Hematopoietic Stem- and Progenitor-Cell Gene Therapy for Hurler Syndrome.

BACKGROUND: Allogeneic hematopoietic stem-cell transplantation is the standard of care for Hurler syndrome (mucopolysaccharidosis type I, Hurler variant [MPSIH]). However, this treatment is only partially curative and is associated with complications. METHODS: We are conducting an ongoing study involving eight children with MPSIH. At enrollment, the children lacked a suitable allogeneic donor and had a Developmental Quotient or Intelligence Quotient score above 70 (i.e., none had moderate or severe cognitive impairment). The children received autologous hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with an α-L-iduronidase (IDUA)-encoding lentiviral vector after myeloablative conditioning. Safety and correction of blood IDUA activity up to supraphysiologic levels were the primary end points. Clearance of lysosomal storage material as well as skeletal and neurophysiological development were assessed as secondary and exploratory end points. The planned duration of the study is 5 years. RESULTS: We now report interim results. The children's mean (±SD) age at the time of HSPC gene therapy was 1.9±0.5 years. At a median follow-up of 2.10 years, the procedure had a safety profile similar to that known for autologous hematopoietic stem-cell transplantation. All the patients showed prompt and sustained engraftment of gene-corrected cells and had supraphysiologic blood IDUA activity within a month, which was maintained up to the latest follow-up. Urinary glycosaminoglycan (GAG) excretion decreased steeply, reaching normal levels at 12 months in four of five patients who could be evaluated. Previously undetectable levels of IDUA activity in the cerebrospinal fluid became detectable after gene therapy and were associated with local clearance of GAGs. Patients showed stable cognitive performance, stable motor skills corresponding to continued motor development, improved or stable findings on magnetic resonance imaging of the brain and spine, reduced joint stiffness, and normal growth in line with World Health Organization growth charts. CONCLUSIONS: The delivery of HSPC gene therapy in patients with MPSIH resulted in extensive metabolic correction in peripheral tissues and the central nervous system. (Funded by Fondazione Telethon and others; ClinicalTrials.gov number, NCT03488394; EudraCT number, 2017-002430-23.).

Expanded circulating hematopoietic stem/progenitor cells as novel cell source for the treatment of TCIRG1 osteopetrosis.

Allogeneic hematopoietic stem cell transplantation is the treatment of choice for autosomal recessive osteopetrosis caused by defects in the TCIRG1 gene. Despite recent progress in conditioning, a relevant number of patients are not eligible for allogeneic stem cell transplantation because of the severity of the disease and significant transplant-related morbidity. We exploited peripheral CD34+ cells, known to circulate at high frequency in the peripheral blood of TCIRG1-deficient patients, as a novel cell source for autologous transplantation of gene corrected cells. Detailed phenotypical analysis showed that circulating CD34+ cells have a cellular composition that resembles bone marrow, supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPCs). To overcome the limit of bone marrow harvest/ HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex-vivo expansion of HSPCs coupled with lentiviral gene transfer. Circulating CD34+ cells from TCIRG1-defective patients were transduced with a clinically-optimized lentiviral vector (LV) expressing TCIRG1 under the control of phosphoglycerate promoter and expanded ex vivo. Expanded cells maintained long-term engraftment capacity and multi-lineage repopulating potential when transplanted in vivo both in primary and secondary NSG recipients. Moreover, when CD34+ cells were differentiated in vitro, genetically corrected osteoclasts resorbed the bone efficiently. Overall, we provide evidence that expansion of circulating HSPCs coupled to gene therapy can overcome the limit of stem cell harvest in osteopetrotic patients, thus opening the way to future gene-based treatment of skeletal diseases caused by bone marrow fibrosis.

A role for miR-155 in enabling tumor-infiltrating innate immune cells to mount effective antitumor responses in mice.

A productive immune response requires transient upregulation of the microRNA miR-155 in hematopoietic cells mediating innate and adaptive immunity. In order to investigate miR-155 in the context of tumor-associated immune responses, we stably knocked down (KD) miR-155 in the myeloid compartment of MMTV-PyMT mice, a mouse model of spontaneous breast carcinogenesis that closely mimics tumor-host interactions seen in humans. Notably, miR-155/KD significantly accelerated tumor growth by impairing classic activation of tumor-associated macrophages (TAMs). This created an imbalance toward a protumoral microenvironment as evidenced by a lower proportion of CD11c(+) TAMs, reduced expression of activation markers, and the skewing of immune cells within the tumor toward an macrophage type 2/T helper 2 response. This study highlights the importance of tumor-infiltrating hematopoietic cells in constraining carcinogenesis and establishes an antitumoral function of a prototypical oncomiR.

Dual-regulated lentiviral vector for gene therapy of X-linked chronic granulomatosis.

