In vivo targeting of hematopoietic stem cells to treat sickle cell disease
Final Report Abstract
Sickle cell disease (SCD) is an inherited disorder of red blood cells (RBC) affecting millions of people worldwide. Polymerization of sickle hemoglobin results in hemolysis and microvascular occlusions, leading to painful sickle cell crisis, stroke, acute chest syndrome and chronic organ damage. Therapeutic options consist of lifelong chronic RBC transfusions or hydroxyurea. The only curative treatment option for SCD patients is hematopoietic stem cell (HSC) transplantation. However, the risk of graft-versus-host disease is considerable and suitable HLA-matched donors are only available to 15% of the SCD-patients in the US. Autologous stem cell gene therapy eliminates these limitations, providing a potentially curative therapy for all patients. However, the cost and required infrastructure preclude the more widespread use of gene therapy especially in developing countries. In vivo gene therapy, i.e. the direct injection of a gene modifying agent, could eliminate the need for hospitalization, cell harvest, cell correction and conditioning of the patient. The aim of this work is to explore the potential of retroviral particles enveloped with the baboon endogenous retrovirus glycoprotein (BaEV) for in vivo HSC gene therapy. In contrast to the commonly used VSVg envelope, BaEV mediates efficient gene transfer into quiescent HSCs and is serum resistant. First, we generated a stable BaEV packaging cell line to produce hightiter and high-quality BaEV pseudotyped retroviral particles. Using this engineered cell line, we tested BaEV in the context of the two retroviral vector systems capable of infecting quiescent cells, lentiviral and alpha-retroviral vectors. Competitive in vivo gene transfer experiments in humanized mice revealed that both lenti- and alpha-retroviral systems can transduce HSC at similar rates. Next, we tested if mobilization of HSCs into the peripheral blood could enhance gene transfer efficacy. The combination of AMD3100 and Gro-beta efficiently mobilizes HSCs and led to initial gene marking rates of 3.5% of the human hCD45+ cells. To increase the genemodified cells to the therapeutic threshold required for SCD, which is estimated to be in the range of 20-30% of RBC, we tested a chemoselection strategy based on the expression of MGMT-P140K, which mediates resistance to the alkylating agent BCNU. After three cycles of BCNU treatment, we observed a 20-fold to 144-fold enrichment of gene-marked cells in the peripheral blood using either a low escalating or a high dose regimen, respectively. In the bone marrow, gene marking reached 6-7% transduced cells in lymphoid and myeloid lineages and 18.1% in hCD34+ cells long-term. The analysis of barcoded viral vectors revealed transduction and expansion of multiple clones in primary and secondary recipients. Finally, we combined the MGMT-P140K overexpression with a therapeutic payload by targeting the transcription factors BCL11A and ZNF410 using miRNA-embedded shRNAs (shmiR) containing vectors. Downregulation of BCL11A and ZNF410 in erythroid cells leads to sustained reactivation of gamma-globin and HbF induction, which largely attenuates the hematologic effects of sickle cell disease. Following in vivo gene transfer and BCNU selection, we observed a significant upregulation of gamma-globin and HbF in gene marked cells, reaching 35% and 25%, respectively. In summary, we demonstrate the proof-of-principle that BaEVRLess-pseudotyped retroviral particles are capable of in vivo HSC gene transfer.
Publications
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Genetic engineering of a virus producer cell line to generate high-titer and high-quality BaEVRlesspseudotyped alpha-retroviral particles. ESGCT Collaborative Virtual Congress 19-22 October 2021. Human Gene Therapy.
Klatt D., Wright K.E., Wolf S., Schambach A., Verhoeyen E., Williams D.A. & Brendel C.
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Stable BaEVRless producer cell line for the production and in vivo application of alpharetroviral particles. ASGCT Annual Meeting. Washington DC 16-19 May 2022. Molecular Therapy.
Klatt D., Mucci A., Zhang C.Y., Wright K.E., Wolf S., Schambach A., Verhoeyen .E, Williams D.A. & Brendel C.
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In vivo HSC gene therapy using BaEVRLess-pseudotyped retroviral vectors. ASGCT Annual Meeting. Los Angeles 16-20 May 2023. Molecular Therapy.
Klatt D., Liu B., Mucci A., Peschers M., Schiroli G., Schambach A., Harris C., Pellin D., Manis J., Armant M., Scadden D.T., Verhoeyen E, Williams D.A. & Brendel C.
