineage and temporal specific enhancer responsible for regulating the RAG1 gene throughout specific timepoints of T and B lymphocyte differentiation. Using these key elements, we developed a series of lead candidate vectors for future preclinical development for RAG1 Severe Combined Immunodeficiency (RAG1 SCID) gene therapy. Collectively, the work here describes the development of a novel approach to design endogenously regulated lentiviral vectors which can be applied to other gene targets. This approach has the ability to revolutionize the field of vector design and expand the potential targets of gene therapy viral vectors.