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Background: Cerebral adrenoleukodystrophy is a severe form of X-linked adrenoleukodystrophy characterized by white-matter disease, loss of neurologic function, and early death. Elivaldogene autotemcel (eli-cel) gene therapy, which consists of autologous CD34+ cells transduced with Lenti-D lentiviral vector containing ABCD1 complementary DNA, is being tested in persons with cerebral adrenoleukodystrophy., Methods: In a phase 2-3 study, we evaluated the efficacy and safety of eli-cel therapy in boys with early-stage cerebral adrenoleukodystrophy and evidence of active inflammation on magnetic resonance imaging (MRI). The primary efficacy end point was survival without any of six major functional disabilities at month 24. The secondary end points included overall survival at month 24 and the change from baseline to month 24 in the total neurologic function score., Results: A total of 32 patients received eli-cel; 29 patients (91%) completed the 24-month study and are being monitored in the long-term follow-up study. At month 24, none of these 29 patients had major functional disabilities; overall survival was 94%. At the most recent assessment (median follow-up, 6 years), the neurologic function score was stable as compared with the baseline score in 30 of 32 patients (94%); 26 patients (81%) had no major functional disabilities. Four patients had adverse events that were directly related to eli-cel. Myelodysplastic syndrome (MDS) with excess blasts developed in 1 patient at month 92; the patient underwent allogeneic hematopoietic stem-cell transplantation and did not have MDS at the most recent follow-up., Conclusions: At a median follow-up of 6 years after lentiviral gene therapy, most patients with early cerebral adrenoleukodystrophy and MRI abnormalities had no major functional disabilities. However, insertional oncogenesis is an ongoing risk associated with the integration of viral vectors. (Funded by Bluebird Bio; ALD-102 and LTF-304 ClinicalTrials.gov numbers NCT01896102 and NCT02698579, respectively.)., (Copyright © 2024 Massachusetts Medical Society.)

Background: In X-linked adrenoleukodystrophy, mutations in ABCD1 lead to loss of function of the ALD protein. Cerebral adrenoleukodystrophy is characterized by demyelination and neurodegeneration. Disease progression, which leads to loss of neurologic function and death, can be halted only with allogeneic hematopoietic stem-cell transplantation., Methods: We enrolled boys with cerebral adrenoleukodystrophy in a single-group, open-label, phase 2-3 safety and efficacy study. Patients were required to have early-stage disease and gadolinium enhancement on magnetic resonance imaging (MRI) at screening. The investigational therapy involved infusion of autologous CD34+ cells transduced with the elivaldogene tavalentivec (Lenti-D) lentiviral vector. In this interim analysis, patients were assessed for the occurrence of graft-versus-host disease, death, and major functional disabilities, as well as changes in neurologic function and in the extent of lesions on MRI. The primary end point was being alive and having no major functional disability at 24 months after infusion., Results: A total of 17 boys received Lenti-D gene therapy. At the time of the interim analysis, the median follow-up was 29.4 months (range, 21.6 to 42.0). All the patients had gene-marked cells after engraftment, with no evidence of preferential integration near known oncogenes or clonal outgrowth. Measurable ALD protein was observed in all the patients. No treatment-related death or graft-versus-host disease had been reported; 15 of the 17 patients (88%) were alive and free of major functional disability, with minimal clinical symptoms. One patient, who had had rapid neurologic deterioration, had died from disease progression. Another patient, who had had evidence of disease progression on MRI, had withdrawn from the study to undergo allogeneic stem-cell transplantation and later died from transplantation-related complications., Conclusions: Early results of this study suggest that Lenti-D gene therapy may be a safe and effective alternative to allogeneic stem-cell transplantation in boys with early-stage cerebral adrenoleukodystrophy. Additional follow-up is needed to fully assess the duration of response and long-term safety. (Funded by Bluebird Bio and others; STARBEAM ClinicalTrials.gov number, NCT01896102 ; ClinicalTrialsRegister.eu number, 2011-001953-10 .).

