Transduced CD34+ cells from adrenoleukodystrophy patients with HIV-derived vector mediate long-term engraftment of NOD/SCID mice

Abstract

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.

Human CD34+cells differentiate into microglia and express recombinant therapeutic protein

“…CD34 ϩ cells were isolated from UCB of normal individuals or from granulocyte colonystimulating factor MPB from ALD patients after obtaining their informed consent and in accordance with approved institutional guidelines. CD34 ϩ cells were purified by immunomagnetic selection (17). Fluorescence-activated cell sorting (FACS) analysis performed on a FACStar (Becton Dickinson) showed that the CD34 ؉ population had a purity of over 90%.…”

“…Human UCB and MPB CD34 ϩ cells were transduced according to previously described procedures (17,18) with a self-inactivating lentiviral͞HIV vector (TRIP⌬U3) carrying expression cassettes for the human ALD protein or the enhanced GFP (EGFP) under the control of the elongation factor 1␣ promoter. Briefly, CD34 ϩ cells were incubated with lentiviral vector in the presence of four recombinant human cytokines (stem cell factor, Flt3-ligand, IL-3, pegylatedmegakaryocyte growth and differentiation factor; UCB ϭ 100͞ 10͞100͞60 ng͞ml, MPB ϭ all 10 ng͞ml).…”

“…Virus titers were 10 9 units͞ml. Monocytes͞macrophages were identified by using monoclonal anti-CD68 KP1 (DAKO) (17). Transduction efficiency in CD45 ϩ or CD68 ϩ cells was tested by determining the percentage of EGFP-and ALD-positive cells by FACS or immunocytochemistry (17).…”

Therapy of X-linked adrenoleukodystrophy

“…The clinical efficacy of lovastatin therapy in X-ALD patients remains to be proven. of ALD protein in monocytes/macrophages derived from engrafted stem cells [126]. Additionally, it was shown that cells derived from human CD34+ cells isolated from cord blood or peripheral blood migrate into the brain after infusion into immunodeficient mice and differentiate into perivascular and ramified microglia.…”

Stem cell-transplantation therapy for adrenoleukodystrophy: current perspectives

“…After laying the preclinical groundwork for efficient ex vivo lentiviral vector-mediated transduction of human hematopoietic stem cells with wild type ABCD1, a French group described the initial human experience with this gene-therapy approach. [65][66][67][68] Two boys with ALD, both aged 7 years, lacked HLA-matched donors or UCB units for allogeneic HCT. Both demonstrated progressive cerebral disease on MRI.…”