Gene Therapy Of Metachromatic Leukodystrophy Research Articles

Engineered arylsulfatase A with increased activity, stability and brain delivery for therapy of metachromatic leukodystrophy

A deficiency of human arylsulfatase A (hASA) causes metachromatic leukodystrophy (MLD), a lysosomal storage disease characterized by sulfatide accumulation and central nervous system (CNS) demyelination. Efficacy of enzyme replacement therapy (ERT) is increased by genetic engineering of hASA to elevate its activity and transfer across the blood-brain barrier (BBB), respectively. To further improve the enzyme's bioavailability in the CNS, we mutated a cathepsin cleavage hot spot and obtained hASAs with substantially increased half-lives. We then combined the superstabilizing exchange E424A with the activity-promoting triple substitution M202V/T286L/R291N and the ApoEII-tag for BBB transfer in a trimodal modified neoenzyme called SuPerTurbo-ASA. Compared with wild-type hASA, half-life, activity, and M6P-independent uptake were increased more than 7-fold, about 3-fold, and more than 100-fold, respectively. ERT of an MLD-mouse model with immune tolerance to wild-type hASA did not induce antibody formation, indicating absence of novel epitopes. Compared with wild-type hASA, SuPerTurbo-ASA was 8- and 12-fold more efficient in diminishing sulfatide storage of brain and spinal cord. In both tissues, storage was reduced by ∼60%, roughly doubling clearance achieved with a 65-fold higher cumulative dose of wild-type hASA previously. Due to its enhanced therapeutic potential, SuPerTurbo-ASA might be a decisive advancement for ERT and gene therapy of MLD.

Gene therapy for metachromatic leukodystrophy: Lead candidate optimization

Gene therapy for metachromatic leukodystrophy: Lead candidate optimization

Lentiviral haematopoietic stem cell gene therapy for metachromatic leukodystrophy: Results in 5 patients treated under nominal compassionate use

Lentiviral haematopoietic stem cell gene therapy for metachromatic leukodystrophy: Results in 5 patients treated under nominal compassionate use

Lentiviral haematopoietic stem cell gene therapy for metachromatic leukodystrophy: Results in nine patients treated with a cryopreserved formulation of OTL-200

Lentiviral haematopoietic stem cell gene therapy for metachromatic leukodystrophy: Results in nine patients treated with a cryopreserved formulation of OTL-200

Toward Reference Intervals of ARSA Activity in the Cerebrospinal Fluid: Implication for the Clinical Practice of Metachromatic Leukodystrophy.

Cerebrospinal fluid (CSF) has emerged as a sensitive matrix for the screening of biomarkers for diagnosis and clinical follow-up of diseases with neurological manifestations, including some lysosomal storage disorders. In this study, we assessed the range of values of arylsulfatase A (ARSA) activity in the CSF of pediatric and adult donors, and in pediatric patients who underwent gene therapy for metachromatic leukodystrophy (MLD). A cohort of 56 CSF samples was included in the study: pediatric donors (n = 36), adult donors (n = 9), and MLD patients (n = 11) at different timepoints [pre-gene therapy (GT), post-GT + 1 Year, post-GT + 2 Years, post-GT + 3 Years]. We have used our fluorometric assay for the determination of ARSA activity. The total protein content in the samples was also evaluated. We discovered that ARSA activity was higher in pediatric donors (geometric mean: 1.039 nmol/mg/h; 95% range: 0.859-1.258 nmol/mg/h) compared to adults (geometric mean: 0.305 nmol/mg/h; 95% range: 0.214-0.435 nmol/mg/h). No ARSA activity was detected in the CSF of MLD patients pre-GT, whereas ARSA activity was stably expressed and almost restored to range of values of pediatric donors in MLD patients post-GT + 3 Years with a geometric mean of 0.822 nmol/mg/h (95% range: 0.580-1.165 nmol/mg/h). This study establishes range of values of ARSA activity in the CSF for MLD clinical practice. The observed ranges of ARSA activities in CSF exhibited an unpredicted age dependence and, in turn, revealed the need of using pediatric ARSA activity for evaluating the restoration of the enzyme activity during the therapy of MLD.