Adeno-associated virus (AAV) gene therapy constitutes a powerful tool for the treatment of neurodegenerative diseases. While AAVs are generally administered systemically to newborns in preclinical studies of neurological disorders, in adults the maturity of the blood-brain barrier (BBB) must be considered when selecting the route of administration. Delivery of AAVs into the cerebrospinal fluid (CSF) represents an attractive approach to target the central nervous system (CNS) and bypass the BBB. In this study, we investigated the efficacy of intra-CSF delivery of a single-stranded (ss) AAV9-CAG-GFP vector in adult mice via intracisternal (iCist) or intralumbar (it-Lumb) administration. It-Lumb ssAAV9 delivery resulted in greater diffusion throughout the entire spinal cord and green fluorescent protein (GFP) expression mainly in the cerebellum, cortex and olfactory bulb. By contrast, iCist delivery led to strong GFP expression throughout the entire brain. Comparison of the transduction efficiency of ssAAV9-CAG-GFP versus ssAAV9-SYN1-GFP following it-Lumb administration revealed widespread and specific GFP expression in neurons and motoneurons of the spinal cord and brain when the neuron-specific synapsin 1 (SYN1) promoter was used. Our findings demonstrate that it-Lumb ssAAV9 delivery is a safe and highly efficient means of targeting the CNS in adult mice.

Leukodystrophies (LDs) are rare, often devastating genetic disorders with neurologic symptoms. There are currently no disease-specific therapeutic approaches for these diseases. In this review we use metachromatic leukodystrophy as an example to outline in the brief the therapeutic approaches to MLD that have been tested in animal models and in clinical trials, such as enzyme-replacement therapy, bone marrow/umbilical cord blood transplants, ex vivo transplantation of genetically modified hematopoietic stem cells, and gene therapy. These studies suggest that to be successful the ideal therapy for MLD must provide persistent and high level expression of the deficient gene, arylsulfatase A in the CNS. Gene therapy using adeno-associated viruses is therefore the ideal choice for clinical development as it provides the best balance of potential for efficacy with reduced safety risk. Here we have summarized the published preclinical data from our group and from others that support the use of a gene therapy with AAVrh.10 serotype for clinical development as a treatment for MLD, and as an example of the potential of gene therapy for LDs especially for Krabbe disease, which is the focus of this special issue. © 2016 Wiley Periodicals, Inc., Competing Interests: None for all authors, (© 2016 Wiley Periodicals, Inc.)

Gene therapy for rare inherited neurologic diseases has entered the clinics. One strategy relies upon the replacement of brain microglia using hematopoietic stem cell gene therapy with lentiviral vectors. Therapeutic success using this approach has been obtained in X-linked adrenoleukodystrophy and metachromatic leukodystrophy. The other strategy relies upon the intracerebral administration of adeno-associated virus vectors encoding lysosomal enzymes. Therapeutic trials are ongoing in Batten's disease, metachromatic leukodystrophy, and Sanfilippo type A and B diseases., (© 2016 S. Karger AG, Basel.)

Metachromatic leukodystrophy (MLD), a fatal disorder caused by deficiency of the lysosomal enzyme arylsulfatase A (ARSA), is associated with an accumulation of sulfatides, causing widespread demyelination in both central and peripheral nervous systems. On the basis of prior studies demonstrating that adeno-associated virus AAVrh.10 can mediate widespread distribution in the CNS of a secreted lysosomal transgene, and as a prelude to human trials, we comparatively assessed the optimal CNS delivery route of an AAVrh.10 vector encoding human ARSA in a large animal model for broadest distribution of ARSA enzyme. Five routes were tested (each total dose, 1.5 × 10(12) genome copies of AAVrh.10hARSA-FLAG): (1) delivery to white matter centrum ovale; (2) deep gray matter delivery (putamen, thalamus, and caudate) plus overlying white matter; (3) convection-enhanced delivery to same deep gray matter locations; (4) lateral cerebral ventricle; and (5) intraarterial delivery with hyperosmotic mannitol to the middle cerebral artery. After 13 weeks, the distribution of ARSA activity subsequent to each of the three direct intraparenchymal administration routes was significantly higher than in phosphate-buffered saline-administered controls, but administration by the intraventricular and intraarterial routes failed to demonstrate measurable levels above controls. Immunohistochemical staining in the cortex, white matter, deep gray matter of the striatum, thalamus, choroid plexus, and spinal cord dorsal root ganglions confirmed these results. Of the five routes studied, administration to the white matter generated the broadest distribution of ARSA, with 80% of the brain displaying more than a therapeutic (10%) increase in ARSA activity above PBS controls. No significant toxicity was observed with any delivery route as measured by safety parameters, although some inflammatory changes were seen by histopathology. We conclude that AAVrh.10-mediated delivery of ARSA via CNS administration into the white matter is likely to be safe and yields the widest distribution of ARSA, making it the most suitable route of vector delivery.

