Retroviral Transfer and Long-Term Expression of the Adrenoleukodystrophy Gene in Human CD34⁺Cells

Abstract

Adrenoleukodystrophy (ALD) is a demyelinating disease of the central nervous system that results from a genetic deficiency of ALDP, an ABC protein involved in the transport of very long-chain fatty acids (VLCFAs). The cloning of the ALD gene and the positive effects of allogeneic bone marrow transplantation support the feasibility of a gene therapy approach. We report the retroviral transfer of the ALD cDNA to peripheral blood and bone marrow CD34+ cells from control donors and ALD patients. Prestimulation of these cells with cytokines, followed by infection with the M48-ALD retroviral vector, resulted in 20% transduction efficiency (4-40%) and expression of the vector-encoded ALDP in 20% of CD34+ cells (7.3-50%). Long-term culture (LTC) of transduced CD34+ cells from two ALD patients showed efficient transduction (24-28%) and stable expression (25-32%) of ALDP in derived clonogenic progenitors at 3 weeks of culture. The expression of ALDP in CFU cells derived from 5 and 6 weeks of LTC confirmed the effective transduction of LTC-initiating cells. Expression of ALDP was observed in CD68+ CFU-derived cells, suggesting that monocyte-macrophages, the target bone marrow cells in ALD, were produced from transduced progenitor cells. VL-CFA content was corrected in LTC and CFU-derived cells in proportion to the percentage of transduced cells, indicating that the vector-encoded ALDP was functional. Although not efficient yet to allow a clinical perspective, these results demonstrate the feasibility of ALD gene transfer into CD34+ cells of ALD patients.

Highly Efficient Lentiviral Gene Transfer in CD34 + and CD34 + /38 − /lin − Cells from Mobilized Peripheral Blood after Cytokine Prestimulation

“…However, for most of the monogenic diseases, there is no selective advantage of corrected cells, and the main problem resides in the very low percentage of corrected cells (usually <1%) in blood after several months. This is the case for adrenoleukodystrophy [29], Gaucher disease [30], and chronic granulomatous disease [31].…”

Hematopoietic stem cell gene therapy of murine protoporphyria by methylguanine-DNA-methyltransferase-mediated in vivo drug selection

“…3,35 However, for most of the monogenic diseases, there is no selective advantage of corrected cells, and the main problem resides in the low percentage of corrected cells in the blood after several months. [36][37][38] Lentiviral vectors based on HIV have inherent advantages in transducing nondividing cells including quiescent HSCs and hold the promise of curing congenital gene deficiencies. 12 Three recent studies have demonstrated the power of lentiviral vectors expressing the mutant MGMT gene in HSC selection and expansion Gene therapy of EPP with in vivo drug-based selection E Richard et al in mice, dogs and humans.…”

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

“…(iii) rabbit antiglial fibrillary acidic protein (GFAP) antibody (dilution 1:200, DAKO). The expression of EGFP and human ALD protein was visualized by using a rabbit anti-EGFP antibody [dilution 1:1,000, Abcam, UK) and a rabbit anti-ALD protein antibody (dilution 1:4,000 (23)]. The expression of mouse ALD protein was studied by using a specific rabbit anti-ALD protein antibody [dilution of 1:1,000, (24)].…”

“…CD34 ϩ cells were seeded in methylcellulose and macrophage colonies (colony-forming units-macrophages) were assessed 15 days after for VLCFA analysis. VLCFA were extracted and measured by gas͞liquid chromatography͞mass spectrometry (23). Results are expressed as C26:0͞C22:0 ratios.…”