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• W2898845079 abstract "Sickle cell disease (SCD) is caused by a mutation (E6V) in the hemoglobin (Hb) β-chain that induces polymerization of Hb tetramers, red blood cell deformation, ischemia, anemia, and multiple organ damage. Gene therapy is a potential alternative to human leukocyte antigen (HLA)-matched allogeneic hematopoietic stem cell transplantation, available to a minority of patients. We developed a lentiviral vector expressing a β-globin carrying three anti-sickling mutations (T87Q, G16D, and E22A) inhibiting axial and lateral contacts in the HbS polymer, under the control of the β-globin promoter and a reduced version of the β-globin locus-control region. The vector (GLOBE-AS3) transduced 60%–80% of mobilized CD34+ hematopoietic stem-progenitor cells (HSPCs) and drove βAS3-globin expression at potentially therapeutic levels in erythrocytes differentiated from transduced HSPCs from SCD patients. Transduced HSPCs were transplanted in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG)-immunodeficient mice to analyze biodistribution, chimerism, and transduction efficiency in bone marrow (BM), spleen, thymus, and peripheral blood 12–14 weeks after transplantation. Vector integration site analysis, performed in pre-transplant HSPCs and post-transplant BM cells from individual mice, showed a normal lentiviral integration pattern and no evidence of clonal dominance. An in vitro immortalization (IVIM) assay showed the low genotoxic potential of GLOBE-AS3. This study enables a phase I/II clinical trial aimed at correcting the SCD phenotype in juvenile patients by transplantation of autologous hematopoietic stem cells (HSC) transduced by GLOBE-AS3. Sickle cell disease (SCD) is caused by a mutation (E6V) in the hemoglobin (Hb) β-chain that induces polymerization of Hb tetramers, red blood cell deformation, ischemia, anemia, and multiple organ damage. Gene therapy is a potential alternative to human leukocyte antigen (HLA)-matched allogeneic hematopoietic stem cell transplantation, available to a minority of patients. We developed a lentiviral vector expressing a β-globin carrying three anti-sickling mutations (T87Q, G16D, and E22A) inhibiting axial and lateral contacts in the HbS polymer, under the control of the β-globin promoter and a reduced version of the β-globin locus-control region. The vector (GLOBE-AS3) transduced 60%–80% of mobilized CD34+ hematopoietic stem-progenitor cells (HSPCs) and drove βAS3-globin expression at potentially therapeutic levels in erythrocytes differentiated from transduced HSPCs from SCD patients. Transduced HSPCs were transplanted in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG)-immunodeficient mice to analyze biodistribution, chimerism, and transduction efficiency in bone marrow (BM), spleen, thymus, and peripheral blood 12–14 weeks after transplantation. Vector integration site analysis, performed in pre-transplant HSPCs and post-transplant BM cells from individual mice, showed a normal lentiviral integration pattern and no evidence of clonal dominance. An in vitro immortalization (IVIM) assay showed the low genotoxic potential of GLOBE-AS3. This study enables a phase I/II clinical trial aimed at correcting the SCD phenotype in juvenile patients by transplantation of autologous hematopoietic stem cells (HSC) transduced by GLOBE-AS3. Sickle cell disease (SCD) is caused by a mutation in the sixth amino acid of the hemoglobin (Hb) β-chain (E6V), which causes polymerization of Hb tetramers upon deoxygenation. As a consequence, red blood cells (RBCs) lose flexibility and adopt the characteristic sickle shape in the capillary circulation, causing ischemia, stroke, multi-organ damage, severe pain, chronic hemolytic anemia, and reduced life expectancy.1Stamatoyannopoulos G. The Molecular Basis of Blood Diseases. Third Edition. W.B. Saunders Co., 2001Google Scholar SCD is endemic in Africa and frequent in the Western world: approximately 100,000 individuals in the USA and 10,000 in France are affected by the disease, with an incidence of 1:5,000 and 1:2,500, respectively. Current treatment includes regular blood transfusions and induction of fetal Hb (HbF) synthesis by hydroxyurea.2Madigan C. Malik P. Pathophysiology and therapy for haemoglobinopathies. Part I: sickle cell disease.Expert