Though heavily hyped in the early 1990s as the answer to many genetic diseases, gene therapy has largely failed to live up to its promises up to now. The scientific challenges of conveying replacement genes into the brain have prevented it from having an impact on more than a handful of conditions. In addition, gene therapy has been beset by safety problems. During the last 5 years, new vectors and techniques have started to resolve not only safety issues that once held gene therapy back but also markedly to improve efficient gene delivery within the brain and even the spinal cord. Brain gene therapy will not be suitable for every neurological disease, but considerable progress made in the field means we can now envisage that CNS gene therapy will be able to treat many neurological conditions that are not obviously treatable in any other way., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Clonality analysis of viral vector-transduced cell populations represents a convincing approach to dissect the physiology of tissue and organ regeneration, to monitor the fate of individual gene-corrected cells in vivo, and to assess vector biosafety. With the decoding of mammalian genomes and the introduction of next-generation sequencing technologies, the demand for automated bioinformatic analysis tools that can rapidly process and annotate vector integration sites is rising. Here, we provide a publicly accessible, graphical user interface-guided automated bioinformatic high-throughput integration site analysis pipeline. Its performance and key features are illustrated on pyrosequenced linear amplification-mediated PCR products derived from one patient previously enrolled in the first lentiviral vector clinical gene therapy study. Analysis includes trimming of vector genome junctions, alignment of genomic sequence fragments to the host genome for the identification of integration sites, and the annotation of nearby genomic elements. Most importantly, clinically relevant features comprise the determination of identical integration sites with respect to different time points or cell lineages, as well as the retrieval of the most prominent cell clones and common integration sites. The resulting output is summarized in tables within a convenient spreadsheet and can be further processed by researchers without profound bioinformatic knowledge.

X-linked adrenoleukodystrophy (X-ALD) is a severe genetic demyelinating disease caused by a deficiency in ALD protein, an adenosine triphosphate-binding cassette transporter encoded by the ABCD1 gene. When performed at an early stage of the disease, allogeneic hematopoietic stem cell transplantation (HCT) can arrest the progression of cerebral demyelinating lesions. To overcome the limitations of allogeneic HCT, hematopoietic stem cell (HSC) gene therapy strategy aiming to perform autologous transplantation of lentivirally corrected cells was developed. We demonstrated the preclinical feasibility of HSC gene therapy for ALD based on the correction of CD34+ cells from X-ALD patients using an HIV1-derived lentiviral vector. These results prompted us to initiate an HSC gene therapy trial in two X-ALD patients who had developed progressive cerebral demyelination, were candidates for allogeneic HCT, but had no HLA-matched donors or cord blood. Autologous CD34+ cells were purified from the peripheral blood after G-CSF stimulation, genetically corrected ex vivo with a lentiviral vector encoding wild-type ABCD1 cDNA, and then reinfused into the patients after they had received full myeloablative conditioning. Over 3 years of follow-up, the hematopoiesis remained polyclonal in the two patients treated with 7-14% of granulocytes, monocytes, and T and B lymphocytes expressing the lentivirally encoded ALD protein. There was no evidence of clonal dominance or skewing based on the retrieval of lentiviral insertion repertoire in different hematopoietic lineages by deep sequencing. Cerebral demyelination was arrested 14 and 16months, respectively, in the two treated patients, without further progression up to the last follow-up, a clinical outcome that is comparable to that observed after allogeneic HCT. Longer follow-up of these two treated patients and HSC gene therapy performed in additional ALD patients are however needed to evaluate the safety and efficacy of lentiviral HSC gene therapy in cerebral forms of X-ALD., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A recent clinical trial for adrenoleukodystrophy (ALD) showed the efficacy and safety of lentiviral vector (LV) gene transfer in hematopoietic stem progenitor cells. However, several common insertion sites (CIS) were found in patients' cells, suggesting that LV integrations conferred a selective advantage. We performed high-throughput LV integration site analysis on human hematopoietic stem progenitor cells engrafted in immunodeficient mice and found the same CISs reported in patients with ALD. Strikingly, most CISs in our experimental model and in patients with ALD cluster in megabase-wide chromosomal regions of high LV integration density. Conversely, cancer-triggering integrations at CISs found in tumor cells from γ-retroviral vector-based clinical trials and oncogene-tagging screenings in mice always target a single gene and are contained in narrow genomic intervals. These findings imply that LV CISs are produced by an integration bias toward specific genomic regions rather than by oncogenic selection.

