Efficient CNS targeting in adult mice by intrathecal infusion of single-stranded AAV9-GFP for gene therapy of neurological disorders.

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.

Gene therapy in metachromatic leukodystrophy.

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.

Adrenoleukodystrophy and other peroxisomal diseases.

The gene predisposing for X-linked adrenoleukodystrophy (ALD), the most common peroxisomal disorder, has been identified recently by positional cloning. The ALD protein is a 75 kDa peroxisomal membrane protein belonging to the family of ATP-binding cassette transporter proteins. With the combination of genetic complementation and candidate gene approaches, two genes responsible for Zellweger syndrome, a group of genetically heterogeneous disorders affecting peroxisome biogenesis, have also been identified.

Identification of mutations in the putative ATP-binding domain of the adrenoleukodystrophy gene.

The recently identified adrenoleukodystrophy (ALD) gene is predicted to encode a peroxisomal protein of 745 amino acids that includes one domain for ATP-binding, termed nucleotide-binding fold (NBF). To determine whether mutations occur in the putative NBF of ALD protein, we analyzed by denaturing gradient gel electrophoresis (DGGE) exon 6 and 8 that encode most part of this domain in 50 ALD patients. Four amino acid substitutions, three frameshift mutations leading to premature termination signal, and a splicing mutation were identified. These amino acid substitutions occurred at residues highly conserved in other ATP-binding cassette (ABC) proteins. In addition, a nonsense mutation was detected in exon 4.

Enzyme, cell and gene-based therapies for metachromatic leukodystrophy.

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.

[Subtle brain abnormalities in adrenomyeloneuropathy]. / Anomalies cérébrales subtiles de l'Adrénomyéloneuropathie.

PURPOSE: To describe subtle brain abnormalities detected on MRI in adult patients with adrenomyeloneuropathy (AMN). Materials and methods. Retrospective evaluation of data acquired prospectively as part of a clinical trial (Riluzole) in 66 adult patients with AMN without obvious brain lesion on MR. All patients underwent brain MR including T1W, T2W, FLAIR and spectroscopy. After a review had been validated by three different reviewers, review of MR images was performed by consensus using a semi-quantitative scale. RESULTS: Preliminary analysis of MR images confirmed the presence of signal abnormalities involving the corticospinal tracts in 36 patients (54.6%). Additional subtle abnormalities were also detected: white matter palor, mainly parieto-occipital in location, with patchy hyperintensity in 36 patients (54.6%), hyperintense pontocerebellar fibers on T2W and FLAIR in 25 patients (41.7%). The presence of elevated Cho/Cr and mI/Cr ratios, described in the literature, were confirmed. CONCLUSION: This retrospective study allows the description of an AMN pattern on MRI in patients without white matter or callosal abnormalities.

[X-linked adrenoleukodystrophy]. / Adrénoleucodystrophie liée à l'X.

X-linked adrenoleukodystrophy (ALD) is a severe neurodegenerative disorder. ALD is characterized by progressive demyelination within the central and peripheral nervous system, adrenal insufficiency (Addison's disease) and accumulation of very-long-chain fatty acids (VLCFA) in plasma, fibroblasts and tissues. The overall incidence of ALD is 1:17,000 including hemizygotes and heterozygotes who are frequently symptomatic. There are two main ALD phenotypes: 1) a cerebral demyelinating form which affects boys between 5-12 years, but also 35% of adult males; 2) a form that mainly involves the spinal cord (adrenomyeloneuropathy, AMN) in adult males between 20-50 years and 50% of heterozygous women after the age of 40 years. AMN presents with progressive spastic paraparesis. Addison's disease may be the first symptom of ALD in boys and adult males. These patients are at risk to develop cerebral ALD or AMN for life. ALD results from mutations in the ABCD1 gene without correlation between genotype and phenotype. The diagnosis of ALD relies upon the measurement of plasma VLCFA levels that allows the identification of 100% affected males and of 80-95% heterozygous women. Because of these false-negative, it is therefore mandatory to search for a mutation in the ABCD1 gene in all women at risk to be heterozygous for ALD. The ABCD1 gene encodes a peroxisomal transmembrane protein (ALD protein) with the structure of an half ATP-binding cassette transporter. It is possible that ALD protein imports VLCFA or VLCFA-CoA into peroxisomes in which they are degraded by a peroxisomal beta-oxidation system. Elongation of VLCFAs is enhanced in fibroblasts from ALD patients and likely contributes to the load of VLCFA in tissues. The underlying mechanisms that lead to cerebral demyelination, axonal degeneration in spinal cord and adrenal insufficiency are unknown. The "toxic" role of VLCFA accumulation remains to be demonstrated. The mechanisms that lead to the inflammatory reaction in cerebral ALD might involve abnormal acylation of gangliosides and phospholipids by VLCFA that would result in immune reaction of brain macrophages and astrocytes bearing CD1 molecules that recognize lipid antigens. De novo mutation of ABCD1 occurs in less than 8% of ALD patients. The genetic counseling aims to identify: 1) women who are at risk to be heterozygous; 2) neurologically asymptomatic boys. It is only at this stage that allogeneic bone marrow transplantation has clinical benefit; 3) ALD patients who have Addison's disease that can lead to sudden death. Prenatal diagnosis (chorionic villus samples, cultured amniotic fluid cells) relies upon DNA based mutation detection techniques, expression of ALD protein and measurement of VLCFA levels. Allogeneic bone marrow transplantation is the only treatment that provides a permanent cure when the procedure is performed at an early stage of cerebral demyelination, i.e when the patients are asymptomatic despite abnormal brain MRI. Treatment of Addison's disease is mandatory but does not modify the course of neurological symptoms. Dietary therapy failed to halt the neurologic progression in cerebral ALD and AMN. It might have a partial preventive effect in boys treated before 6 years of age.

