scholarly journals Development of a forward-oriented therapeutic lentiviral vector for hemoglobin disorders

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Naoya Uchida ◽  
Matthew M. Hsieh ◽  
Lydia Raines ◽  
Juan J. Haro-Mora ◽  
Selami Demirci ◽  
...  
Keyword(s):  
Gene Therapy ◽  
High Efficiency ◽  
Lentiviral Vector ◽  
Transduction Efficiency ◽  
Specific Expression ◽  
Hematopoietic Stem ◽  
Rev Response Element ◽  
High Level ◽  
Intron 2

Abstract Hematopoietic stem cell (HSC) gene therapy is being evaluated for hemoglobin disorders including sickle cell disease (SCD). Therapeutic globin vectors have demanding requirements including high-efficiency transduction at the HSC level and high-level, erythroid-specific expression with long-term persistence. The requirement of intron 2 for high-level β-globin expression dictates a reverse-oriented globin-expression cassette to prevent its loss from RNA splicing. Current reverse-oriented globin vectors can drive phenotypic correction, but they are limited by low vector titers and low transduction efficiencies. Here we report a clinically relevant forward-oriented β-globin-expressing vector, which has sixfold higher vector titers and four to tenfold higher transduction efficiency for long-term hematopoietic repopulating cells in humanized mice and rhesus macaques. Insertion of Rev response element (RRE) allows intron 2 to be retained, and β-globin production is observed in transplanted macaques and human SCD CD34+ cells. These findings bring us closer to a widely applicable gene therapy for hemoglobin disorders.

scholarly journals Long-Term Transfer and Expression of the Human β-Globin Gene in a Mouse Transplant Model

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3414-3422 ◽  
Author(s):  
Harry Raftopoulos ◽  
Maureen Ward ◽  
Philippe Leboulch ◽  
Arthur Bank
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Bone Marrow Cells ◽  
High Titer ◽  
Globin Gene ◽  
High Level Expression ◽  
Hematopoietic Stem ◽  
High Level ◽  
Transplant Model

Abstract Somatic gene therapy of hemoglobinopathies depends initially on the demonstration of safe, efficient gene transfer and long-term, high-level expression of the transferred human β-globin gene in animal models. We have used a β-globin gene/β-locus control region retroviral vector containing several modifications to optimize gene transfer and expression in a mouse transplant model. In this report we show that transplantation of β-globin–transduced hematopoietic cells into lethally irradiated mice leads to the continued presence of the gene up to 8 months posttransplantation. The transferred human β-globin gene is detected in 3 of 5 mice surviving long term (>4 months) transplanted with bone marrow cells transduced with high-titer virus. Southern blotting confirms the presence of the unrearranged 5.1-kb human β-globin gene-containing provirus in 2 of these mice. In addition, long-term expression of the transferred gene is seen in 2 mice at levels of 5% and 20% that of endogenous murine β-globin at 6 and 8 months posttransplantation. We further document stem cell transduction by the successful transfer and high-level expression of the human β-globin gene from mice transduced 9 months earlier into irradiated secondary recipient mice. These results demonstrate high-level, long-term somatic human β-globin gene transfer into the hematopoietic stem cells of an animal for the first time, and suggest the potential feasibility of a retroviral gene therapy approach to sickle cell disease and the β thalassemias.

Development of a New Generation, Forward-Oriented Therapeutic Vector for Hemoglobin Disorders

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1172-1172 ◽  
Author(s):  
Naoya Uchida ◽  
Matthew M. Hsieh ◽  
Aylin C Bonifacino ◽  
Allen E Krouse ◽  
Mark E Metzger ◽  
...  
Keyword(s):  
Globin Gene ◽  
Transduction Efficiency ◽  
Erythroid Cells ◽  
Cd34 Cells ◽  
Globin Promoter ◽  
High Level ◽  
Viral Components ◽  
Intron 2 ◽  
Viral Titers