Leukodystrophies (LDs) refer to a group on inherited diseases in which molecular abnormalities of glial cells are responsible for exclusive or predominant defects in myelin formation and/or maintenance within the central and, sometimes, the peripheral nervous system. For three of them [X-linked adrenoleukodystrophy (X-ALD), metachromatic (MLD) and globoid cell LDs], a gene therapy strategy aiming at transferring the disease gene into autologous hematopoietic stem cells (HSCs) using lentiviral vectors has been developed and has already entered into the clinics for X-ALD and MLD. Long-term follow-up has shown that HSCs gene therapy can arrest the devastating progression of X-ALD. Brain gene therapy relying upon intracerebral injections of adeno-associated vectors is also envisaged for MLD. The development of new gene therapy viral vectors allowing targeting of the disease gene into oligodendrocytes or astrocytes should soon benefit other forms of LDs.

The β-haemoglobinopathies are the most prevalent inherited disorders worldwide. Gene therapy of β-thalassaemia is particularly challenging given the requirement for massive haemoglobin production in a lineage-specific manner and the lack of selective advantage for corrected haematopoietic stem cells. Compound β(E)/β(0)-thalassaemia is the most common form of severe thalassaemia in southeast Asian countries and their diasporas. The β(E)-globin allele bears a point mutation that causes alternative splicing. The abnormally spliced form is non-coding, whereas the correctly spliced messenger RNA expresses a mutated β(E)-globin with partial instability. When this is compounded with a non-functional β(0) allele, a profound decrease in β-globin synthesis results, and approximately half of β(E)/β(0)-thalassaemia patients are transfusion-dependent. The only available curative therapy is allogeneic haematopoietic stem cell transplantation, although most patients do not have a human-leukocyte-antigen-matched, geno-identical donor, and those who do still risk rejection or graft-versus-host disease. Here we show that, 33 months after lentiviral β-globin gene transfer, an adult patient with severe β(E)/β(0)-thalassaemia dependent on monthly transfusions since early childhood has become transfusion independent for the past 21 months. Blood haemoglobin is maintained between 9 and 10 g dl(-1), of which one-third contains vector-encoded β-globin. Most of the therapeutic benefit results from a dominant, myeloid-biased cell clone, in which the integrated vector causes transcriptional activation of HMGA2 in erythroid cells with further increased expression of a truncated HMGA2 mRNA insensitive to degradation by let-7 microRNAs. The clonal dominance that accompanies therapeutic efficacy may be coincidental and stochastic or result from a hitherto benign cell expansion caused by dysregulation of the HMGA2 gene in stem/progenitor cells.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only therapeutic approach that can arrest cerebral demyelination of X-linked adrenoleukodystrophy (ALD) in boys and results in long-term in a good quality of life, provided the procedure is performed at an early stage of disease. Similar benefits of allogeneic HSCT have been demonstrated in adults with cerebral ALD. However, it is not yet known whether allogeneic HSCT can prevent or rescue adrenomyeloneuropathy. Allogeneic HSCT remains associated with significant morbidity and mortality risks, particularly in adults, and not all ALD patients have donors despite the availability of cord blood. The absence of biological markers that can predict the evolutivity of cerebral disease is a major limitation to propose in due time allogeneic HSCT to ALD patients. Recently, HSC gene therapy using lentiviral vector was shown to have comparable efficacy than allogeneic HSCT in two boys with cerebral ALD who had no Human-leukocyte-antigen (HLA)-matched donor. If these results are confirmed in an extended series of patients, HSC gene therapy may become the first therapeutic option for all ALD male patients who develop cerebral demyelination.