Regulated transgene expression may improve the safety and efficacy of hematopoietic stem cell (HSC) gene therapy. Clinical trials for X-linked chronic granulomatous disease (X-CGD) employing gammaretroviral vectors were limited by insertional oncogenesis or lack of persistent engraftment. Our novel strategy, based on regulated lentiviral vectors (LV), targets gp91(phox) expression to the differentiated myeloid compartment while sparing HSC, to reduce the risk of genotoxicity and potential perturbation of reactive oxygen species levels. Targeting was obtained by a myeloid-specific promoter (MSP) and posttranscriptional, microRNA-mediated regulation. We optimized both components in human bone marrow (BM) HSC and their differentiated progeny in vitro and in a xenotransplantation model, and generated therapeutic gp91(phox) expressing LVs for CGD gene therapy. All vectors restored gp91(phox) expression and function in human X-CGD myeloid cell lines, primary monocytes, and differentiated myeloid cells. While unregulated LVs ectopically expressed gp91(phox) in CD34(+) cells, transcriptionally and posttranscriptionally regulated LVs substantially reduced this off-target expression. X-CGD mice transplanted with transduced HSC restored gp91(phox) expression, and MSP-driven vectors maintained regulation during BM development. Combining transcriptional (SP146.gp91-driven) and posttranscriptional (miR-126-restricted) targeting, we achieved high levels of myeloid-specific transgene expression, entirely sparing the CD34(+) HSC compartment. This dual-targeted LV construct represents a promising candidate for further clinical development.

Genotoxic effects of base and prime editing in human hematopoietic stem cells.

Base and prime editors (BEs and PEs) may provide more precise genetic engineering than nuclease-based approaches because they bypass the dependence on DNA double-strand breaks. However, little is known about their cellular responses and genotoxicity. Here, we compared state-of-the-art BEs and PEs and Cas9 in human hematopoietic stem and progenitor cells with respect to editing efficiency, cytotoxicity, transcriptomic changes and on-target and genome-wide genotoxicity. BEs and PEs induced detrimental transcriptional responses that reduced editing efficiency and hematopoietic repopulation in xenotransplants and also generated DNA double-strand breaks and genotoxic byproducts, including deletions and translocations, at a lower frequency than Cas9. These effects were strongest for cytidine BEs due to suboptimal inhibition of base excision repair and were mitigated by tailoring delivery timing and editor expression through optimized mRNA design. However, BEs altered the mutational landscape of hematopoietic stem and progenitor cells across the genome by increasing the load and relative proportions of nucleotide variants. These findings raise concerns about the genotoxicity of BEs and PEs and warrant further investigation in view of their clinical application.

miR-126 identifies a quiescent and chemo-resistant human B-ALL cell subset that correlates with minimal residual disease.

Complete elimination of B-cell acute lymphoblastic leukemia (B-ALL) by a risk-adapted primary treatment approach remains a clinical key objective, which fails in up to a third of patients. Recent evidence has implicated subpopulations of B-ALL cells with stem-like features in disease persistence. We hypothesized that microRNA-126, a core regulator of hematopoietic and leukemic stem cells, may resolve intratumor heterogeneity in B-ALL and uncover therapy-resistant subpopulations. We exploited patient-derived xenograft (PDX) models with B-ALL cells transduced with a miR-126 reporter allowing the prospective isolation of miR-126(high) cells for their functional and transcriptional characterization. Discrete miR-126(high) populations, often characterized by MIR126 locus demethylation, were identified in 8/9 PDX models and showed increased repopulation potential, in vivo chemotherapy resistance and hallmarks of quiescence, inflammation and stress-response pathway activation. Cells with a miR-126(high) transcriptional profile were identified as distinct disease subpopulations by single-cell RNA sequencing in diagnosis samples from adult and pediatric B-ALL. Expression of miR-126 and locus methylation were tested in several pediatric and adult B-ALL cohorts, which received standardized treatment. High microRNA-126 levels and locus demethylation at diagnosis associate with suboptimal response to induction chemotherapy (MRD > 0.05% at day +33 or MRD+ at day +78).

Longitudinal single-cell profiling of chemotherapy response in acute myeloid leukemia.

Acute myeloid leukemia may be characterized by a fraction of leukemia stem cells (LSCs) that sustain disease propagation eventually leading to relapse. Yet, the contribution of LSCs to early therapy resistance and AML regeneration remains controversial. We prospectively identify LSCs in AML patients and xenografts by single-cell RNA sequencing coupled with functional validation by a microRNA-126 reporter enriching for LSCs. Through nucleophosmin 1 (NPM1) mutation calling or chromosomal monosomy detection in single-cell transcriptomes, we discriminate LSCs from regenerating hematopoiesis, and assess their longitudinal response to chemotherapy. Chemotherapy induced a generalized inflammatory and senescence-associated response. Moreover, we observe heterogeneity within progenitor AML cells, some of which proliferate and differentiate with expression of oxidative-phosphorylation (OxPhos) signatures, while others are OxPhos (low) miR-126 (high) and display enforced stemness and quiescence features. miR-126 (high) LSCs are enriched at diagnosis in chemotherapy-refractory AML and at relapse, and their transcriptional signature robustly stratifies patients for survival in large AML cohorts.

