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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 alpha-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, ; EudraCT number, .)Hematopoietic Gene Therapy for Hurler Syndrome Eight patients with Hurler syndrome who lacked suitable allogeneic donors received autologous hematopoietic stem and progenitor cells transduced ex vivo with an alpha-L-iduronidase-encoding lentiviral vector. This therapy resulted in extensive metabolic correction in peripheral tissues and the central nervous system.

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, an important 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 (BM), supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPC). To overcome the limit of BM harvest/HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex vivo expansion of HSPC coupled with lentiviral gene transfer. Circulating CD34(+). cells from TCIRG1-defective patients were transduced with a clinically-optimized lentiviral vector 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 NOD scid gamma common chain (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 HSPC 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 BM fibrosis.

Allogeneic hematopoietic stem cell transplantation (HSCT) performed early in life is the current standard of care for patients with severe type 1 mucopolysaccharidosis (Hurler disease), a metabolic disorder caused by mutations in the alpha-L-iduronidase (IDUA) gene, leading to impaired breakdown of glycosaminoglycans (GAG). Secretion of IDUA by donor-derived hematopoietic cells may cross-correct non-hematopoietic cells, slowing progression of tissue damage and cognitive decline. Nevertheless, Hurler patients undergoing HSCT manifest substantial residual disease burden, e.g. on the skeleton and central nervous system (CNS). We conducted a phase I/II clinical study (NCT03488394) to test whether infusion of autologous CD34+ hematopoietic stem and progenitor cells (HSPC) transduced ex vivo with a lentiviral vector coding for the IDUA gene was feasible, safe and capable of restoring enzymatic activity in the patients' blood and tissues, up to supraphysiologic levels. The trial originally planned to enroll 6 Hurler patients with preserved neurocognitive function (DQ/IQ>70) that had no access to a suitable allogeneic donor. Sample size has recently been increased to 8 patients. By July 2019, six patients have been treated at a median age of 24 months (range: 14-34), with a median follow up of 4 months (range: 1-13). In all patients, we collected a high number of autologous HSPC by leukapheresis following mobilization with lenograstim and plerixafor, resulting in drug products with a median of 21 million CD34+ cells/kg (range: 13-29). Transduction efficiency was high with a median above 80% and a vector copy number (VCN) of 1.7 (range: 1.0-5.2), employing a shortened, 2 day transduction protocol that included prostaglandin E2. All patients showed rapid hematopoietic recovery following myeloablative conditioning with busulfan (targeted to an AUC of 80mg*h/L), fludarabine (160mg/sqm) and rituximab (375mg/sqm). Median duration of grade 4 neutropenia associated with conditioning was 15.5 days (range: 13-19). Also associated with conditioning, Grade 3 thrombocytopenia lasted 4 days, while only 2 out of 6 patients experienced a platelet drop below 20,000/mcL on a single day, in the absence of transfusion support. Adverse events were mild and compatible with myeloablative conditioning, with the exception of patient 3 who experienced an anaphylactic reaction on day+12, which promptly responded to antihistamines, IV fluids and steroids. All evaluable patients showed sustained, supraphysiologic blood IDUA activity (dried blood spot), which was on average 3 fold above the upper limit of normal (evaluable patients: n=5 at 1 month, n=4 at 2 months, n=3 at 3 months). Notably, in n=4 Hurler patients treated with allogeneic HSCT, we detected IDUA activity that ranged within the lowest quartile of normal in spite of full donor chimerism, suggesting substantial gain achieved by overexpressing IDUA in ex vivo genetically-modified autologous HSPC. Urinary GAG excretion fell to normal levels within 3-6 months. IDUA activity was also detected in the cerebrospinal fluid (CSF) of treated patients, accompanied by a logfold reduction in CSF GAGs in the 2 patients with longest follow up. This suggests that gene therapy accomplishes full metabolic correction of tissues, including the CNS. Gene therapy did not induce antibodies against the IDUA protein, while pre-existing antibodies induced by enzyme replacement therapy before gene therapy rapidly disappeared. Patient 1 who reached the 1-year follow-up demonstrated a stable cognitive score, improved findings on brain and spine MRI, resumed growth velocity and an improvement of his skeletal phenotype. The preliminary results from our phase I/II study compare favorably with the standard of care in terms of safety and efficacy, and highlight the potential of genetic engineering of HSPC grafts for therapeutic gain-of-function. Disclosures Gentner: Genenta Science: Consultancy, Equity Ownership, Research Funding. Parini:Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; BioMarin: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; Ultragenyx: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; SOBI: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; Orphan Europe: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; Sanofi-Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support. Naldini:San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Wiskott-Aldrich Syndrome (WAS) gene therapy was licensed to GlaxoSmithKline (GSK) in 2014. It was then licensed to Orchard Therapeutics (OTL) in April 2018. OTL is the current sponsor of the clinical trial.; Genenta Science: Consultancy, Equity Ownership; Magenta Therapeutics: Equity Ownership. Aiuti:San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Wiskott-Aldrich Syndrome (WAS) gene therapy was licensed to GlaxoSmithKline (GSK) in 2014. It was than licensed to Orchard Therapeutics (OTL) in April 2018. OTL is the current sponsor of the clinical trial.; San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Study PI.