Abstract Ameliorating hemoglobin disorders such as sickle cell disease (SCD) using hematopoietic stem cell (HSC) gene therapy is under development. Unlike in other diseases, therapeutic globin vectors have demanding requirements including high-level β-globin expression, tissue specificity among erythroid cells, long-term persistence, and high-level modification at the HSC level. These demanding requirements necessitate the inclusion of complex genetic elements including the locus control region (LCR), β-globin promoter, β-globin gene, and the 3' untranslated region (3'UTR), all now feasible using lentiviral vectors. The additional requirement of intron 2 for high-level β-globin expression dictates a reverse-oriented globin-expression cassette to prevent loss by RNA splicing during viral preparation. This reverse-orientation is in contrast to all other therapeutic vectors under clinical development. Current reverse-oriented globin vectors can drive phenotypic correction in mouse models for both b-thalassemia and SCD, while they are limited by lower viral titers and lower transduction efficiency in primary human HSCs, limiting their prospects, especially in SCD. We hypothesized that the reverse-orientation impedes both viral preparation and vector transduction, as despite deletion of cryptic polyadenylation (polyA) signals to optimize a conventional reverse-oriented globin vector, titers were still 10-fold lower than a standard GFP-vector. We thus designed a forward-oriented globin-expressing vector, which was further optimized by minimizing the size of the LCR, inclusion of a large segment of the β-globin promoter and an enhancer region of the 3'UTR lacking the polyA signal. Viral titers of the forward-oriented vectors (1.0±0.2x10e9 IU/mL) were 6-fold higher than the optimized vector in the reverse orientation (1.6±0.2x10e8 IU/mL, p<0.01), and comparable to a standard GFP-marking vector (1.9±0.2x10e9 IU/mL, p<0.01). The forward-oriented vector demonstrated 3-4 fold higher transduction efficiency among human erythroid cells derived from transduced CD34+ cells in in vitro culture (34±0% vs 13±1%, p<0.01) and in xenografted mice (31±9% vs 7±5%, p<0.05). To evaluate transduction efficiency for long-term HSCs, we transduced rhesus CD34+ cells with our optimized reverse-oriented vector and our forward-oriented vector including GFP or YFP genes (instead of β-globin gene) in a competitive repopulation assay following 10 Gy total body irradiation. In two animals, GFP and YFP signals from both vectors were detected exclusively in red blood cells, documenting tissue specificity. Gene marking levels were 10-fold higher out to 4 years with the forward-oriented vector, compared to the reverse-oriented vector, and were comparable to standard GFP or YFP-marking vectors in 2 other animals. We then replaced the GFP gene with the β-globin gene containing intron 2 in the forward-oriented vector construct. To positively select intron-2-containing β-globin vectors, essential viral components (packaging signal, rev response element (RRE), or central polypurine tract (cPPT)) were deleted in the backbone of the forward-oriented vector, and the deleted viral components were inserted into intron 2 of the β-globin gene. We observed that half of the forward-oriented vectors lost intron 2 during vector preparation when no elements were included into intron 2. Insertion of the RRE resulted in positive selection of intron-2-containing β-globin vectors. We confirmed β-globin expression from the forward-oriented vector by hemoglobin A production in human erythroid cells derived from transduced peripheral blood mononuclear cells from SCD patients. Finally, human β-globin expression was detected in rhesus erythroid cells following transplantation of transduced CD34+ cells in 2 animals. In summary, we have developed a clinically relevant forward-oriented globin-expressing vector, which has 6 fold higher viral titers and 4-10 fold higher transduction efficiency for hematopoietic repopulating cells, as compared to the optimized reverse-oriented vector. RRE insertion allowed positive selection of intron-2-containing β-globin vectors, and human β-globin production was observed in transplanted rhesus macaques with the forward-oriented β-globin vector transduction. These findings bring us closer to a curative gene therapy for hemoglobin disorders. Disclosures No relevant conflicts of interest to declare.

scholarly journals Stromal support enhances cell-free retroviral vector transduction of human bone marrow long-term culture-initiating cells

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1393-1399 ◽  
Author(s):  
KA Moore ◽  
AB Deisseroth ◽  
CL Reading ◽  
DE Williams ◽  
JW Belmont
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
Long Term Culture ◽  
Stromal Support