The neurobiology of X-linked adrenoleukodystrophy, a demyelinating peroxisomal disorder.

Adrenoleukodystrophy (ALD) is caused by mutations in an ATP-binding-cassette transporter located in the peroxisomal membrane, which result in a fatal demyelinating disease in boys and a milder phenotype in men and some heterozygous women. There is no molecular signature to indicate a particular clinical course. The underlying molecular mechanisms of this disease have yet to be targeted clinically. Is the increase in very-long-chain fatty acids (VLCFA) the disease trigger? Why is there no phenotype in ALD null mice that show this increase? Do VLCFA destabilize human myelin, once formed, and lead to the inflammation seen in this genetic disease? Bone-marrow transplantation might save a child by providing normal brain macrophages and allowing myelin regeneration early in disease. The processes that underlie ALD challenge neuroscientists to elucidate peroxisomal transporter functions in the nervous system and to pursue the gene-transfer strategies leading to remyelination until a preventive therapy emerges.

Adrenoleukodystrophy presenting as addison's disease in children and adults.

X-linked adrenoleukodystrophy (ALD) is a peroxisomal genetic disorder that causes adrenal insufficiency, demyelination in the central nervous system, and increased levels of very long chain fatty acids in tissues and body fluids. Although most cases appear in childhood as a devastating degenerative disorder or in adulthood as a milder disorder affecting the spinal cord, many patients have adrenal insufficiency prior to the onset of their neurologic deterioration for many years. Addison's disease without neurologic involvement may also remain the only clinical manifestation of X-linked ALD. Because of the prognostic implications, the need for genetic counseling, and the potential benefit of therapeutic interventions, any boy or adult male with Addison's disease must be tested for X-linked ALD.

[Adult onset hereditary leukoencephalopathies]. / Leucoencéphalopathies génétiques de l'adulte.

In clinical practice, the term "genetic leukoencephalopathy" refers to a group of genetic diseases whose common point is to give an aspect of diffuse leukoencephalopathy on MRI. With progress in diagnostic techniques including radiology, biochemistry or genetics, a large number of hereditary diseases causing leukoencephalopathy have been identified. Although generally beginning in childhood, these diseases often have more insidious clinical forms which can begin in adulthood. These forms remain poorly known. Some are accessible to treatment so their diagnosis appears essential. The diagnostic steps must be guided by clinical examination (neurological, ophthalmological and systemic), electromyography and MRI. The purpose of this review is to propose a classification of the genetic leukoencephalopathies and to give a progress report applicable in neurological practice.

[X-linked adrenoleukodystrophy in a female proband: clinical presentation, biological diagnosis and family consequences]. / Adrénoleucodystrophie liée à l'X chez une proposante symptomatique: présentation clinique, diagnostic biologique et conséquences familiales.