Summary 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., Highlights • CD34+CD38− cells as an HSC-enriched starting population for ex vivo gene therapy • Reduced culture time (, In this article, Gentner and colleagues undertake a comprehensive strategy to advance ex vivo genetic engineering of HSCs for gene therapy. They experimentally define an optimal strategy to purify HSCs, which allows uncoupling long-term from short-term hematopoietic reconstitution, and implement ex vivo conditions that best preserve their biological properties applying novel transduction-enhancing compounds and pyrimidoindole derivatives to support HSC expansion.

Hematopoietic stem and progenitor cell (HSPC) expansion remains an important unmet goal for ex vivo gene therapy based on gene addition and editing to compensate for the negative impact of the gene transfer procedure enabling faster engraftment and less complications. Additionally, ex vivo expansion of corrected cells may improve efficacy at more sustainable manufacturing costs by downscaling transduction. To date, our knowledge of precise mechanisms of action of expansion compounds is limited, and it remains unclear whether cord blood expansion protocols also maintain stemness of mobilized peripheral blood CD34+ cells (mPB), the preferred HSPC source for gene therapy. We performed serial (day 0,4,8) droplet-based single cell RNA sequencing (scRNAseq) on lentivirally transduced mPB expanded with UM171 to dissect cellular heterogeneity, monitor population dynamics over time and identify a transcriptional profile of primitive cells in culture. By associating published HSPC gene expression profiles to our scRNAseq dataset from uncultured mPB, we found that 45% of cells harbored a myelo-lymphoid signature. Smaller cell clusters expressed a shared erythroid (ERY) and megakaryocytic (MK) signature (20%), or a more primitive multipotent HSC-like signature (15%) characterized by enhanced JAK/STAT signaling and expression of HSC associated genes (AVP, HOPX, ID3). Unsupervised ordering of cells within pseudotime separated emerging MK/ERYpoiesis (FCER1A, HBD) from lympho-myelopoiesis (CD52, JUN), with intermediate states of more primitive progenitors located in between. After 4 days in culture, we noted a general increase in nuclear and mitochondrial gene transcription with activation of oxidative metabolism, paralleled by cell cycle activation, as expected from cytokine stimulation. By d8 of culture these changes leveled off but remained higher than uncultured cells. Of note, cells at d8 revealed an activation of cellular stress response pathways (e.g. TNFa, IFNg) hinting towards a compromised culture that may eventually exhaust HSC. Unsupervised clustering of cultured mPB highlighted a dramatic expansion (70-80%) of MK/ERY progenitor cells with high cycling activity with only 20-30% cells showing myelo-lymphoid transcriptional features. In line, pseudotime analysis highlighted a main ERY and MK trajectory separated from that of cells characterized by the expression of HSPC genes (HOPX, SPINK2) and of an emerging myeloid trajectory (MPO). To profile HSC in culture, we sorted and sequenced CD34+90+201+ cells from d4 expansion culture (3% of total cells), which we show to contain >70% of SCID repopulating potential. ScRNAseq revealed transcriptional similarity with the myelo-lymphoid progenitor cluster identified in the unsorted d4 culture. Unsupervised clustering of the CD34+90+201+ population revealed cell cycle dependent heterogeneity, identifying a highly quiescent cluster with expression of HSC-like signatures. This cluster was also characterized by relatively low gene expression, possibly reflecting a non-activated cell state consistent with primitive HSPC. Pseudotime analysis produced a four-branched minimum spanning tree, which retained a clear cell cycle and metabolic effect. Top variable genes included cell cycle, glycolytic, mitochondrial and ribosomal genes, identifying different metabolic modules along the branched trajectory. These results highlight that cell heterogeneity within a purified, HSC-enriched population is driven mainly by metabolic activation and cell cycle status. As a complementary approach, we purified LT-HSC from uncultured mPB (CD34+38-90+45RA-49f+), marked them with CFSE and expanded them in UM171 culture. LT-HSCs expanded on average 3.5 fold in 7 days, with the following distribution: 0 divisions: 3%; 1: 26%; 2: 47%; 3: 21%; 4: 3%. We performed scRNAseq on LT-HSC pre culture and after 7d separating a highly proliferative (≥2 divisions) and quiescent (0 - 1 division) fraction, allowing us to obtain unprecedented insight into the response of engrafting cells to ex vivo culture and set a framework to dissect self-renewal (HSC expansion), HSC maintenance and loss through differentiation as potential culture outcomes. Our combined functional/transcriptomic approach will define new HSC markers in culture and greatly facilitate side-by-side comparison of different expansion protocols towards rapid clinical translation. Disclosures No relevant conflicts of interest to declare.