X-linked adrenoleukodystrophy (ALD) is a severe demyelinating disease of the brain caused by a deficiency in ALD protein, an adenosine triphosphate--binding cassette (ABC) transporter encoded by the ABCD1 gene. ALD progression can be halted by allogeneic hematopoietic cell transplantation (HCT). We have developed a gene therapy strategy based on ABCD1 gene transfer to autologous hematopoietic stem cells (CD34+) by a lentiviral vector derived from HIV-1. We initiated a clinical trial involving three ALD patients for whom no matched donor was available. Autologous CD34+ cells were transduced ex vivo with an HIV derived vector the wild-type ABCD1 gene then re-infused after myeloablative treatment. Polyclonal reconstitution was detected up to 24 to 30 months, with between 9% and 14% of granulocytes, monocytes, and T and B lymphocytes expressing the ALD protein, strongly suggesting that the patients' hematopoietic stem cells have been successfully transduced. Cerebral demyelination halted after 14 to 16 months in two first treated patients an outcome similar to that achieved by allogeneic HCT These results suggest that lentiviral vectors are suitable for transferring therapeutic genes to hematopoietic stem cells, and provide the first example of successful gene therapy for a severe neurodegenerative disease.

The development of Alzheimer's disease (AD) is closely connected with cholesterol metabolism. Cholesterol increases the production and deposition of amyloid-beta (Abeta) peptides that result in the formation of amyloid plaques, a hallmark of the pathology. In the brain, cholesterol is synthesized in situ but cannot be degraded nor cross the blood-brain barrier. The major exportable form of brain cholesterol is 24S-hydroxycholesterol, an oxysterol generated by the neuronal cholesterol 24-hydroxylase encoded by the CYP46A1 gene. We report that the injection of adeno-associated vector (AAV) encoding CYP46A1 in the cortex and hippocampus of APP23 mice before the onset of amyloid deposits markedly reduces Abeta peptides, amyloid deposits and trimeric oligomers at 12 months of age. The Morris water maze (MWM) procedure also demonstrated improvement of spatial memory at 6 months, before the onset of amyloid deposits. AAV5-wtCYP46A1 vector injection in the cortex and hippocampus of amyloid precursor protein/presenilin 1 (APP/PS) mice after the onset of amyloid deposits also reduced markedly the number of amyloid plaques in the hippocampus, and to a less extent in the cortex, 3 months after the injection. Our data demonstrate that neuronal overexpression of CYP46A1 before or after the onset of amyloid plaques significantly reduces Abeta pathology in mouse models of AD.

X-linked adrenoleukodystrophy (ALD) is a severe brain demyelinating disease in boys that is caused by a deficiency in ALD protein, an adenosine triphosphate-binding cassette transporter encoded by the ABCD1 gene. ALD progression can be halted by allogeneic hematopoietic cell transplantation (HCT). We initiated a gene therapy trial in two ALD patients for whom there were no matched donors. Autologous CD34+ cells were removed from the patients, genetically corrected ex vivo with a lentiviral vector encoding wild-type ABCD1, and then re-infused into the patients after they had received myeloablative treatment. Over a span of 24 to 30 months of follow-up, we detected polyclonal reconstitution, with 9 to 14% of granulocytes, monocytes, and T and B lymphocytes expressing the ALD protein. These results strongly suggest that hematopoietic stem cells were transduced in the patients. Beginning 14 to 16 months after infusion of the genetically corrected cells, progressive cerebral demyelination in the two patients stopped, a clinical outcome comparable to that achieved by allogeneic HCT. Thus, lentiviral-mediated gene therapy of hematopoietic stem cells can provide clinical benefits in ALD.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by deficiency of the lysosomal enzyme arylsulfatase A. Deficiency of this enzyme results in intralysosomal storage of sphingolipid cerebroside 3-sulfates (sulfatides), which are abundant in myelin and neurons. A pathological hallmark of MLD is demyelination and neurodegeneration, causing various and ultimately lethal neurological symptoms. This review discusses the potential therapeutic application of hematopoietic stem cell gene therapy and intracerebral gene transfer (brain gene therapy) in patients with MLD.