Gene transfer into hematopoietic stem cells by cell-free virions is a goal for gene therapy of hematolymphoid disorders. Because the hematopoietic microenvironment provided by the stroma is required for stem cell maintenance both in vivo and in vitro, we reasoned that cell- free transduction of bone marrow cells (BMC) may be aided by stromal support. We used two high-titer replication-defective retroviral vectors to differentially mark progenitor cells. The transducing vector was shown to be a specific DNA fragment by polymerase chain reaction of colony-forming cells derived from progenitors maintained in long-term culture (LTC). BMC were infected separately by cell-free virions with or without pre-established, irradiated, allogeneic stromal layers, and in the presence or absence of exogenous growth factors (GF). The GF assessed were interleukin-3 (IL-3) and IL-6 in combination, leukemia inhibitory factor (LIF), mast cell growth factor (MGF), and LIF and MGF in combination. In addition, we developed a competitive LTC system to directly assess the effect of infection conditions on the transduction of clonogenic progenitors as reflected by the presence of a predominate provirus after maintenance in the same microenvironment. The results show gene transfer into human LTC-initiating cells by cell-free retroviral vector and a beneficial effect of stromal support allowing a transduction efficiency of 64.6% in contrast to 15.8% without a supporting stromal layer. A high transduction rate was achieved independent of stimulation with exogenous GF. We propose that autologous marrow stromal support during the transduction period may have application in clinical gene therapy protocols.

Hematopoietic Stem Cell Gene Therapy with Lentiviral Vector in X-Linked Adrenoleukodystrophy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 163-163
Author(s):  
Patrick Aubourg ◽  
Salima Hacein-Bey-Abina ◽  
Cynthia Bartholomae ◽  
Manfred Schmidt ◽  
Ina Kutschera ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Conflicts Of Interest ◽  
Lentiviral Vector ◽  
Biological Data ◽  
Hematopoietic Stem ◽  
Matched Donor

Abstract Abstract 163FN2 The most severe form of X-linked adrenoleukodystrophy (ALD) is characterized by rapidly progressive and lethal cerebral demyelination in childhood. The progression of cerebral demyelination of ALD can be arrested by allogeneic hematopoietic stem cell (HSC) transplantation (HCT) within 12–18 months, provided the procedure is performed at an early stage of the disease. The long term beneficial effects of HCT in ALD are likely due to the progressive turn-over of brain microglia that are derived from myeloid progenitors in the bone-marrow. Despite the increased availability of cord blood, not all boys with cerebral ALD and who are candidate for HCT have a suitable HLA-matched donor. In addition, allogeneic HCT remains associated with significant mortality risk. In late 2009, we reported that HSC gene therapy with lentiviral vector was able to arrest the progression of cerebral ALD in two boys who have no HLA-matched donor to perform HCT. ALD protein expression in myeloid and lymphoid lineages as well as the identification of identical lentiviral insertion sites in myeloid and lymphoid lineages strongly suggested that multi-potent long-term repopulating hematopoietic cells were transduced. In those 2 treated patients, hematopoiesis has remained polyclonal without evidence of clonal skewing or dominance up to the last follow-up. Data on clinical efficacy, gene marking and lentiviral integration studies with a longer follow-up (4 years ½) will be presented. HSC gene therapy however failed to arrest the progression of cerebral ALD in a third treated ALD patient, 36 months after gene therapy. Hematopoiesis remained also polyclonal in this patient and biological data that could explain failure of gene therapy in this patient will be presented. At last, data on a fourth patient who has been treated more recently (12 months) by HSC gene therapy will also be presented. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Stromal support enhances cell-free retroviral vector transduction of human bone marrow long-term culture-initiating cells

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1393-1399 ◽  
Author(s):  
KA Moore ◽  
AB Deisseroth ◽  
CL Reading ◽  
DE Williams ◽  
JW Belmont
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
Long Term Culture ◽  
Stromal Support

Abstract Gene transfer into hematopoietic stem cells by cell-free virions is a goal for gene therapy of hematolymphoid disorders. Because the hematopoietic microenvironment provided by the stroma is required for stem cell maintenance both in vivo and in vitro, we reasoned that cell- free transduction of bone marrow cells (BMC) may be aided by stromal support. We used two high-titer replication-defective retroviral vectors to differentially mark progenitor cells. The transducing vector was shown to be a specific DNA fragment by polymerase chain reaction of colony-forming cells derived from progenitors maintained in long-term culture (LTC). BMC were infected separately by cell-free virions with or without pre-established, irradiated, allogeneic stromal layers, and in the presence or absence of exogenous growth factors (GF). The GF assessed were interleukin-3 (IL-3) and IL-6 in combination, leukemia inhibitory factor (LIF), mast cell growth factor (MGF), and LIF and MGF in combination. In addition, we developed a competitive LTC system to directly assess the effect of infection conditions on the transduction of clonogenic progenitors as reflected by the presence of a predominate provirus after maintenance in the same microenvironment. The results show gene transfer into human LTC-initiating cells by cell-free retroviral vector and a beneficial effect of stromal support allowing a transduction efficiency of 64.6% in contrast to 15.8% without a supporting stromal layer. A high transduction rate was achieved independent of stimulation with exogenous GF. We propose that autologous marrow stromal support during the transduction period may have application in clinical gene therapy protocols.