Lentiviral (LV)-based hematopoietic stem and progenitor cell (HSPC) gene therapy is becoming a promising alternative to allogeneic stem cell transplantation for curing genetic diseases. To potentially improve the efficacy, safety and economic sustainability of HSPC transduction, we reasoned to genetically manipulate only the more potent CD34+CD38- HSPC, thereby improving HSPC maintenance in culture in the absence of differentiating cells and downscaling the cell therapy product by a factor of ten without compromising long-term engraftment. This approach would also decrease the total load of vector integration infused in the patients, thus improving its overall safety.First, we determined the engraftment kinetics of CD34+ mobilized peripheral blood (mPB) subpopulations over a 24wk xenotransplantation period. We sorted CD34+ mPB into 4 fractions with increasing expression levels of CD38 and marked each fraction with a specific fluorescent protein allowing to track the population of origin driving hematopoietic reconstitution. Differentially labeled fractions were mixed, and various combinations of CD38-, CD38int and CD38hi HSPC were injected into NSG mice (2 exp, n=30). Almost all long-term repopulating capacity (>90%) was contained within CD34+CD38- cells, and these cells took over hematopoiesis by 9wks. Instead, early reconstitution was mainly driven by CD34+CD38int progenitor cells.In the prospect of a clinical translation, we then modeled the co-administration of gene-modified CD34+CD38- mPB cells with uncultured CD34+CD38int/+ supporter cells aimed to drive fast hematopoietic recovery (3 exp, n=38). Repopulation by gene-modified CD34+CD38- cells was slower (15wks) and incomplete ( 5 wks and re-established long-term (24wks) gene marking up to 85%, thus allowing to benefit from prompt hematopoietic recovery driven by transiently repopulating CD38int/+ supporter cells.Last, we optimized LV transduction in the framework of an improved culture protocol. Exposing CD34+ or CD34+CD38- mPB cells to prostaglandin E2 (PGE2) increased transduction efficiency 1.5-2.5x, allowing to markedly reduce pre-stimulation and LV exposure times better preserving HSC functions. Importantly, the higher gene-transfer efficiency was maintained for up to 24 wks following xenotransplantation (n=33), suggesting that PGE2 facilitated LV transduction in long-term HSC.In summary, these results support the clinical development of novel HSPC gene therapy protocols based on the modification of highly purified HSC subsets, with the prospect to improve the efficacy, safety and feasibility of future ex vivo gene therapy studies.