Efficient Ex Vivo Engineering and Expansion of Highly Purified Human Hematopoietic Stem and Progenitor Cell Populations for Gene Therapy.

Ex vivo gene therapy based on CD34+ hematopoietic stem cells (HSCs) has shown promising results in clinical trials, but genetic engineering to high levels and in large scale remains challenging. We devised a sorting strategy that captures more than 90% of HSC activity in less than 10% of mobilized peripheral blood (mPB) CD34+ cells, and modeled a transplantation protocol based on highly purified, genetically engineered HSCs co-infused with uncultured progenitor cells. Prostaglandin E2 stimulation allowed near-complete transduction of HSCs with lentiviral vectors during a culture time of less than 38 hr, mitigating the negative impact of standard culture on progenitor cell function. Exploiting the pyrimidoindole derivative UM171, we show that transduced mPB CD34+CD38- cells with repopulating potential could be expanded ex vivo. Implementing these findings in clinical gene therapy protocols will improve the efficacy, safety, and sustainability of gene therapy and generate new opportunities in the field of gene editing.

Targeting the ANG2/TIE2 axis inhibits tumor growth and metastasis by impairing angiogenesis and disabling rebounds of proangiogenic myeloid cells.

Tumor-infiltrating myeloid cells convey proangiogenic programs that counteract the efficacy of antiangiogenic therapy. Here, we show that blocking angiopoietin-2 (ANG2), a TIE2 ligand and angiogenic factor expressed by activated endothelial cells (ECs), regresses the tumor vasculature and inhibits progression of late-stage, metastatic MMTV-PyMT mammary carcinomas and RIP1-Tag2 pancreatic insulinomas. ANG2 blockade did not inhibit recruitment of MRC1(+) TIE2-expressing macrophages (TEMs) but impeded their upregulation of Tie2, association with blood vessels, and ability to restore angiogenesis in tumors. Conditional Tie2 gene knockdown in TEMs was sufficient to decrease tumor angiogenesis. Our findings support a model wherein the ANG2-TIE2 axis mediates cell-to-cell interactions between TEMs and ECs that are important for tumor angiogenesis and can be targeted to induce effective antitumor responses.

Tumor-targeted interferon-alpha delivery by Tie2-expressing monocytes inhibits tumor growth and metastasis.

The use of type I interferons (IFNs) in cancer therapy has been limited by ineffective dosing and significant toxicity. Here, we exploited the tumor-homing ability of proangiogenic Tie2-expressing monocytes (TEMs) to deliver IFN-alpha to tumors. By transplanting hematopoietic progenitors transduced with a Tie2 promoter/enhancer-driven Ifna1 gene, we turned TEMs into IFN-alpha cell vehicles that efficiently targeted the IFN response to orthotopic human gliomas and spontaneous mouse mammary carcinomas and obtained significant antitumor responses and near complete abrogation of metastasis. TEM-mediated IFN-alpha delivery inhibited tumor angiogenesis and activated innate and adaptive immune cells but did not impair myelopoiesis and wound healing detectably. These results illustrate the therapeutic potential of gene- and cell-based IFN-alpha delivery and should allow the development of IFN treatments that more effectively treat cancer.

Identification of proangiogenic TIE2-expressing monocytes (TEMs) in human peripheral blood and cancer.

Tumor-infiltrating myeloid cells, including tumor-associated macrophages (TAMs), have been implicated in tumor progression. We recently described a lineage of mouse monocytes characterized by expression of the Tie2 angiopoietin receptor and required for the vascularization and growth of several tumor models. Here, we report that TIE2 expression in human blood identifies a subset of monocytes distinct from classical inflammatory monocytes and comprised within the less abundant "resident" population. These TIE2-expressing monocytes (TEMs) accounted for 2% to 7% of blood mononuclear cells in healthy donors and were distinct from rare circulating endothelial cells and progenitors. In human cancer patients, TEMs were observed in the blood and, intriguingly, within the tumors, where they represented the main monocyte population distinct from TAMs. Conversely, TEMs were hardly detected in nonneoplastic tissues. In vitro, TEMs migrated toward angiopoietin-2, a TIE2 ligand released by activated endothelial cells and angiogenic vessels, suggesting a homing mechanism for TEMs to tumors. Purified human TEMs, but not TEM-depleted monocytes, markedly promoted angiogenesis in xenotransplanted human tumors, suggesting a potentially critical role of TEMs in human cancer progression. Human TEMs may provide a novel, biologically relevant marker of angiogenesis and represent a previously unrecognized target of cancer therapy.