scholarly journals Efficient transduction of pigtailed macaque hematopoietic repopulating cells with HIV-based lentiviral vectors

Blood ◽  
2008 ◽  
Vol 111 (12) ◽  
pp. 5537-5543 ◽  
Author(s):  
Grant D. Trobridge ◽  
Brian C. Beard ◽  
Christina Gooch ◽  
Martin Wohlfahrt ◽  
Philip Olsen ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Ex Vivo ◽  
High Titer ◽  
Lentiviral Vectors ◽  
Macaca Nemestrina ◽  
Hematopoietic Stem ◽  
Host Restriction ◽  
Vector Integration ◽  
High Level

AbstractLentiviral vectors are attractive for hematopoietic stem cell (HSC) gene therapy because they do not require mitosis for nuclear entry, they efficiently transduce hematopoietic repopulating cells, and self-inactivating (SIN) designs can be produced at high titer. Experiments to evaluate HIV-derived lentiviral vectors in nonhuman primates prior to clinical trials have been hampered by low transduction frequencies due in part to host restriction by TRIM5α. We have established conditions for efficient transduction of pigtailed macaque (Macaca nemestrina) long-term repopulating cells using VSV-G–pseudotyped HIV-based lentiviral vectors. Stable, long-term, high-level gene marking was observed in 3 macaques using relatively low MOIs (5-10) in a 48-hour ex vivo transduction protocol. All animals studied had rapid neutrophil engraftment with a median of 10.3 days to a count greater than 0.5 × 109/L (500/μL). Expression was detected in all lineages, with long-term marking levels in granulocytes at approximately 20% to 30%, and in lymphocytes at approximately 12% to 23%. All animals had polyclonal engraftment as determined by analysis of vector integration sites. These data suggest that lentiviral vectors should be highly effective for HSC gene therapy, particularly for diseases in which maintaining the engraftment potential of stem cells using short-term ex vivo transduction protocols is critical.

scholarly journals Fully Closed, Large-Scale, and Clinical Grade Cell Sorting of Hematopoietic Stem Cell (HSC)-Enriched CD90+ Cells for Transplantation and Gene Therapy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3246-3246
Author(s):  
Stefan Radtke ◽  
Margaret Cui ◽  
Anai M Perez ◽  
Yan-Yi Chan ◽  
Stefanie Schmuck ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Transduction Efficiency ◽  
Target Cells ◽  
Gene Modification ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Cell Fractions