In addition to its role in invasion and metastasis of several tumors, the multifunctional urokinase receptor uPAR (urokinase plasminogen activator receptor) is directly involved in the growth of several cancer cells in vitro and in vivo. We have compared growth rate and oncogenic transformation in wild-type (wt) or uPAR(-/-) mouse embryonic fibroblasts (MEFs). Surprisingly, uPAR(-/-) MEFs grew faster than wt MEFs. This agreed with elevated levels of cell cycle mediators like extracellular signal-regulated protein kinase, p38, AP1 and Cyclin D1. Infection with a uPAR retrovirus reverted the effect, decreasing the growth rate.When MEFs were transformed with H-Ras(V12) and E1A oncogenes, the efficiency of transformation in uPAR(-/-) MEFs was higher than in wt. UPAR(-/-) MEFs grew faster at low serum, produced more colonies in agar and produced tumors in vivo in nude mice with a lower latency period. The properties of the heterozygous uPAR(+/-) MEFs were always intermediate. We conclude therefore that in MEFs uPAR concentration controls cell proliferation and the transforming activity of some oncogenes.Oncogene advance online publication, 31 July 2006; doi:10.1038/sj.onc.1209833

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.

Transplantation of genetically engineered, autologous hematopoietic stem and progenitor cells (HSPC) is becoming a promising alternative to allogeneic stem cell transplantation for curing genetic diseases, avoiding the risks of graft versus host disease and prolonged immunosuppression. Most clinical gene therapy protocols are based on CD34+ HSPC engineered during >2 days of ex vivo culture. By xenotransplanting mobilized peripheral blood (mPB) CD34+ HSPC, which were lentivirally (LV) marked with different fluorescent proteins according to CD38/CD90 expression levels allowing quantitative assessment of the contribution of CD38/CD90 subpopulations to hematopoietic reconstitution (n=48 NSG mice, 3 experiments), we identified 2 distinct waves of reconstitution: (1) short term repopulation (up to 2 months) mostly driven by CD34+CD38intCD90+/- cells and (2) long-term repopulation driven by CD34+CD38-CD90+ (70%) and CD34+CD38-CD90- cells (30%). Notably, an intermediate wave extending from 2 to 4 months driven by CD34+CD38low cells was selectively eliminated by prolonged ex vivo culture and could be rescued when culture time was reduced to 1 day. We therefore developed a novel LV transduction protocol able to provide curative levels of gene transfer during a single day of ex vivo culture. Stimulating CD34+ cells or CD34+CD38- cells with Prostaglandin E2 (PGE2) increased gene transfer with VSVg-pseudotyped LVs by 1.5-2 fold acting on early steps of transduction, an effect that was further potentiated by the late-acting compound Cyclosporin A. Using large-scale vector preparations for gene therapy of mucopolysaccharidosis type 1, chronic granulomatous disease or beta-thalassemia, we show by in vitro and xenotransplantation assays that a 1-day PGE2 protocol achieved similar transduction efficiencies into BM or MPB HSPC from healthy donors and patients as our 62h benchmark protocol. PGE2 treatment did not result in toxicity or skewed multi-lineage differentiation. However, shortening ex vivo culture increased engraftment levels in the NSG mouse model. To entirely avoid culturing progenitor cells, we explored the feasibility to limit ex vivo manipulation to HSC-enriched CD34+CD38- cells that may be co-transplanted with unmanipulated CD34+ progenitor cells devoid of long-term engraftment potential. This could further improve hematopoietic reconstitution, increase safety by reducing the LV integration load infused into the patient and downscale ex vivo manipulation making the process more efficient and economically sustainable. To this end, we optimized a sequential bead-based, GMP-compatible selection procedure to separate mPB into a CD34+CD38- stem and CD38+ progenitor cell fraction. We reached high purity (87+/-6.6% CD34+) and recovery of CD34+CD38- cells (37.3+/-8.7%), making their isolation clinically viable. Bead-selected CD34+CD38- cells showed higher engraftment potential than equivalent numbers of FACS-sorted cells. Co-infusion of unmanipulated (culture-sensitive) CD38+ supporter cells with genetically-engineered CD34+CD38- cells into NSG mice resulted in rapid engraftment followed by near-complete replacement of untransduced short-term repopulating progenitors by gene-marked HSPC deriving from CD34+CD38- cells after the 3rd month post-transplant. Finally, we explored ex vivo expansion of mPB CD34+CD38- cells with arylhydrocarbon receptor antagonists and/or pyrimido-indole-derivatives. These cells expanded 3-10 fold in a 7-14 d time-window, far less than seen for total CD34+ cells, thereby facilitating culture handling and reducing cost. Unlike CD34+ cells, expanded mPB CD34+CD38- cells largely maintained their SCID-repopulating potential providing proof-of-concept for the expansion of gene-modified HSC. This clinically applicable platform will improve the efficacy, safety and sustainability of ex vivo gene addition and open up new opportunities in the field of gene editing. Disclosures Ciceri: MolMed SpA: Consultancy.