Gene therapy for blood cell diseases with autologous hematopoietic stem cells

Gene therapy is under development to treat genetic diseases of blood cells, including primary immune deficiencies (PID), lysosomal storage and related metabolic disorders, and hemoglobinopathies. Based on the experience with allogeneic hematopoietic stem cell (HSC) transplantation to successfully cure these disorders, gene therapy using autologous HSC holds the potential for safer treatments, avoiding immune complications. The key technical challenge is to achieve highly efficient gene correction in HSC, without impairing the long-term reconstituting capacity of the stem cells nor causing genotoxicity by the genetic manipulations. Initial trials using gamma-retroviral vectors showed clinical efficacy for ADA-deficient SCID, X-SCID, X-Chronic Granulomatous Disease and Wiskott-Aldrich Syndrome, although insertional oncogenesis led to leukoproliferative complications in the latter three conditions. Subsequent trials using retroviral or lentiviral vectors from which the long-terminal repeat enhancers are deleted (SIN vectors) have led to further demonstration of efficacy for these PID and no clinical genotoxic events. X-linked Adrenoleukodystrophy was the first disorder approached using lentiviral vectors to transduce HSC and results in have been were equivalent to those with allogeneic HSCT, without GVHD. Autologous HSC gene therapy for Metachromatic Leukodystrophy produced outcomes that were better than those previously observed using allogeneic HSCT, with over-expression of the relevant gene product (arylsulfatase A) leading to cross-correction and clinically meaningful neuroprotection and excellent development outcomes. For hemoglobinopathies, early reports from clinical trials for beta-thalassemia and sickle cell disease indicate some degree of efficacy, although it remains uncertain whether current vector designs yield sufficient beta-globin gene transfer and expression to ameliorate severe forms of the diseases. The potential game-changer in this area is correction of the underlying defective gene in HSC, using site-specific endonucleases (ZFN, TALEN, Meganucleases and CRISPR/Cas9) to augment homology-driven gene repair. There have been rapid advances in this approach, although the frequency of engrafting HSC that are gene-corrected and without off-target effects are not yet up to levels that would be therapeutic for most disorders. One disorder at a time, autologous HSC gene therapy is advancing to provide effective and safe therapies for genetic diseases of blood cells.

P05.4 Brain gene therapy for Metachromatic Leukodystrophy: from bench to bedside

P05.4 Brain gene therapy for Metachromatic Leukodystrophy: from bench to bedside

Hematopoietic stem cell gene therapy for metachromatic leukodystrophy and other lysosomal storage diseases

Hematopoietic stem cell gene therapy for metachromatic leukodystrophy and other lysosomal storage diseases

13 Local and systemic gene therapy for metachromatic leukodystrophy

13 Local and systemic gene therapy for metachromatic leukodystrophy

Timing of Therapeutic Intervention Determines Functional and Survival Outcomes in a Mouse Model of Late Infantile Batten Disease

Classical late infantile neuronal ceroid lipofuscinosis (cLINCL) is a monogenic disorder caused by the loss of tripeptidyl peptidase 1 (TPP1) activity as a result of mutations in CLN2. Absence of TPP1 results in lysosomal storage with an accompanying axonal degeneration throughout the central nervous system (CNS), which leads to progressive neurodegeneration and early death. In this study, we compared the efficacies of pre- and post-symptomatic injections of recombinant adeno-associated virus (AAV) for treating the cellular and functional abnormalities of CLN2 mutant mice. Intracranial injection of AAV1-hCLN2 resulted in widespread human TPP1 (hTPP1) activity in the brain that was 10-100-fold above wild-type levels. Injections before disease onset prevented storage and spared neurons from axonal degeneration, reflected by the preservation of motor function. Furthermore, the majority of CLN2 mutant mice treated pre-symptomatically lived for at least 330 days, compared with a median survival of 151 days in untreated CLN2 mutant controls. In contrast, although injection after disease onset ameliorated lysosomal storage, there was evidence of axonal degeneration, motor function showed limited recovery, and the animals had a median lifespan of 216 days. These data illustrate the importance of early intervention for enhanced therapeutic benefit, which may provide guidance in designing novel treatment strategies for cLINCL patients.

