High-throughput transcriptome analyses from ASPIRO, a phase 1/2/3 study of gene replacement therapy for X-linked myotubular myopathy.

X-linked myotubular myopathy (XLMTM) is a severe congenital disease characterized by profound muscle weakness, respiratory failure, and early death. No approved therapy for XLMTM is currently available. Adeno-associated virus (AAV)-mediated gene replacement therapy has shown promise as an investigational therapeutic strategy. We aimed to characterize the transcriptomic changes in muscle biopsies of individuals with XLMTM who received resamirigene bilparvovec (AT132; rAAV8-Des-hMTM1) in the ASPIRO clinical trial and to identify potential biomarkers that correlate with therapeutic outcome. We leveraged RNA-sequencing data from the muscle biopsies of 15 study participants and applied differential expression analysis, gene co-expression analysis, and machine learning to characterize the transcriptomic changes at baseline (pre-dose) and at 24 and 48 weeks after resamirigene bilparvovec dosing. As expected, MTM1 expression levels were significantly increased after dosing (p < 0.0001). Differential expression analysis identified upregulated genes after dosing that were enriched in several pathways, including lipid metabolism and inflammatory response pathways, and downregulated genes were enriched in cell-cell adhesion and muscle development pathways. Genes involved in inflammatory and immune pathways were differentially expressed between participants exhibiting ventilator support reduction of either greater or less than 6 h/day after gene therapy compared to pre-dosing. Co-expression analysis identified similarly regulated genes, which were grouped into modules. Finally, the machine learning model identified five genes, including MTM1, as potential RNA biomarkers to monitor the progress of AAV gene replacement therapy. These findings further extend our understanding of AAV-mediated gene therapy in individuals with XLMTM at the transcriptomic level.

Nuclear architecture dictates HIV-1 integration site selection.

Long-standing evidence indicates that human immunodeficiency virus type 1 (HIV-1) preferentially integrates into a subset of transcriptionally active genes of the host cell genome. However, the reason why the virus selects only certain genes among all transcriptionally active regions in a target cell remains largely unknown. Here we show that HIV-1 integration occurs in the outer shell of the nucleus in close correspondence with the nuclear pore. This region contains a series of cellular genes, which are preferentially targeted by the virus, and characterized by the presence of active transcription chromatin marks before viral infection. In contrast, the virus strongly disfavours the heterochromatic regions in the nuclear lamin-associated domains and other transcriptionally active regions located centrally in the nucleus. Functional viral integrase and the presence of the cellular Nup153 and LEDGF/p75 integration cofactors are indispensable for the peripheral integration of the virus. Once integrated at the nuclear pore, the HIV-1 DNA makes contact with various nucleoporins; this association takes part in the transcriptional regulation of the viral genome. These results indicate that nuclear topography is an essential determinant of the HIV-1 life cycle.

Induction of fetal hemoglobin synthesis by CRISPR/Cas9-mediated editing of the human ß-globin locus.

Naturally occurring, large deletions in the ß-globin locus result in hereditary persistence of fetal hemoglobin, a condition that mitigates the clinical severity of sickle cell disease (SCD) and ß-thalassemia. We designed a clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) strategy to disrupt a 13.6-kb genomic region encompassing the δ- and ß-globin genes and a putative γ-δ intergenic fetal hemoglobin (HbF) silencer. Disruption of just the putative HbF silencer results in a mild increase in γ-globin expression, whereas deletion or inversion of a 13.6-kb region causes a robust reactivation of HbF synthesis in adult erythroblasts that is associated with epigenetic modifications and changes in chromatin contacts within the ß-globin locus. In primary SCD patient-derived hematopoietic stem/progenitor cells, targeting the 13.6-kb region results in a high proportion of γ-globin expression in erythroblasts, increased HbF synthesis, and amelioration of the sickling cell phenotype. Overall, this study provides clues for a potential CRISPR/Cas9 genome editing approach to the therapy of ß-hemoglobinopathies.

Optimization of CRISPR/Cas9 Delivery to Human Hematopoietic Stem and Progenitor Cells for Therapeutic Genomic Rearrangements.