Ex vivo gene addition into CD34+ hematopoietic stem and progenitor cells (HSPC) followed by autologous transplantation has proven a safe and efficacious therapy for immunodeficiencies, storage diseases and hemoglobinopathies. According to xenograft models and population size estimates of vector-marked cells in gene therapy-treated patients, less than 0.01% of infused CD34+ cells drive long-term (LT) repopulation. Advances in clinical-grade cell sorting technology may make HSC-enriched CD34+ subpopulations accessible for gene therapy, with advantages in terms of lentiviral vector (LV) cost, safety (lowering of integration load) and, potentially, efficacy. By differentially marking mobilized peripheral blood (mPB) CD34 subpopulations distinguished by increasing levels of CD38 expression in order to quantitatively assess their hematopoietic output in an NSG xenograft model over 6 months, we previously mapped most (>90%) LT repopulating capacity to CD34+CD38- cells (lowest 10% CD38 staining), while CD34+CD38int cells drove short-term (ST) reconstitution during the first 2 months after transplantation. We now characterize these subpopulations in terms of CD90 expression, a marker, which has been used in conjunction with CD34 for HSC purification in past clinical trials. CD34+ mPB cells were sorted into CD38-90+ (5% of CD34+), CD38-90- (5%), CD38+90+ (30%) and CD38+90- (60%) fractions and exhaustively transduced with GFP-, OFP-, BFP- and mCherry-expressing LVs, respectively. Differentially marked subfractions were pooled maintaining their original proportions and transplanted into NSG mice. ST engraftment mainly came from CD38+ cells, with equal contribution from the CD90+ and CD90- compartment. LT engraftment was almost exclusively derived from CD34+CD38- cells, of which 70% came from CD90+ and 30% from CD90- cells. Hence, CD34+CD38- is a more sensitive and specific marker combination than CD34+CD90+ to purify LT-HSC. CD34+CD38- cells can be purified by a sequential bead-based selection (CD34 selection of CD38-depleted cells) potentially applicable to clinical practice. We show that CD34+CD38- cells can be efficiently transduced with clinical grade LVs using shortened ex vivo manipulation protocols, reaching similar gene marking levels as with the standard protocol currently used in clinical trials that comprises a double dose of LV. Transduction was stable for at least 5 months when serially measured in xenotransplanted mice, and mice showed multi-lineage hematopoiesis indistinguishable from CD34+ grafts. Based on these results, we are aiming towards clinical development of a new gene therapy protocol based on CD34+CD38-HSPC efficiently transduced with minimum ex vivo culture time (

SummaryTumor-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.

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.

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 anglopoletin 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 enclothelial 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 Iigand released by activated enclothelial cells and anglogenic vessels, suggesting a homing mechanism for TEMs to tumors. Purified human TEMs, but not TEM-clepleted 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. 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 anglopoletin 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 enclothelial 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 Iigand released by activated enclothelial cells and anglogenic vessels, suggesting a homing mechanism for TEMs to tumors. Purified human TEMs, but not TEM-clepleted 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.

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.