INTRODUCTION: X-linked adrenoleukodystrophy (ALD) is the most frequent type of leukodystrophy (1/17 000 males). The phenotypic range in male patients varies from the severe cerebral presentations in children to the milder myeloneuropathy and to isolate adrenal insufficiency. More than a half of the carrier females display clinical symptoms over the age of 40 years. OBSERVATION: Diagnosis of ALD was raised in a 40 year-old female who presented with spastic paraparesis and sphincterian dysfunction, occurring after the delivery of her first child. There was no family history of ALD. Very long-chain fatty acids (VLFCA) were assayed in her one-year-old son in order to propose appropriate hormonal and neurological survey. His dosage was abnormal and an adrenal insufficiency was subsequently found. A brain MRI will be proposed biannually when he reaches to age of for years. The proband's mother had an increased level of VLCFA, showing that she was a carrier. Family screening was extended to the proband's sisters and maternal aunt who already had children, but also to her brother, who may express a mild form of the disease later on, and to her maternal uncles who might be asymptomatic carriers. A frameshift mutation was found in the ABCD1 gene and will allow accurate carrier identification and prenatal diagnosis in the family. CONCLUSION: ALD diagnosis should be evoked in a woman affected by myelopathy despite the lack of family history. Such a diagnosis has severe consequences since some of the related males may carry the mutation although they do not display any symptom at time of diagnosis, and because carrier females have a risk to both have a clinical expression of the disease and give birth to an affected boy.

Bone marrow-derived mesenchymal stem cells remain host-derived despite successful hematopoietic engraftment after allogeneic transplantation in patients with lysosomal and peroxisomal storage diseases.

Human bone marrow contains mesenchymal stem cells (MSCs) that can differentiate into various cells of mesenchymal origin. We developed an efficient method of isolating and culture expanding a homogenous population of MSCs from bone marrow and determined that MSCs express alpha-L-iduronidase, arylsulfatase-A and B, glucocerebrosidase, and adrenoleukodystrophy protein. These findings raised the possibility that MSCs may be useful in the treatment of storage disorders. To determine if donor derived MSCs are transferred to the recipients with lysosomal or peroxisomal storage diseases by allogeneic hematopoietic stem cell (HSC) transplantation, we investigated bone marrow derived MSCs of 13 patients 1-14 years after allogeneic transplantation. Highly purified MSCs were genotyped either by fluorescence in situ hybridization using probes for X and Y-chromosomes in gender mis-matched recipients or by radiolabeled PCR amplification of polymorphic simple sequence repeats. Phenotype was determined by the measurement of disease specific protein/enzyme activity in purified MSCs. We found that MSCs isolated from recipients of allogeneic HSC transplantation are not of donor genotype and have persistent phenotypic defects despite successful donor type hematopoietic engraftment. Whether culture expanded normal MSCs can be successfully transplanted into patients with storage diseases and provide therapeutic benefit needs to be determined.

Retroviral transfer and long-term expression of the adrenoleukodystrophy gene in human CD34+ cells.

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.

Mirror expression of adrenoleukodystrophy and adrenoleukodystrophy related genes in mouse tissues and human cell lines.

The adrenoleukodystrophy and adrenoleukodystrophy related proteins belong to a new family of half ATP-binding cassette transporters which are localized within the peroxisomal membrane and whose functions are still unknown. They could possibly homo- or heterodimerize resulting in transporters with similar or distinct functions. The expression of adrenoleukodystrophy and adrenoleukodystrophy related genes was studied at the mRNA and protein levels in adult mouse tissues and several human cell lines. We found that adrenoleukodystrophy and adrenoleukodystrophy related genes have strikingly different expression in most mouse tissues and human cell lines analyzed, indicating that adrenoleukodystrophy and adrenoleukodystrophy related proteins do not function as obligatory partners but might rather fulfill similar metabolic functions in different tissues.

X-linked adrenoleukodystrophy is a frequent cause of idiopathic Addison's disease in young adult male patients.