Editing the ß-globin locus in hematopoietic stem cells is an alternative therapeutic approach for gene therapy of ß-thalassemia and sickle cell disease. Using the CRISPR/Cas9 system, we genetically modified human hematopoietic stem and progenitor cells (HSPCs) to mimic the large rearrangements in the ß-globin locus associated with hereditary persistence of fetal hemoglobin (HPFH), a condition that mitigates the clinical phenotype of patients with ß-hemoglobinopathies. We optimized and compared the efficiency of plasmid-, lentiviral vector (LV)-, RNA-, and ribonucleoprotein complex (RNP)-based methods to deliver the CRISPR/Cas9 system into HSPCs. Plasmid delivery of Cas9 and gRNA pairs targeting two HPFH-like regions led to high frequency of genomic rearrangements and HbF reactivation in erythroblasts derived from sorted, Cas9+ HSPCs but was associated with significant cell toxicity. RNA-mediated delivery of CRISPR/Cas9 was similarly toxic but much less efficient in editing the ß-globin locus. Transduction of HSPCs by LVs expressing Cas9 and gRNA pairs was robust and minimally toxic but resulted in poor genome-editing efficiency. Ribonucleoprotein (RNP)-based delivery of CRISPR/Cas9 exhibited a good balance between cytotoxicity and efficiency of genomic rearrangements as compared to the other delivery systems and resulted in HbF upregulation in erythroblasts derived from unselected edited HSPCs.

Gene therapy for Wiskott-Aldrich syndrome in a severely affected adult.

Until recently, hematopoietic stem cell transplantation was the only curative option for Wiskott-Aldrich syndrome (WAS). The first attempts at gene therapy for WAS using a ϒ-retroviral vector improved immunological parameters substantially but were complicated by acute leukemia as a result of insertional mutagenesis in a high proportion of patients. More recently, treatment of children with a state-of-the-art self-inactivating lentiviral vector (LV-w1.6 WASp) has resulted in significant clinical benefit without inducing selection of clones harboring integrations near oncogenes. Here, we describe a case of a presplenectomized 30-year-old patient with severe WAS manifesting as cutaneous vasculitis, inflammatory arthropathy, intermittent polyclonal lymphoproliferation, and significant chronic kidney disease and requiring long-term immunosuppressive treatment. Following reduced-intensity conditioning, there was rapid engraftment and expansion of a polyclonal pool of transgene-positive functional T cells and sustained gene marking in myeloid and B-cell lineages up to 20 months of observation. The patient was able to discontinue immunosuppression and exogenous immunoglobulin support, with improvement in vasculitic disease and proinflammatory markers. Autologous gene therapy using a lentiviral vector is a viable strategy for adult WAS patients with severe chronic disease complications and for whom an allogeneic procedure could present an unacceptable risk. This trial was registered at www.clinicaltrials.gov as #NCT01347242.

Efficient Non-viral Gene Delivery into Human Hematopoietic Stem Cells by Minicircle Sleeping Beauty Transposon Vectors.

The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform that combines simplicity, inexpensive manufacture, and favorable safety features in the context of human applications. However, efficient correction of hematopoietic stem and progenitor cells (HSPCs) with non-viral vector systems, including SB, demands further refinement of gene delivery techniques. We set out to improve SB gene transfer into hard-to-transfect human CD34+ cells by vectorizing the SB system components in the form of minicircles that are devoid of plasmid backbone sequences and are, therefore, significantly reduced in size. As compared to conventional plasmids, delivery of the SB transposon system as minicircle DNA is ∼20 times more efficient, and it is associated with up to a 50% reduction in cellular toxicity in human CD34+ cells. Moreover, providing the SB transposase in the form of synthetic mRNA enabled us to further increase the efficacy and biosafety of stable gene delivery into hematopoietic progenitors ex vivo. Genome-wide insertion site profiling revealed a close-to-random distribution of SB transposon integrants, which is characteristically different from gammaretroviral and lentiviral integrations in HSPCs. Transplantation of gene-marked CD34+ cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution, which was most efficient when the SB transposase was supplied as mRNA and nucleofected cells were maintained for 4-8 days in culture before transplantation. Collectively, implementation of minicircle and mRNA technologies allowed us to further refine the SB transposon system in the context of HSPC gene delivery to ultimately meet clinical demands of an efficient and safe non-viral gene therapy protocol.

