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.

Targeted genome editing in hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematological diseases. However, the limited efficiency of homology-directed editing in primitive HSPCs constrains the yield of corrected cells and might affect the feasibility and safety of clinical translation. These concerns need to be addressed in stringent preclinical models and overcome by developing more efficient editing methods. We generated a humanized X-linked severe combined immunodeficiency (SCID-X1) mouse model and evaluated the efficacy and safety of hematopoietic reconstitution from limited input of functional HSPCs, establishing thresholds for full correction upon different types of conditioning. Unexpectedly, conditioning before HSPC infusion was required to protect the mice from lymphoma developing when transplanting small numbers of progenitors. We then designed a one-size-fits-all (interleukin-2 receptor common γ-chain) gene correction strategy and, using the same reagents suitable for correction of human HSPC, validated the edited human gene in the disease model in vivo, providing evidence of targeted gene editing in mouse HSPCs and demonstrating the functionality of the-edited lymphoid progeny. Finally, we optimized editing reagents and protocol for human HSPCs and attained the threshold of editing in long-term repopulating cells predicted to safely rescue the disease, using clinically relevant HSPC sources and highly specific zinc finger nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9). Overall, our work establishes the rationale and guiding principles for clinical translation of SCID-X1 gene editing and provides a framework for developing gene correction for other diseases.

The recent hypothesis that postnatal microglia are maintained independently of circulating monocytes by local precursors that colonize the brain before birth has relevant implications for the treatment of various neurological diseases, including lysosomal storage disorders (LSDs), for which hematopoietic cell transplantation (HCT) is applied to repopulate the recipient myeloid compartment, including microglia, with cells expressing the defective functional hydrolase. By studying wild-type and LSD mice at diverse time-points after HCT, we showed the occurrence of a short-term wave of brain infiltration by a fraction of the transplanted hematopoietic progenitors, independently from the administration of a preparatory regimen and from the presence of a disease state in the brain. However, only the use of a conditioning regimen capable of ablating functionally defined brain-resident myeloid precursors allowed turnover of microglia with the donor, mediated by local proliferation of early immigrants rather than entrance of mature cells from the circulation.

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...

Changes in podocyte number or density have been suggested to play an important role in renal disease progression. Here , we investigated the temporal relationship between glomerular podocyte number and development of proteinuria and glomerulosclerosis in the male Munich Wistar Fromter (MWF) rat. We also assessed whether changes in podocyte number affect podocyte function and focused specifically on the slit diaphragm-associated protein nephrin. Age-matched Wistar rats were used as controls. Estimation of podocyte number per glomerulus was determined by digital morphometry of WT1-positive cells. MWF rats developed moderate hypertension , massive proteinuria , and glomerulosclerosis with age. Glomerular hypertrophy was already observed at 10 weeks of age and progressively increased thereafter. By contrast , mean podocyte number per glomerulus was lower than normal in young animals and further decreased with time. As a consequence , the capillary tuft volume per podocyte was more than threefold increased in older rats. Electron microscopy showed important changes in podocyte structure of MWF rats , with expansion of podocyte bodies surrounding glomerular filtration membrane. Glomerular nephrin expression was markedly altered in MWF rats and inversely correlated with both podocyte loss and proteinuria. Our findings suggest that reduction in podocyte number is an important determinant of podocyte dysfunction and progressive impairment of the glomerular permselectivity that lead to the development of massive proteinuria and ultimately to renal scarring. Proteinuric nephropathies progress toward end-stage renal failure independently of the primary insult. Proteinuria is the leakage of plasma proteins into the urine due to dysfunction of the glomerular barrier, which loses its permselective properties. Increasing evidence suggests that the visceral glomerular epithelial cell is a key determinant in the maintenance of the permselective function of the glomerular capillary.1-5 Podocytes are highly differentiated and specialized epithelial cells anchored to the glomerular basement membrane (GBM). Foot processes of neighboring podocytes interdigitate each other over the capillary wall and are bridged by the slit diaphragm forming the filtration barrier. The most characteristic structural change of damaged podocytes, concomitant with proteinuria, consists of foot process effacement, reorganization of actin cytoskeleton, and apical dislocation of the slit diaphragm. [5][6][7][8] We have recently demonstrated that in a genetic model of spontaneous glomerulosclerosis, the male Munich Wistar Fromter (MWF) rat, 9 proteinuria paralleled redistribution of the slit diaphragm protein zonula occludens-1 in the absence of changes in the ultrastructure of the podocyte foot processes as measured by mean foot process width.