X-Linked adrenoleukodystrophy (ALD) is a genetic disease associated with demyelination of the central nervous system, adrenal insufficiency, and accumulation of very long chain fatty acids in tissue and body fluids. ALD is due to mutation of a gene located in Xq28 that encodes a peroxisomal transporter protein of unknown function. The most common phenotype of ALD is the cerebral form (45%) that develops in boys between 5-12 yr. Adrenomyeloneuropathy (AMN) involves the spinal cord and peripheral nerves in young adults (35%). Adrenal insufficiency (Addison's disease) is frequently associated with AMN or cerebral ALD and may remain the only clinical expression of ALD (8% of cases). The prevalence of ALD among adults with Addison's disease remains unknown. To evaluate this prevalence, we performed biochemical analysis of very long chain fatty acids in 14 male patients (age ranging from 12-45 yr at diagnosis) previously diagnosed as having primary idiopathic adrenocortical insufficiency. In 5 of 14 patients (35%), elevated plasma concentrations of very long chain fatty acids were detected. None of these patients had adrenocortical antibodies. By electrophysiological tests and magnetic resonance imaging it was determined that two patients had cerebral ALD, one had adrenomyeloneuropathy with cerebral involvement, and two had preclinical AMN. Our data support the hypothesis that ALD is a frequent cause of idiopathic Addison's disease in children and adults.

Etiological diagnosis of primary adrenal insufficiency using an original flowchart of immune and biochemical markers.

Approximately 70-80% of cases of primary adrenal insufficiency are classified as idiopathic. An effective protocol for the etiological diagnosis of primary adrenal insufficiency is needed to ensure correct patient management. With the aim of developing an algorithm for the etiological diagnosis of primary adrenal insufficiency, we studied 56 Italian patients with nonsurgical primary adrenal insufficiency and 24 French patients with X-linked adrenoleukodystrophy (ALD) for serum levels of adrenal cortex, steroid-21-hydroxylase (21OHAb), islet cell (ICA), glutamate decarboxylase (GAD65Ab), IA2/ICA512 (ICA512Ab), thyroid peroxidase (TPOAb) autoantibodies, and plasmatic concentrations of very long chain fatty acids (VLCFA). High levels of 21OH and adrenal cortex antibodies were found in 35/42 (83%) and 17/42 (40%) Italian patients with idiopathic adrenal insufficiency, respectively. Levels of adrenal autoantibodies correlated inversely with disease duration (P < 0.0001). Elevated VLCFA were found in 4/42 (10%) idiopathic patients. A total of 34/35 (97%) idiopathic patients with a disease duration of less than 20 yr was positive for either 21OHSAb or elevated levels of VLCFA. None of 14 patients with posttuberculosis adrenal insufficiency had elevated levels of either adrenal antibodies or VLCFA. ICA, GAD65Ab, ICA512Ab, and TPOAb were found in 6/56 (11%), 8/56 (14%), 4/56 (7%), and 23/56 (41%) patients, respectively. None of 24 French ALD patients with adrenal insufficiency was positive for organ-specific autoantibodies. The measuring of 21OH antibodies and plasma VLCFA levels enabled a correct diagnosis of autoimmune (89%) and ALD (8%) in 97% of patients with idiopathic primary adrenal insufficiency of less than 20 yr of duration. The results of our study have important therapeutic and prognostic implications.

Simvastatin does not normalize very long chain fatty acids in adrenoleukodystrophy mice.

X-linked adrenoleukodystrophy (ALD) is a genetic demyelinating disorder characterized by accumulation of very long chain fatty acid (VLCFA) in tissues. Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, normalizes VLCFA in fibroblasts and plasma from ALD patients. We dietary treated ALD mice with simvastatin, an analog of lovastatin with similar pharmacokinetics and effects on plasma VLCFA in ALD patients at 20 or 60 mg/kg/day for 6-12 weeks. No decrease of VLCFA content was observed in mouse tissues, including the brain. A significant increase of VLCFA was rather observed in the brain of ALD mice at 60 mg/kg/day.

Retroviral-mediated adrenoleukodystrophy-related gene transfer corrects very long chain fatty acid metabolism in adrenoleukodystrophy fibroblasts: implications for therapy.

X-linked adrenoleukodystrophy is a demyelinating disorder of the central nervous system with an impaired very long chain fatty acid metabolism. The adrenoleukodystrophy gene encodes a peroxisomal membrane protein that is part of a family of related ATP-binding transporters including the adrenoleukodystrophy-related protein. The adrenoleukodystrophy protein and adrenoleukodystrophy-related protein show 66% identity and have a mirror expression in most mouse tissues. We show that retroviral-mediated adrenoleukodystrophy-related gene transfer corrects very long chain fatty acid accumulation in adrenoleukodystrophy fibroblasts, irrespective of the presence or absence of adrenoleukodystrophy protein. Pharmacological approaches aiming at overexpressing the adrenoleukodystrophy-related gene in the central nervous system of adrenoleukodystrophy patients might thus offer new therapeutic leads.