Systemic AAV8-Mediated Gene Therapy Drives Whole-Body Correction of Myotubular Myopathy in Dogs.

X-linked myotubular myopathy (XLMTM) results from MTM1 gene mutations and myotubularin deficiency. Most XLMTM patients develop severe muscle weakness leading to respiratory failure and death, typically within 2 years of age. Our objective was to evaluate the efficacy and safety of systemic gene therapy in the p.N155K canine model of XLMTM by performing a dose escalation study. A recombinant adeno-associated virus serotype 8 (rAAV8) vector expressing canine myotubularin (cMTM1) under the muscle-specific desmin promoter (rAAV8-cMTM1) was administered by simple peripheral venous infusion in XLMTM dogs at 10 weeks of age, when signs of the disease are already present. A comprehensive analysis of survival, limb strength, gait, respiratory function, neurological assessment, histology, vector biodistribution, transgene expression, and immune response was performed over a 9-month study period. Results indicate that systemic gene therapy was well tolerated, prolonged lifespan, and corrected the skeletal musculature throughout the body in a dose-dependent manner, defining an efficacious dose in this large-animal model of the disease. These results support the development of gene therapy clinical trials for XLMTM.

IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors.

Long-living memory stem T cells (T(SCM)) with the ability to self-renew and the plasticity to differentiate into potent effectors could be valuable weapons in adoptive T-cell therapy against cancer. Nonetheless, procedures to specifically target this T-cell population remain elusive. Here, we show that it is possible to differentiate in vitro, expand, and gene modify in clinically compliant conditions CD8(+) T(SCM) lymphocytes starting from naive precursors. Requirements for the generation of this T-cell subset, described as CD62L(+)CCR7(+)CD45RA(+)CD45R0(+)IL-7Rα(+)CD95(+), are CD3/CD28 engagement and culture with IL-7 and IL-15. Accordingly, T(SCM) accumulates early after hematopoietic stem cell transplantation. The gene expression signature and functional phenotype define this population as a distinct memory T-lymphocyte subset, intermediate between naive and central memory cells. When transplanted in immunodeficient mice, gene-modified naive-derived T(SCM) prove superior to other memory lymphocytes for the ability to expand and differentiate into effectors able to mediate a potent xenogeneic GVHD. Furthermore, gene-modified T(SCM) are the only T-cell subset able to expand and mediate GVHD on serial transplantation, suggesting self-renewal capacity in a clinically relevant setting. These findings provide novel insights into the origin and requirements for T(SCM) generation and pave the way for their clinical rapid exploitation in adoptive cell therapy.

Outcomes following gene therapy in patients with severe Wiskott-Aldrich syndrome.

IMPORTANCE: Wiskott-Aldrich syndrome is a rare primary immunodeficiency associated with severe microthrombocytopenia. Partially HLA antigen-matched allogeneic hematopoietic stem cell (HSC) transplantation is often curative but is associated with significant comorbidity. OBJECTIVE: To assess the outcomes and safety of autologous HSC gene therapy in Wiskott-Aldrich syndrome. DESIGN, SETTING, AND PARTICIPANTS: Gene-corrected autologous HSCs were infused in 7 consecutive patients with severe Wiskott-Aldrich syndrome lacking HLA antigen-matched related or unrelated HSC donors (age range, 0.8-15.5 years; mean, 7 years) following myeloablative conditioning. Patients were enrolled in France and England and treated between December 2010 and January 2014. Follow-up of patients in this intermediate analysis ranged from 9 to 42 months. INTERVENTION: A single infusion of gene-modified CD34+ cells with an advanced lentiviral vector. MAIN OUTCOMES AND MEASURES: Primary outcomes were improvement at 24 months in eczema, frequency and severity of infections, bleeding tendency, and autoimmunity and reduction in disease-related days of hospitalization. Secondary outcomes were improvement in immunological and hematological characteristics and evidence of safety through vector integration analysis. RESULTS: Six of the 7 patients were alive at the time of last follow-up (mean and median follow-up, 28 months and 27 months, respectively) and showed sustained clinical benefit. One patient died 7 months after treatment of preexisting drug-resistant herpes virus infection. Eczema and susceptibility to infections resolved in all 6 patients. Autoimmunity improved in 5 of 5 patients. No severe bleeding episodes were recorded after treatment, and at last follow-up, all 6 surviving patients were free of blood product support and thrombopoietic agonists. Hospitalization days were reduced from a median of 25 days during the 2 years before treatment to a median of 0 days during the 2 years after treatment. All 6 surviving patients exhibited high-level, stable engraftment of functionally corrected lymphoid cells. The degree of myeloid cell engraftment and of platelet reconstitution correlated with the dose of gene-corrected cells administered. No evidence of vector-related toxicity was observed clinically or by molecular analysis. CONCLUSIONS AND RELEVANCE: This study demonstrated the feasibility of the use of gene therapy in patients with Wiskott-Aldrich syndrome. Controlled trials with larger numbers of patients are necessary to assess long-term outcomes and safety.

