Anne Dubart-Kupperschmitt

Transduced CD34+ Cells from Adrenoleukodystrophy Patients with HIV-Derived Vector Mediate Long-Term Engraftment of NOD/SCID Mice

X-linked adrenoleukodystrophy (ALD), an inherited demyelinating disorder of the central nervous system, can be corrected by allogeneic bone marrow transplantation, likely due to the turnover of brain macrophages that are bone marrow derived. ALD is characterized by an accumulation of very long chain fatty acids (VLCFA) due to the deficiency of an ATP binding cassette transporter that imports these fatty acids in peroxisomes. Murine retroviral transduction results in metabolic correction of ALD CD34(+) cells in vitro but reinfusion of these cells into ALD patients would not provide clinical benefit owing to the absence of selective advantage conferred by transgene expression. High-efficiency transduction of ALD CD34(+) peripheral blood mobilized cells was achieved using an HIV-based vector driving ALD gene expression under the elongation factor 1 alpha promoter and a protocol without prestimulation of CD34(+) cells with cytokines prior to transduction to preserve their stem cell properties. Efficient expression of the ALD gene was demonstrated in monocytes/macrophages derived from cultures of transduced ALD CD34(+) cells and in long-term culture initiating cells. VLCFA metabolism was corrected in transduced CD34(+), CFU-derived, and LTC-derived cells, indicating that the vector-encoded ALD protein was fully functional. Transplantation of transduced ALD CD34(+) cells into NOD/SCID mice resulted in long-term expression of ALD protein in monocytes/macrophages derived from engrafted stem cells.

Glycoprotein Ibα Promoter Drives Megakaryocytic Lineage‐Restricted Expression After Hematopoietic Stem Cell Transduction Using a Self‐Inactivating Lentiviral Vector

Megakaryocytic (MK) lineage is an attractive target for cell/gene therapy approaches, aiming at correcting platelet protein deficiencies. However, MK cells are short‐lived cells, and their permanent modification requires modification of hematopoietic stem cells with an integrative vector such as a lentiviral vector. Glycoprotein (Gp) IIb promoter, the most studied among the MK regulatory sequences, is also active in stem cells. To strictly limit transgene expression to the MK lineage after transduction of human CD34+ hematopoietic cells with a lentiviral vector, we looked for a promoter activated later during MK differentiation. Human cord blood, bone marrow, and peripheral‐blood mobilized CD34+ cells were transduced with a human immunodeficiency virus‐derived self‐inactivating lentiviral vector encoding the green fluorescent protein (GFP) under the transcriptional control of GpIbα, GpIIb, or EF1α gene regulatory sequences. Both GpIbα and GpIIb promoters restricted GFP expression (analyzed by flow cytometry and immunoelectron microscopy) in MK cells among the maturing progeny of transduced cells. However, only the GpIbα promoter was strictly MK‐specific, whereas GpIIb promoter was leaky in immature progenitor cells not yet engaged in MK cell lineage differentiation. We thus demonstrate the pertinence of using a 328‐base‐pair fragment of the human GpIbα gene regulatory sequence, in the context of a lentiviral vector, to tightly restrict transgene expression to the MK lineage after transduction of human CD34+ hematopoietic cells.