Abstract
We generated an integrating, CD46-targeted, helper-dependent adenovirus HDAd5/35++ vector system for hematopoietic stem cell (HSC) gene therapy. The ∼12-kb transgene cassette included a β-globin locus control region (LCR)/promoter driven human γ-globin gene and an elongation factor alpha-1 (EF1α)-mgmtP140K expression cassette, which allows for drug-controlled increase of γ-globin-expressing erythrocytes. We transduced bone marrow lineage-depleted cells from human CD46-transgenic mice and transplanted them into lethally irradiated recipients. The percentage of γ-globin-positive cells in peripheral blood erythrocytes in primary and secondary transplant recipients was stable and greater than 90%. The γ-globin level was 10%–20% of adult mouse globin. Transgene integration, mediated by a hyperactive Sleeping Beauty SB100x transposase, was random, without a preference for genes. A second set of studies was performed with peripheral blood CD34+ cells from mobilized donors. 10 weeks after transplantation of transduced cells, human cells were harvested from the bone marrow and differentiated ex vivo into erythroid cells. Erythroid cells expressed γ-globin at a level of 20% of adult α-globin. Our studies suggest that HDAd35++ vectors allow for efficient transduction of long-term repopulating HSCs and high-level, almost pancellular γ-globin expression in erythrocytes. Furthermore, our HDAd5/35++ vectors have a larger insert capacity and a safer integration pattern than currently used lentivirus vectors.
Highlights
hematopoietic stem cell (HSC) Gene Therapy Hemoglobinopathies (b-thalassemia and sickle cell disease) are the most frequent monogenic diseases worldwide, with approximately 5% of the world population carrying a hemoglobin disorder trait.[1]
Currently, SIN lentivirus and rAAV6 vectors are used for HSC transduction.[31,32,33]
Integrating HDAd5/35++ vectors have a number of potential advantages over self-inactivating lentivirus (SIN-LV) and rAAV vectors for globin gene therapy
Summary
HSC Gene Therapy Hemoglobinopathies (b-thalassemia and sickle cell disease) are the most frequent monogenic diseases worldwide, with approximately 5% of the world population carrying a hemoglobin disorder trait.[1]. Therapeutic genes are under the control of b-globin locus control region (LCR) versions for erythroid-specific, position-independent expression. For HSC transduction, SIN-LV-globin vectors are incubated ex vivo with CD34+ cells for 2 to 3 days under conditions that support cell cycling. Because the vectors require an erythroid-specific LCR, SIN-LV vectors for globin gene therapy are relatively large and difficult to produce at high titers. This in turn influences the cost for gene therapy. Because of the structure and/or size of globin SIN-LV vectors, the HSC transduction frequency is relatively low.[5] no leukemic events have been found in patients treated with SIN-LV vectors, their preference for integrating into active genes and transformation events seen in vitro after HSC transduction[7] create a challenge for HSC gene therapy
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