Introduction: Hematopoietic stem cell (HSC) gene therapy/editing is a viable treatment option for various hematological diseases and disorders including hemoglobinopathies and HIV/AIDS. Most if not all currently available approaches target CD34-enriched cell fractions, a heterogeneous mix of mostly committed progenitor cells and only very few true HSCs with long-term multilineage engraftment potential. As a consequence, gene therapy/editing approaches are currently limited in their HSC targeting efficiency, very expensive consuming huge quantities of modifying reagents, and can lead to unwanted side-effects in non-target cells. We recently described a novel HSC-enriched CD34 subset (CD90+CD45RA-) that is exclusively responsible for rapid recovery onset, robust long-term multilineage engraftment, as well as entire reconstitution of the bone marrow stem cell compartment in the nonhuman primate (NHP) stem cell transplantation and gene therapy model (Radtke et al. 2017, STM). Most importantly, we demonstrate that this CD34 subset reduces the number of target cells, modifying reagents and costs by more than 10-fold without compromising the long-term efficiency of gene-modification in the NHP (Humbert and Radtke et al. 2019, STM). Here, we aimed to develop a clinical protocol to reliably purify and efficiently gene-modify human HSC-enriched CD90+ cell fractions. Methods: Large-scale enrichment of CD34+ cells from GCSF-mobilized leukapheresis products was initially performed on the Miltenyi CliniMACS Prodigy according to previously established protocols (Adair et al. 2017, Nat. Comm.). Yield, purity, quality, and feasibility of CD90 sorting was then comprehensively tested on two different commercially available cell sorting systems comparing the jet-in-air sorter FX500 from Sony and the cartridge-based closed-system sorter MACSQuant Tyto from Miltenyi Biotech with our clinically approved gold-standard CD34-mediated gene therapy approach. Sorted CD90+ and bulk CD34+ cells were transduced with a clinical-grade lentivirus encoding for GFP and the multilineage differentiation as well as engraftment potential tested using in vitro assays and the NSG mouse xenograft model, respectively. Results: Flow-cytometric sort-purification of CD90+ cells was similarly efficient in purity and yield using either the FX500 or Tyto (Figure A,B). Both approaches reliably reduced the overall target cell count by 10 to 15-fold without impacting the cells viability and in vitro colony-forming cell potential. Unexpectedly, the transduction efficiency of sort-purified CD90+ cells was significantly improved compared to bulk-transduced CD34+ cells and especially the CD34+CD90+ subset (Figure C). All cell fractions demonstrated robust mouse xenograft potential (Figure D). Most importantly, significantly higher levels of GFP+ expression in the peripheral blood, bone marrow, spleen and thymus were observed after transplantation of gene-modified CD90+ compared to bulk CD34+ cells in NSG mice (Figure E). Conclusion: Here, we show that sort-purification of our HSC-enriched CD34+CD90+ cell subset is technically feasible and highly reproducible in two different systems. Purification of human CD90+ cell fractions significantly increased the gene-modification efficiency of primitive human HSCs with multilineage mouse engraftment potential. These findings should have important implications for currently available as well as future HSC gene therapy and gene editing protocols. Isolation of an HSC-enriched phenotype will allow more targeted gene modification and thus likely reduce unwanted off target effects. Our approach further reduced the overall costs for gene modifying reagents, can be combined with a closed transduction system, increase the portability and ultimately make HSC gene therapy GMP-facility independent and affordable. Finally, this stem cell selection strategy may also allow efficient and effective depletion of donor T cells in the setting of allogeneic stem cell or organ transplantation. Figure: A) Purity and B) yield of CD90+ cells after sort-purification. C) Transduction efficiency of bulk-transduced CD34+CD90+ cells and sort-purified CD90+ cells. Frequency of D) human chimerism and E) GFP+ human CD45+ cells in the peripheral blood (PB), bone marrow, spleen and thymus after transplantation of gene-modified bulk CD34+ or sort-purified CD90+ cells. Figure Disclosures Kiem: CSL Behring: Consultancy; Rocket Pharma: Consultancy, Equity Ownership; Homology Medicines: Consultancy, Equity Ownership; Magenta Therapeutics: Consultancy.

scholarly journals Hematopoietic Stem Cell Gene Therapy for Cystinosis: From Bench-to-Bedside

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3273
Author(s):  
Stephanie Cherqui
Keyword(s):  
Clinical Trial ◽  
Gene Therapy ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
The Body ◽  
Hematopoietic Stem ◽  
Tissue Integration ◽  
Toxicological Studies ◽  
The Impact

Cystinosis is an autosomal recessive metabolic disease that belongs to the family of lysosomal storage disorders. The gene involved is the CTNS gene that encodes cystinosin, a seven-transmembrane domain lysosomal protein, which is a proton-driven cystine transporter. Cystinosis is characterized by the lysosomal accumulation of cystine, a dimer of cysteine, in all the cells of the body leading to multi-organ failure, including the failure of the kidney, eye, thyroid, muscle, and pancreas, and eventually causing premature death in early adulthood. The current treatment is the drug cysteamine, which is onerous and expensive, and only delays the progression of the disease. Employing the mouse model of cystinosis, using Ctns−/− mice, we first showed that the transplantation of syngeneic wild-type murine hematopoietic stem and progenitor cells (HSPCs) led to abundant tissue integration of bone marrow-derived cells, a significant decrease in tissue cystine accumulation, and long-term kidney, eye and thyroid preservation. To translate this result to a potential human therapeutic treatment, given the risks of mortality and morbidity associated with allogeneic HSPC transplantation, we developed an autologous transplantation approach of HSPCs modified ex vivo using a self-inactivated lentiviral vector to introduce a functional version of the CTNS cDNA, pCCL-CTNS, and showed its efficacy in Ctns−/− mice. Based on these promising results, we held a pre-IND meeting with the Food and Drug Administration (FDA) to carry out the FDA agreed-upon pharmacological and toxicological studies for our therapeutic candidate, manufacturing development, production of the GMP lentiviral vector, design Phase 1/2 of the clinical trial, and filing of an IND application. Our IND was cleared by the FDA on 19 December 2018, to proceed to the clinical trial using CD34+ HSPCs from the G-CSF/plerixafor-mobilized peripheral blood stem cells of patients with cystinosis, modified by ex vivo transduction using the pCCL-CTNS vector (investigational product name: CTNS-RD-04). The clinical trial evaluated the safety and efficacy of CTNS-RD-04 and takes place at the University of California, San Diego (UCSD) and will include up to six patients affected with cystinosis. Following leukapheresis and cell manufacturing, the subjects undergo myeloablation before HSPC infusion. Patients also undergo comprehensive assessments before and after treatment to evaluate the impact of CTNS-RD-04 on the clinical outcomes and cystine and cystine crystal levels in the blood and tissues for 2 years. If successful, this treatment could be a one-time therapy that may eliminate or reduce renal deterioration as well as the long-term complications associated with cystinosis. In this review, we will describe the long path from bench-to-bedside for autologous HSPC gene therapy used to treat cystinosis.