935. Brain Targeted Gene Therapy for Metachromatic Leukodystrophy: In Vitro Expression and Activity of a Melanotransferrin-Arylsulfatase A Fusion Protein

935. Brain Targeted Gene Therapy for Metachromatic Leukodystrophy: In Vitro Expression and Activity of a Melanotransferrin-Arylsulfatase A Fusion Protein

Gene therapy of metachromatic leukodystrophy reverses neurological damage and deficits in mice

Metachromatic leukodystrophy (MLD) is a demyelinating lysosomal storage disorder for which new treatments are urgently needed. We previously showed that transplantation of gene-corrected hematopoietic stem progenitor cells (HSPCs) in presymptomatic myeloablated MLD mice prevented disease manifestations. Here we show that HSC gene therapy can reverse neurological deficits and neuropathological damage in affected mice, thus correcting an overt neurological disease. The efficacy of gene therapy was dependent on and proportional to arylsulfatase A (ARSA) overexpression in the microglia progeny of transplanted HSPCs. We demonstrate a widespread enzyme distribution from these cells through the CNS and a robust cross-correction of neurons and glia in vivo. Conversely, a peripheral source of enzyme, established by transplanting ARSA-overexpressing hepatocytes from transgenic donors, failed to effectively deliver the enzyme to the CNS. These results indicate that the recruitment of gene-modified, enzyme-overexpressing microglia makes the enzyme bioavailable to the brain and makes therapeutic efficacy and disease correction attainable. Overall, our data provide a strong rationale for implementing HSPC gene therapy in MLD patients.

Ex vivo cell-mediated gene therapy for metachromatic leukodystrophy using neurospheres

Metachromatic leukodystrophy (MLD) is an autosomal recessive disease caused by mutations in the gene encoding the lysosomal enzyme arylsulfatase A (ASA). In MLD, accumulation of the substrate, sulfated glycoprotein, in the central and peripheral nervous systems results in progressive motor and mental deterioration. Neural progenitor cells are thought to be useful for cell replacement therapy and for cell-mediated gene therapy in neurodegenerative diseases. In the present study, we examined the feasibility of ex vivo gene therapy for MLD using neural progenitor cells. Neural progenitor cells (neurospheres) were prepared from the striatum of E14 embryo MLD knockout mice or GFP transgenic mice and were transduced with the VSV pseudotyped HIV vector carrying the ASA gene (HIV-ASA). For in vivo study, neurospheres from GFP mice were transduced with HIV-ASA and inoculated into the brain parenchyma of adult MLD mice. HIV vector-transduced progenitor cells retained the potential for differentiation into neurons, astrocytes and oligodendrocytes in vitro. Expression of ASA in neurospheres transduced with HIV-ASA was confirmed by spectrophotometric enzyme assay and Western blotting. In vivo, GFP-positive cells were detectable 1 month after injection. These cells included GFAP- and MAP2-positive cells. Immunohistochemistry using anti-ASA antibody demonstrated localization of ASA in both GFP-positive and -negative cells. Partial clearance of accumulated sulfatide was confirmed in vivo in MLD knockout mice. The present findings suggest that ASA enzyme is released from migrated neurospheres and is able to digest sulfatide in surrounding cells. Our results suggest the potential of genetically engineered neural progenitor cells (neurospheres) for ex vivo therapy in MLD.

407. A Single Unilateral Injection of AAV1-ASA and AAV1-FGE Vectors into the Hippocampus Results in Bilateral Expression and Widespread Distribution of ASA and Prevention of Sulfatide Storage in the Whole Brain of MLD Model Mice

