Hematopoietic Stem Cell Transposon Therapy for Severe Combined Immunodeficiency

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DESCRIPTION (provided by applicant): Our goal, and the main challenge in the treatment of severe combined immunodeficiency (SCID) today, is to develop a robust alternative to allogeneic hematopoietic stem cell (HSC) transplantation and to gene correction of autologous HSC with viral vectors, as both these strategies have been associated with unacceptable morbidity and mortality in SCID patients. Non-viral gene therapy represents a genuine alternative to HSC transplantation and viral gene therapy. To correct SCID (caused by a point mutation in exon 85 of the DNA protein kinase catalytic subunit, DNA-PKcs, gene, ~13 kB in length), we will focus on non-viral Tol2 transposon-mediated delivery of DNA-PKcs into hematopoietic cell line and fresh HSC derived from wild type and SCID mice. Transposon-mediated gene delivery has several advantages. Foremost among these are: simple error-free design, reduced immunogenicity and risk of contamination with replication competent virus, and, critically, the ability of the high cargo Tol2 transposons to stably deliver large genes (>10 kB) into mammalian cells. As currently available methods for naked DNA delivery, such as nucleofection, result in cellular injury that is significantly more toxic to the stem cells than to their more differentiated progeny, electromagnetic charge based methods are not useful for our primary goal, stem cell gene therapy in murine model of SCID. Therefore, a liposome-mediated HSC-specific gene delivery with Stem Cell Factor conjugated liposomes will be used instead. The challenges that warrant exploration in the field of SCID treatment are delivery of non-viral vectors to HSC without compromising its functional abilities, and correction of large genes (such as DNA-PKcs). With our expertise in non-viral stem cell transgenesis on one hand, and in immunobiology readouts on the other, we are well positioned to meet these challenges. We propose incremental data-driven investigations that address the three most important concerns: (i) gene delivery (a prerequisite for genomic transposition); (ii) functional readouts (establishing the relevance); and (iii) risk of insertional mutagenesis and tumorigenesis (critical for pre-clinical safety assessment of the proposed approaches). We focused upon a prototypic immune disorder offering a high likelihood of success due to small numbers of corrected cells needed. We trust that we present a compelling argument that testing of non-viral gene therapy strategy in particular is critical for prioritization of primary immunodeficiency gene therapy approaches for clinical translation in general. PUBLIC HEALTH RELEVANCE: Our goal is to develop clinically relevant approaches that will facilitate stem cell gene therapy to treat patients with primary immunodeficiencies. The fundamental insights gained from these studies will have broad implications relevant to both gene therapy and treatment of blood diseases.