Hematopoietic Stem Cell Gene Therapy Trial with Lentiviral Vector in X-Linked Adrenoleukodystrophy

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 821-821 ◽  
Author(s):  
Marina Cavazzana-Calvo ◽  
Nathalie Cartier ◽  
Salima Hacein-Bey Abina ◽  
Gabor Veres ◽  
Manfred Schmidt ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Cd34 Cells ◽  
Hsc Transplantation

Abstract We report preliminary results in 3 children with cerebral X-linked adrenoleukodystrophy (ALD) who received in September 2006, January 2007 and June 2008 lentiviral vector transduced autologous hematopoietic stem cell (HSC). We have previously demonstrated that cerebral demyelination associated with cerebral ALD can be stopped or reversed within 12–18 months by allogeneic HSC transplantation. The long term beneficial effects of HCT transplantation in ALD are due to the progressive turn-over of brain macrophages (microglia) derived from bone-marrow cells. For the current HSC gene therapy procedure, we used mobilized peripheral blood CD34+ cells that were transduced ex vivo for 18 hours with a non-replicative HIV1-derived lentiviral vector (CG1711 hALD) at MOI25 and expressing the ALD cDNA under the control of the MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substituted) promoter, and in the presence of 4 human recombinant cytokines (Il- 3, Stem Cell Factor [SCF], Flt3-ligand and Megakaryocyte Growth and Differentiation Factor [MGDF]) and CH-296 retronectine. Transduced cells were frozen to perform the required (RCL) safety tests. After thawing and prior to reinjection, 50%, 30% and 40% of transduced CD34+ cells expressed the ALD protein with a mean of 0.7, 0.6 and 0.65 copies of integrated provirus per cell. Transduced CD34+ cells were infused to ALD patients after a conditioning regimen including full doses of cyclophosphamide and busulfan. Hematopoietic recovery occured at day 13–15 post-transplant and the procedure was uneventful. In patient P1 and P2, the percentage of lymphocytes and monocytes expressing the ALD protein declined from day 60 to 6 months after gene therapy (GT) and remained stable up to 16 months post-GT. In P1, 9 to 13% of CD14+, CD3+, CD19+ and CD15+ cells expressed ALD protein 16 months post-transplant. In P2 and at the same time-point after transplant, 10 to 18% of CD14+, CD3+, CD19+ and CD15+ cells expressed ALD protein. ALD protein was expressed in 18–20% of bone marrow CD34+ cells from patients P1 and P2, 12 months post-transplant. In patient P3, 20 to 23% of CD3+, CD14+ and CD15+ cells expressed ALD protein 2 months after transplant. Tests assessing vector-derived RCL and vector mobilization were negative up to the last followups in the 3 patients. Integration of the vector was polyclonal and studies of integration sites arein progress. At 16 months post-transplant, HSC gene therapy resulted in neurological effects comparable with allogeneic HSC transplantation in patient P1 and P2. These results support that: ex-vivo HSC gene therapy using HIV1-derived lentiviral vector is not associated with the emergence of RCL and vector mobilization; a high percentage of hematopoietic progenitors were transduced expressing ALD protein in long term; no early evidence of selective advantage of the transduced ALD cells nor clonal expansion were observed. (This clinical trial is sponsored by Institut National de la Santé et de la Recherche Médicale and was conducted in part under a R&D collaboration with Cell Genesys, Inc., South San Francisco, CA)

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