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by a deficiency of arylsulfatase A (ASA). The disease is characterized by progressive demyelination and widespread deposition of sulfatide in both the central and peripheral nervous systems. Therefore, direct injection of viral vector through the blood brain barrier is a possible approach for gene therapy of MLD. Lysosomal enzymes including ASA are known to be secreted and recaptured by surrounding cells through the mannose-6 phosphate receptor mediated pathway. Despite of this unique property, to treat the whole brain, it is likely that a large number of vector particles should be repeatedly injected into multiple sites. High efficient gene transfer and expression are critical for direct gene therapy of the brain of MLD. We have previously reported that co-expression of formylglycine generating enzyme (FGE), which is a post-translational modifying enzyme essential for activating all sulfatases, is essential for over-expression of functional ASA both in vitro and in vivo. In this study, we examined the utility of FGE co-expression in AAV1 mediated gene therapy of ASA knockout (MLD) mice. A preliminary experiment showed that stereotactic injection of AAV1-GFP into the CA3 region of the hippocampus resulted in strong GFP expression in both ipsilateral and contralateral hippocampi, suggesting that AAV1 vector is efficiently transported through axons to distal cell bodies. In a treatment experiment, AAV1-ASA alone or AAV1- ASA plus AAV1-FGE (total 7.5 |[times]| 109particles) were injected into the right hippocampus CA3 region of 8 month-old ASA knockout mice. The treated mice were analyzed 7 months after injection. Colorimetric assay of brain homogenates showed that injection of AAV1-ASA alone increased ASA activity in the injected hemisphere along with a slight increase in ASA in the non-injected hemisphere. Co-introduction of AAV1-ASA and AAV1-FGE significantly increased ASA activity in both sides. Widespread distribution of ASA was also confirmed by immunohistochemical analysis using anti-ASA antibody. Alcian blue staining and chemical analysis showed that sulfatide in the injected hemisphere was decreased after AAV1- ASA injection. Remarkably, reduction of sulfatide in the whole brain was observed when AAV1-ASA and AAV1-FGE were co-introduced. Rotarod test and walking pattern revealed significant improvement of neurological functions. These results indicate that AAV1 mediated expression of ASA and FGE is highly efficient for long-term expression and secretion of functional ASA in the brain. Unexpectedly, ASA could be distributed to the brain areas distant from the site of gene transfer by axonal transfer and diffusion. Extensive distribution of ASA in the brain may rationalize direct gene therapy of MLD.

864. Characterization of Transgenic Mice Over_ Expressing Arylsulfatase A: Implications for Gene Therapy and Sulfatases Activation Mechanisms

We have recently shown that in order to attain therapeutic efficacy by HSC gene therapy for Metachromatic Leukodystrophy (MLD), Arylsulfatase A (ARSA) over_expression in HSC and their progeny is required. The recent identification of Sulfatase Modifying Factor 1 (SUMF1) as a common activator of sulfatases and a rate_limiting factor in the biological activation of these enzymes, raises concerns for possible adverse effects of enzyme over_expression within certain cell type (Cosma M.P. et al., Cell 2003).

968. Co-Expression of FGE Is Essential for Gene Therapy of MLD Based on Cross-Correction of ASA Deficiency

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder with inherited deficiency of arylsulfatase A (ASA). The disease is characterized by progressive demyelination and widespread deposition of sulfatide in both central and peripheral nervous systems. Direct injection of viral vector through the blood brain barrier is a possible approach of gene therapy for MLD. However, to treat a wide area of brain, it is essential to efficiently secrete functional ASA from limited numbers of transduced cells. In this study, we tested the utility of FGE (formylglycine generating enzyme) for over-expression of functional ASA. FGE is a recently identified post-translational modifying enzyme which converts cystein common in the active site of all sulfatases to formylglycine and is thought to be essential for all sulfatases including ASA. COS-7 cells were transfected with the ASA and FGE expression plasmids. ASA activity was increased up to 20 folds in cell lysates and 70 folds in conditioned media by co-expression of FGE. When the expression plasmids were injected into the MLD knock-out mice by the hydrodynamics-based procedure, a striking synergistic increase of ASA activity was also observed in both liver and serum. Blot hybridization analysis of FGE mRNA demonstrated that expression of endogenous FGE is particularly low in human brain. These results suggest that co-expression of FGE and ASA should be essential for gene therapy of MLD targeted whole brain.