Targeted gene addition in human epithelial stem cells by zinc-finger nuclease-mediated homologous recombination.

Preclinical and clinical studies showed that autologous transplantation of epidermis derived from genetically modified epithelial stem cells (EpSCs) leads to long-term correction of inherited skin adhesion defects. These studies were based on potentially genotoxic retroviral vectors. We developed an alternative gene transfer strategy aimed at targeting a "safe harbor" locus, the adeno-associated virus integration site 1 (AAVS1), by zinc-finger nuclease (ZFN)-induced homologous recombination (HR). Delivery of AAVS1-specific ZFNs and a GFP-expressing HR cassette by integration-defective lentiviral (LV) vectors (IDLVs) or adenoviral (Ad) vectors resulted in targeted gene addition with an efficiency of > 20% in a human keratinocyte cell line, > 10% in immortalized keratinocytes, and < 1% in primary keratinocytes. Deep sequencing of the AAVS1 locus showed that ZFN-induced double-strand breaks are mostly repaired by nonhomologous end joining (NHEJ) in primary cells, indicating that poor induction of the HR-dependent DNA repair pathway may be a significant limitation for targeted gene integration. Skin equivalents derived from unselected keratinocyte cultures coinfected with a GFP-IDLV and a ZFN-Ad vector were grafted onto immunodeficient mice. GFP-positive clones were observed in all grafts up to 18 weeks post-transplantation. By histological and molecular analysis, we were able to demonstrate highly efficient targeting of the AAVS1 locus in human repopulating EpSCs.

Genotoxic signature in cord blood cells of newborns exposed in utero to a Zidovudine-based antiretroviral combination.

BACKGROUND: The genotoxicity of zidovudine has been established in experimental models. The objective of the study was to identify genotoxicity markers in cord blood cells from newborns exposed in utero to antiretroviral (ARV) combinations containing zidovudine. METHODS: Cells were investigated by karyotyping and gene expression analysis of the CD34(+) hematopoietic stem/progenitor cell (HPC) compartment. RESULTS: Karyotyping of the cord blood cells from 15 ARV-exposed newborns and 12 controls revealed a higher proportion of aneuploid cells in the exposed group (median, 18.8% [interquartile range, 10.0%-26.7%] vs 6.6% [interquartile range, 3.1%-11.7%]; P < .001). All chromosomes were involved, with a random distribution of these alterations. Gene expression profiling of CD34(+) HPCs from 7 ARV-exposed and 6 control newborns revealed that >300 genes were significantly upregulated or downregulated by at least 1.5-fold in the exposed group (P < .05 for all comparisons). Significant alterations of genes involved in cell cycle control, mitotic checkpoints, and DNA repair were identified. Although this study does not allow discrimination between the roles of each of the 3 drugs, both cytogenetic and transcriptional findings are similar to those in cellular experiments that used zidovudine alone. CONCLUSIONS: The cord blood cells, including hematopoietic stem cells, from newborns exposed in utero to a zidovudine-based ARV combination present cytogenetic and transcriptional abnormalities compatible with DNA damage.

Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells.

