Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disease caused by arylsulfatase A (ARSA) deficiency. Patients with MLD exhibit progressive motor and cognitive impairment and die within a few years of symptom onset. We used a lentiviral vector to transfer a functional ARSA gene into hematopoietic stem cells (HSCs) from three presymptomatic patients who showed genetic, biochemical, and neurophysiological evidence of late infantile MLD. After reinfusion of the gene-corrected HSCs, the patients showed extensive and stable ARSA gene replacement, which led to high enzyme expression throughout hematopoietic lineages and in cerebrospinal fluid. Analyses of vector integrations revealed no evidence of aberrant clonal behavior. The disease did not manifest or progress in the three patients 7 to 21 months beyond the predicted age of symptom onset. These findings indicate that extensive genetic engineering of human hematopoiesis can be achieved with lentiviral vectors and that this approach may offer therapeutic benefit for MLD patients.

IntroductionStable genetic modification of hematopoietic stem/progenitor cells (HSPCs) is achieved with retroviral vectors (RVs) that integrate into the cell genome and express a therapeutic transgene. 1 Transplantation of genetically modified autologous HSPCs provides a therapeutic option for patients with genetic disorders. [1][2][3] However, in clinical trials for X-linked severe combined immunodeficiency (X-SCID) and chronic granulomatous disease (CGD) oncogenesis triggered by ␥RV-mediated insertional mutagenesis has occurred. Leukemic or myelodysplastic cell clones in patients from these trials harbored RV integrations at common insertion sites (CISs) targeting recurrently LMO2 or MDS1-EVI1, PRDM16, SETBP1, and other genes. [4][5][6][7] Alternative to ␥RVs, HIV-derived self-inactivating lentiviral vectors (LVs) transduce human HSPCs efficiently and display a superior safety profile with respect to ␥RVs as shown in in vitro and in vivo preclinical mouse models. [8][9][10][11] Moreover, good efficacy and safety of LVs has also been documented in a recent HSPC-based clinical trial for X-linked adrenoleukodystrophy (ALD). 3 However, a careful LV integration site analysis in derived cells from patients with ALD showed that relevant numbers of CISs were present. 3 This observation raises concerns 12 because the detection of CISs is a well-established hallmark of insertional mutagenesis in mice 13,14 and clinical trials. 5,7,15 Thus, it is possible that the occurrence of CISs in the ALD clinical trial is a still silent effect of genotoxicity. To understand whether CISs generated by LV integrations are the product of genotoxicity we generated our own dataset of LV integrations in human HSPCs and their progeny after engraftment in immunodeficient mice and studied the integration pattern and the clonal repertoire of vector-marked cells in in vitro culture and in vivo. Moreover, we performed an extensive comparison between our dataset and the integrations found in the ALD clinical trial and in other gene therapy trials that reported insertional leukemogenesis, as well as in mice subjected to RV-mediated oncogene tagging. From our own integration data and the meta-analysis of the other integration datasets we provide evidence that the driving force leading to the appearance of CISs in LV-transduced HSPCs from the ALD clinical trial reflects a previously unappreciated bias of LVs in integration site selection rather oncogenic selection. Methods LV production and isolation and transduction of human HSPCsLV.ARSA (arylsulfatase A) and LV.GFP (green fluorescent protein) were produced with the use of the pCCLsin.cPPT.hPGK.hARSA.WPREmut6 and the pCCLsin.cPPT.hPGK.GFP.Wpre transfer plasmids. 16 Vesicular stomatitis virus-pseudotyped LV-concentrated stocks were produced and titered as described. 17 Human HSPCs were obtained by positive selection of CD34-expressing cells (CD34 progenitor cell isolation kit, MACS; Miltenyi Biotec) from BM aspirates, mobilized peripheral blood (MPB), or CB of healthy donors on collection with info...

Metachromatic leukodystrophy is a neurodegenerative disorder characterized by progressive demyelination. The disease is caused by variants in the ARSA gene, which codes for the lysosomal enzyme arylsulfatase A, or, more rarely, in the PSAP gene, which codes for the activator protein saposin B. In this Mutation Update, an extensive review of all the ARSA- and PSAP-causative variants published in the literature to date, accounting for a total of 200 ARSA and 10 PSAP allele types, is presented. The detailed ARSA and PSAP variant lists are freely available on the Leiden Online Variation Database (LOVD) platform at http://www.LOVD.nl/ARSA and http://www.LOVD.nl/PSAP, respectively.

Metachromatic leukodystrophy (MLD) is a rare lysosomal storage disorder resulting from the inherited deficiency of the arylsulfatase A (ARSA) enzyme. Currently, no valid therapeutic options are available for affected patients. A thorough knowledge of disease progression in its diverse clinical variants, together with the identification of reliable prognostic factors, could be instrumental in accurate patient selection for new upcoming therapeutic opportunities, such as enzyme replacement and gene therapy. The described correlation between genotype and clinical presentation proved helpful in predicting patient's prognosis, only in the minority of MLD patients harboring common mutations. Molecular characterization of a cohort of 26 MLD patients allowed us to identify 18 mutations, excluding the common 0 and R alleles, 10 of which are rare and 8 are novel. By categorizing the rare mutations, we were able to confirm a correlation between ARSA gene mutations, age at onset and patterns of disease progression, not only in those patients bearing common mutations, but also in those carrying rare mutant alleles. Moreover, in the case of absent or delayed molecular diagnosis, or of newly identified mutations, the involvement of peripheral nervous system from disease onset proved to be a sensitive prognostic marker predicting a severe progression.

These cases and review of the literature indicate that PRX-related neuropathies have early onset but overall slow progression. Typical features are prominent sensory involvement, often with sensory ataxia; a moderate-to-dramatic reduction of MNCVs and almost invariable absence of SNAPs; and pathologic demyelination with classic onion bulbs, and less commonly myelin folding and basal lamina onion bulbs.