866. Modification of the Expression Vector for Systemic Gene Therapy of MLD

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by a deficiency of arylsulfatase A (ASA). Progressive demyelination associated with accumulation of sulfatide results in a variety of neurologic symptoms. No effective therapy is available at this moment. Gene therapy may be an important option for treatment of MLD. Since sulfatide deposits in both the central and peripheral nervous system, local injection of ASA expression vectors may have limited merit. In order to establish a systemic gene therapy protocol for MLD, we modified the ASA expression vector. First, we tested the utility of SUMF1 for overexpression of functional ASA. SUMF1 is a post-translational modifying enzyme essential for sulfatase activity. Coexpression of SUMF1 with ASA in 293 cells resulted in a several folds increase of ASA activity. When the ASA and SUMF1 expression plasmids were injected into liver by the hydrodynamics-based technique, a striking synergistic increase of ASA activity was observed in both liver and serum. Second, we synthesized the ASA-Tat fusion protein to improve the biodistribution of the enzyme. It was reported that the Tat motif (YGRKKRRQRRR) allows the fusion protein to cross the cell membrane and the blood brain barrier. The ASA-Tat fusion protein had full ASA activity and was secreted from cells transfected with the expression vector. When the conditioned media were incubated with MLD patient's fibroblasts, the ASA-Tat was efficiently taken up to the cells, but its uptake was only partially inhibited by the addition of mannose 6 phosphate (M6P). The M6P independent uptake of ASA-Tat was also confirmed by immunohistochemical staining. These results suggest that combination of SUMF1-coexpression and Tat-modification may be important for systemic delivery of ASA for MLD gene therapy.

865. An Ex Vivo Gene Therapy Approach for Metachromatic Leukodystrophy Using Neural Progenitor Cells

Metachromatic leukodystrophy(MLD) is an autosomal recessive inherited disease caused by mutations of the gene encoding the lysozomal enzyme, arylsulfatase A(ASA). Accumulation of the substrate, sulfated glycoprotein, in both the central and peripheral nervous system results in progressive motor and mental deterioration. No effective therapy has been developed yet. Neural progenitor cells are thought to be useful for cell replacement therapy and also cell mediated gene therapy of neurodegenerative diseases. In the present study, we examined the feasibility of ex vivo gene therapy for MLD using neural progenitor cells. Neural progenitor cells (neurospheres) were prepared from the striatum of E14 embryo MLD knockout mice or GFP transgenic mice and transduced with the VSV pseudotyped HIV vector carrying the ASA gene (HIV-ASA). HIV vector transduced progenitor cells retained the potential for differentiation into neurons, astrocytes, and oligodendrocytes in vitro. Expression of ASA in neurospheres transduced with HIV-ASA was confirmed by the spectrophotometric enzyme assay and Western blotting. When neurospheres from GFP mice were transduced with HIV-ASA and inoculated into the brain parenchyma of adult MLD mice, GFP positive cells were detectable one month after injection. Those cells include GFAP positive astrocytes and undifferentiated cells. Immunohistochemistry using anti-ASA antibody demonstrated that ASA was localized in both GFP positive and negative cells, suggesting that ASA was secreted from the transduced cells and taken up by the surrounding cells. These results hold promise for the use of genetically engineered neural stem cells for ex vivo therapy of MLD.

In vivo gene therapy of metachromatic leukodystrophy by lentiviral vectors: correction of neuropathology and protection against learning impairments in affected mice.

Metachromatic leukodystrophy (MLD) is a lipidosis caused by deficiency of arylsulfatase A (ARSA). Although the genetics of MLD are known, its pathophysiology is not understood. The disease leads to progressive demyelination and early death and no effective treatment is available. We used lentiviral vectors to deliver a functional ARSA gene (human ARSA) into the brain of adult mice with germ-line inactivation of the mouse gene encoding ARSA, As2. We report sustained expression of active enzyme throughout a large portion of the brain, with long-term protection from development of neuropathology and hippocampal-related learning impairments. We show that selective degeneration of hippocampal neurons is a central step in disease pathogenesis, and provide evidence that in vivo transfer of ARSA by lentiviral vectors reverts the disease phenotype in all investigated areas. Therefore, in vivo gene therapy offers a unique option for MLD and other storage diseases affecting the central nervous system.