The continuous renewal of human epidermis is sustained by stem cells contained in the epidermal basal layer and in hair follicles. Cultured keratinocyte stem cells, known as holoclones, generate sheets of epithelium used to restore severe skin, mucosal and corneal defects. Mutations in genes encoding the basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder. Epidermal stem cells from an adult patient affected by LAM5-beta3-deficient JEB were transduced with a retroviral vector expressing LAMB3 cDNA (encoding LAM5-beta3), and used to prepare genetically corrected cultured epidermal grafts. Nine grafts were transplanted onto surgically prepared regions of the patient's legs. Engraftment was complete after 8 d. Synthesis and proper assembly of normal levels of functional LAM5 were observed, together with the development of a firmly adherent epidermis that remained stable for the duration of the follow-up (1 year) in the absence of blisters, infections, inflammation or immune response. Retroviral integration site analysis indicated that the regenerated epidermis is maintained by a defined repertoire of transduced stem cells. These data show that ex vivo gene therapy of JEB is feasible and leads to full functional correction of the disease.

Preclinical corrective gene transfer in xeroderma pigmentosum human skin stem cells.

Xeroderma pigmentosum (XP) is a devastating disease associated with dramatic skin cancer proneness. XP cells are deficient in nucleotide excision repair (NER) of bulky DNA adducts including ultraviolet (UV)-induced mutagenic lesions. Approaches of corrective gene transfer in NER-deficient keratinocyte stem cells hold great hope for the long-term treatment of XP patients. To face this challenge, we developed a retrovirus-based strategy to safely transduce the wild-type XPC gene into clonogenic human primary XP-C keratinocytes. De novo expression of XPC was maintained in both mass population and derived independent candidate stem cells (holoclones) after more than 130 population doublings (PD) in culture upon serial propagation (>10(40) cells). Analyses of retrovirus integration sequences in isolated keratinocyte stem cells suggested the absence of adverse effects such as oncogenic activation or clonal expansion. Furthermore, corrected XP-C keratinocytes exhibited full NER capacity as well as normal features of epidermal differentiation in both organotypic skin cultures and in a preclinical murine model of human skin regeneration in vivo. The achievement of a long-term genetic correction of XP-C epidermal stem cells constitutes the first preclinical model of ex vivo gene therapy for XP-C patients.

Novel lentiviral vectors for gene therapy of sickle cell disease combining gene addition and gene silencing strategies.

Sickle cell disease (SCD) is due to a mutation in the ß-globin gene causing production of the toxic sickle hemoglobin (HbS; α2ßS 2). Transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) transduced with lentiviral vectors (LVs) expressing an anti-sickling ß-globin (ßAS) is a promising treatment; however, it is only partially effective, and patients still present elevated HbS levels. Here, we developed a bifunctional LV expressing ßAS3-globin and an artificial microRNA (amiRNA) specifically downregulating ßS-globin expression with the aim of reducing HbS levels and favoring ßAS3 incorporation into Hb tetramers. Efficient transduction of SCD HSPCs by the bifunctional LV led to a substantial decrease of ßS-globin transcripts in HSPC-derived erythroid cells, a significant reduction of HbS+ red cells, and effective correction of the sickling phenotype, outperforming ßAS gene addition and BCL11A gene silencing strategies. The bifunctional LV showed a standard integration profile, and neither HSPC viability, engraftment, and multilineage differentiation nor the erythroid transcriptome and miRNAome were affected by the treatment, confirming the safety of this therapeutic strategy. In conclusion, the combination of gene addition and gene silencing strategies can improve the efficacy of current LV-based therapeutic approaches without increasing the mutagenic vector load, thus representing a novel treatment for SCD.

Safety and efficacy of gene replacement therapy for X-linked myotubular myopathy (ASPIRO): a multinational, open-label, dose-escalation trial.

