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.
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 ARSAoverexpressing 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.
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...
We report a novel method to generate morphologically, antigenically and transcriptionally dependable microglia-like cells in vivo.
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