Hurler syndrome, metachromatic leukodystrophy, globoid-cell leukodystrophy (Krabbe's disease) and X-linked adrenoleukodystrophy are inherited diseases of the CNS that can be cured or arrested by allogeneic hematopoietic stem-cell transplantation (HSCT). Despite significant progress in medical procedures and the availability of banked umbilical cord blood, HSCT is still associated with significant risks of graft failure or GVHD that can lead to death. Transplantation of autologous hematopoietic stem cells genetically modified to express the missing protein may circumvent the majority of the problems associated with allogeneic HSCT. Promising in concept, these strategies are now at a stage to be tested in phase I/II clinical trials to assess safety and potential efficacy.

Metachromatic leukodystrophy (MLD) is a demyelinating storage disease caused by deficiency of the lysosomal enzyme arylsulfatase A (ARSA). Lack of ARSA activity leads to the accumulation of galactosylceramide-3-O-sulfate (sulfatide) in the central and peripheral nervous systems. Based on the age at onset, the disease is usually classified into three forms: the late-infantile form, which manifests in the second year of life; the juvenile variants (onset between 4 and 12 years), which are subdivided into early-juvenile (EJ, onset before 6 years) and late-juvenile (LJ, onset after 6 years); and the adult form (onset after 12 years of age). Currently, there is no efficient therapy for the late-infantile form of MLD (50% of the patients), death occurring within a few years after onset of neurological symptoms. Allogeneic haematopoietic cell transplantation (HCT), when performed at a very early stage of the disease, may improve selected patients with juvenile or adult forms of MLD. As with other lysosomal storage diseases, the physiopathology of MLD is poorly understood. Demyelination is the main pathological finding, but substantial storage of sulfatides in neurons also occurs, and may contribute to the clinical phenotype. The physiopathological process leading to neuronal and glial cell degeneration and apoptosis involves accumulation of undegraded sulfatides but also secondary abnormalities (storage/mislocalization of unrelated lipids, inflammatory processes). This review summarizes the recent advances in the understanding of the physiopathology of MLD and the new therapeutic perspectives currently under preclinical investigation, including enzyme replacement therapy, gene therapy and cell therapy.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by genetic deficiency of arylsulfatase A (ARSA) enzyme. Failure in catalyzing the degradation of its major substrate, sulfatide (Sulf), in oligodendrocytes and Schwann cells leads to severe demyelination in the peripheral (PNS) and central nervous system (CNS), and early death of MLD patients. The ARSA knockout mice develop a disease that resembles MLD but is milder, without significant demyelination in the PNS and CNS. We showed that adeno-associated virus serotype 5-mediated gene transfer in the brain of ARSA knockout mice reverses Sulf storage and prevents neuropathological abnormalities and neuromotor disabilities when vector injections are performed at a pre-symptomatic stage of disease. Direct injection of viral particles into the brain of ARSA knockout mice at a symptomatic stage results in sustained expression of ARSA, prevention of Sulf storage and neuropathological abnormalities. Despite these significant corrections, the treated mice continue to develop neuromotor disability. We show that more subtle biochemical abnormalities involving gangliosides and galactocerebroside are in fact not corrected.

Metachromatic leukodystrophy (MLD) is a neurodegenerative lysosomal disease caused by a defect of the enzyme arylsulfatase A (ARSA) that disrupts the degradation of sulfatides (Sulf) in neurons and glial cells. Therapy for MLD requires active production of ARSA in the brain to prevent demyelination and neuronal damage, and efficient delivery of ARSA to act faster than disease progression, particularly in the rapidly progressive late infantile form. We used an adeno-associated virus serotype 5 (AAV5) vector to express the human ARSA gene in the brain of MLD mouse model. We achieved rapid, extensive and long-lasting expression of the recombinant ARSA in the brain, cerebellum and brainstem from at least 3 to 15 months post-injection. Analysis of the vector genome and ARSA distribution gave evidence for in vivo cross-correction of many untransduced neurons and astrocytes. ARSA delivery rapidly reversed Sulf storage and prevented neuropathological abnormalities and neuromotor impairment. We believe that AAV5-mediated brain delivery of ARSA is a potentially efficacious therapeutic strategy for MLD patients, especially for those with rapidly progressive form of the disease.

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.