BACKGROUND: X-linked myotubular myopathy is a rare, life-threatening, congenital muscle disease observed mostly in males, which is caused by mutations in MTM1. No therapies are approved for this disease. We aimed to assess the safety and efficacy of resamirigene bilparvovec, which is an adeno-associated viral vector serotype 8 delivering human MTM1. METHODS: ASPIRO is an open-label, dose-escalation trial at seven academic medical centres in Canada, France, Germany, and the USA. We included boys younger than 5 years with X-linked myotubular myopathy who required mechanical ventilator support. The trial was initially in two parts. Part 1 was planned as a safety and dose-escalation phase in which participants were randomly allocated (2:1) to either the first dose level (1·3 × 1014 vector genomes [vg]/kg bodyweight) of resamirigene bilparvovec or delayed treatment, then, for later participants, to either a higher dose (3·5 × 1014 vg/kg bodyweight) of resamirigene bilparvovec or delayed treatment. Part 2 was intended to confirm the dose selected in part 1. Resamirigene bilparvovec was administered as a single intravenous infusion. An untreated control group comprised boys who participated in a run-in study (INCEPTUS; NCT02704273) or those in the delayed treatment cohort who did not receive any dose. The primary efficacy outcome was the change from baseline to week 24 in hours of daily ventilator support. After three unexpected deaths, dosing at the higher dose was stopped and the two-part feature of the study design was eliminated. Because of changes to the study design during its implementation, analyses were done on an as-treated basis and are deemed exploratory. All treated and control participants were included in the safety analysis. The trial is registered with ClinicalTrials.gov, NCT03199469. Outcomes are reported as of Feb 28, 2022. ASPIRO is currently paused while deaths in dosed participants are investigated. FINDINGS: Between Aug 3, 2017 and June 1, 2021, 30 participants were screened for eligibility, of whom 26 were enrolled; six were allocated to the lower dose, 13 to the higher dose, and seven to delayed treatment. Of the seven children whose treatment was delayed, four later received the higher dose (n=17 total in the higher dose cohort), one received the lower dose (n=7 total in the lower dose cohort), and two received no dose and joined the control group (n=14 total, including 12 children from INCEPTUS). Median age at dosing or enrolment was 12·1 months (IQR 10·0-30·9; range 9·5-49·7) in the lower dose cohort, 31·1 months (16·0-64·7; 6·8-72·7) in the higher dose cohort, and 18·7 months (10·1-31·5; 5·9-39·3) in the control cohort. Median follow-up was 46·1 months (IQR 41·0-49·5; range 2·1-54·7) for lower dose participants, 27·6 months (24·6-29·1; 3·4-41·0) for higher dose participants, and 28·3 months (9·7-46·9; 5·7-32·7) for control participants. At week 24, lower dose participants had an estimated 77·7 percentage point (95% CI 40·22 to 115·24) greater reduction in least squares mean hours per day of ventilator support from baseline versus controls (p=0·0002), and higher dose participants had a 22·8 percentage point (6·15 to 39·37) greater reduction from baseline versus controls (p=0·0077). One participant in the lower dose cohort and three in the higher dose cohort died; at the time of death, all children had cholestatic liver failure following gene therapy (immediate causes of death were sepsis; hepatopathy, severe immune dysfunction, and pseudomonal sepsis; gastrointestinal haemorrhage; and septic shock). Three individuals in the control group died (haemorrhage presumed related to hepatic peliosis; aspiration pneumonia; and cardiopulmonary failure). INTERPRETATION: Most children with X-linked myotubular myopathy who received MTM1 gene replacement therapy had important improvements in ventilator dependence and motor function, with more than half of dosed participants achieving ventilator independence and some attaining the ability to walk independently. Investigations into the risk for underlying hepatobiliary disease in X-linked myotubular myopathy, and the need for monitoring of liver function before gene replacement therapy, are ongoing. FUNDING: Astellas Gene Therapies.

High-definition mapping of retroviral integration sites identifies active regulatory elements in human multipotent hematopoietic progenitors.

Integration of retroviral vectors in the human genome follows nonrandom patterns that favor insertional deregulation of gene expression and increase the risk of their use in clinical gene therapy. The molecular basis of retroviral target site selection is still poorly understood. We used deep sequencing technology to build genomewide, high-definition maps of > 60 000 integration sites of Moloney murine leukemia virus (MLV)- and HIV-based retroviral vectors in the genome of human CD34(+) multipotent hematopoietic progenitor cells (HPCs) and used gene expression profiling, chromatin immunoprecipitation, and bioinformatics to associate integration to genetic and epigenetic features of the HPC genome. Clusters of recurrent MLV integrations identify regulatory elements (alternative promoters, enhancers, evolutionarily conserved noncoding regions) within or around protein-coding genes and microRNAs with crucial functions in HPC growth and differentiation, bearing epigenetic marks of active or poised transcription (H3K4me1, H3K4me2, H3K4me3, H3K9Ac, Pol II) and specialized chromatin configurations (H2A.Z). Overall, we mapped 3500 high-frequency integration clusters, which represent a new resource for the identification of transcriptionally active regulatory elements. High-definition MLV integration maps provide a rational basis for predicting genotoxic risks in gene therapy and a new tool for genomewide identification of promoters and regulatory elements controlling hematopoietic stem and progenitor cell functions.

C/EBPdelta regulates cell cycle and self-renewal of human limbal stem cells.

Human limbal stem cells produce transit amplifying progenitors that migrate centripetally to regenerate the corneal epithelium. Coexpression of CCAAT enhancer binding protein delta (C/EBPdelta), Bmi1, and DeltaNp63alpha identifies mitotically quiescent limbal stem cells, which generate holoclones in culture. Upon corneal injury, a fraction of these cells switches off C/EBPdelta and Bmi1, proliferates, and differentiates into mature corneal cells. Forced expression of C/EBPdelta inhibits the growth of limbal colonies and increases the cell cycle length of primary limbal cells through the activity of p27(Kip1) and p57(Kip2). These effects are reversible; do not alter the limbal cell proliferative capacity; and are not due to apoptosis, senescence, or differentiation. C/EBPdelta, but not DeltaNp63alpha, indefinitely promotes holoclone self-renewal and prevents clonal evolution, suggesting that self-renewal and proliferation are distinct, albeit related, processes in limbal stem cells. C/EBPdelta is recruited to the chromatin of positively (p27(Kip1) and p57(Kip2)) and negatively (p16(INK4A) and involucrin) regulated gene loci, suggesting a direct role of this transcription factor in determining limbal stem cell identity.

Estimated comparative integration hotspots identify different behaviors of retroviral gene transfer vectors.

Integration of retroviral vectors in the human genome follows non random patterns that favor insertional deregulation of gene expression and may cause risks of insertional mutagenesis when used in clinical gene therapy. Understanding how viral vectors integrate into the human genome is a key issue in predicting these risks. We provide a new statistical method to compare retroviral integration patterns. We identified the positions where vectors derived from the Human Immunodeficiency Virus (HIV) and the Moloney Murine Leukemia Virus (MLV) show different integration behaviors in human hematopoietic progenitor cells. Non-parametric density estimation was used to identify candidate comparative hotspots, which were then tested and ranked. We found 100 significative comparative hotspots, distributed throughout the chromosomes. HIV hotspots were wider and contained more genes than MLV ones. A Gene Ontology analysis of HIV targets showed enrichment of genes involved in antigen processing and presentation, reflecting the high HIV integration frequency observed at the MHC locus on chromosome 6. Four histone modifications/variants had a different mean density in comparative hotspots (H2AZ, H3K4me1, H3K4me3, H3K9me1), while gene expression within the comparative hotspots did not differ from background. These findings suggest the existence of epigenetic or nuclear three-dimensional topology contexts guiding retroviral integration to specific chromosome areas.

Correction of murine SCID-X1 by lentiviral gene therapy using a codon-optimized IL2RG gene and minimal pretransplant conditioning.

Clinical trials have demonstrated the potential of ex vivo hematopoietic stem cell gene therapy to treat X-linked severe combined immunodeficiency (SCID-X1) using γ-retroviral vectors, leading to immune system functionality in the majority of treated patients without pretransplant conditioning. The success was tempered by insertional oncogenesis in a proportion of the patients. To reduce the genotoxicity risk, a self-inactivating (SIN) lentiviral vector (LV) with improved expression of a codon optimized human interleukin-2 receptor γ gene (IL2RG) cDNA (coγc), regulated by its 1.1 kb promoter region (γcPr), was compared in efficacy to the viral spleen focus forming virus (SF) and the cellular phosphoglycerate kinase (PGK) promoters. Pretransplant conditioning of Il2rg(-/-) mice resulted in long-term reconstitution of T and B lymphocytes, normalized natural antibody titers, humoral immune responses, ConA/IL-2 stimulated spleen cell proliferation, and polyclonal T-cell receptor gene rearrangements with a clear integration preference of the SF vector for proto-oncogenes, contrary to the PGK and γcPr vectors. We conclude that SIN lentiviral gene therapy using coγc driven by the γcPr or PGK promoter corrects the SCID phenotype, potentially with an improved safety profile, and that low-dose conditioning proved essential for immune competence, allowing for a reduced threshold of cell numbers required.