Rev. Mol. Med. 2006; 8: 1-23Crossref Google Scholar, 3Platt O.S. Hydroxyurea for the treatment of sickle cell anemia.N. Engl. J. Med. 2008; 358: 1362-1369Crossref PubMed Scopus (268) Google Scholar The only definitive therapy for SCD is allogeneic transplantation of hematopoietic stem cells (HSCs) from matched sibling donors, with a reported disease-free survival rate of >90% at 6 years after transplantation.4Locatelli F. Kabbara N. Ruggeri A. Ghavamzadeh A. Roberts I. Li C.K. Bernaudin F. Vermylen C. Dalle J.H. Stein J. et al.Eurocord and European Blood and Marrow Transplantation (EBMT) GroupOutcome of patients with hemoglobinopathies given either cord blood or bone marrow transplantation from an HLA-identical sibling.Blood. 2013; 122: 1072-1078Crossref PubMed Scopus (170) Google Scholar Transplants with matched unrelated or mismatched donors carry progressively higher, unacceptable risks for morbidity and mortality.4Locatelli F. Kabbara N. Ruggeri A. Ghavamzadeh A. Roberts I. Li C.K. Bernaudin F. Vermylen C. Dalle J.H. Stein J. et al.Eurocord and European Blood and Marrow Transplantation (EBMT) GroupOutcome of patients with hemoglobinopathies given either cord blood or bone marrow transplantation from an HLA-identical sibling.Blood. 2013; 122: 1072-1078Crossref PubMed Scopus (170) Google Scholar Given the large variability in clinical severity, the limited availability of suitable donors, and the toxicity associated with the procedure, HSC transplantation is not frequently performed on SCD patients, particularly in the adult age. Gene therapy, i.e., transplantation of autologous HSCs genetically corrected with a lentiviral vector (LV) expressing an anti-sickling globin chain, could be a therapeutic option with less treatment-related morbidity, theoretically available to all patients. Hematopoietic stem-progenitor cells (HSPCs) transduced by LVs have been used in clinical trials of gene therapy for immunodeficiencies, lysosomal storage disorders, and hemoglobinopathies, providing strong evidence of clinical efficacy in the absence of treatment-related adverse events.5Naldini L. Gene therapy returns to centre stage.Nature. 2015; 526: 351-360Crossref PubMed Scopus (837) Google Scholar Allogeneic HSC transplantation provides predictions about the minimal level of corrected HSCs and anti-sickling Hb synthesis necessary to achieve clinical benefit in SCD patients. Stable mixed chimerism with donor HSC levels as low as 10%–30% leads to significant clinical improvement, because of the selective survival of normal, donor-derived RBCs with respect to HbS-containing RBCs.6Andreani M. Testi M. Gaziev J. Condello R. Bontadini A. Tazzari P.L. Ricci F. De Felice L. Agostini F. Fraboni D. et al.Quantitatively different red cell/nucleated cell chimerism in patients with long-term, persistent hematopoietic mixed chimerism after bone marrow transplantation for thalassemia major or sickle cell disease.Haematologica. 2011; 96: 128-133Crossref PubMed Scopus (81) Google Scholar, 7Walters M.C. Patience M. Leisenring W. Rogers Z.R. Aquino V.M. Buchanan G.R. Roberts I.A. Yeager A.M. Hsu L. Adamkiewicz T. et al.Stable mixed hematopoietic chimerism after bone marrow transplantation for sickle cell anemia.Biol. Blood Marrow Transplant. 2001; 7: 665-673Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 8Wu C.J. Gladwin M. Tisdale J. Hsieh M. Law T. Biernacki M. Rogers S. Wang X. Walters M. Zahrieh D. et al.Mixed haematopoietic chimerism for sickle cell disease prevents intravascular haemolysis.Br. J. Haematol. 2007; 139: 504-507Crossref PubMed Scopus (48) Google Scholar On the other hand, SCD patients carrying a hereditary persistence of HbF (HPFH) experience a much milder clinical course or are asymptomatic.9Akinsheye I. Alsultan A. Solovieff N. Ngo D. Baldwin C.T. Sebastiani P. Chui D.H. Steinberg M.H. Fetal hemoglobin in sickle cell anemia.Blood. 2011; 118: 19-27Crossref PubMed Scopus (317) Google Scholar, 10Steinberg M.H. Chui D.H. Dover G.J. Sebastiani P. Alsultan A. Fetal hemoglobin in sickle cell anemia: a glass half full?.Blood. 2014; 123: 481-485Crossref PubMed Scopus (148) Google Scholar Together, these data suggest that engraftment of >20% of autologous HSCs producing an RBC progeny with >30% anti-sickling Hb levels could ameliorate the SCD pathology. Pre-clinical and clinical studies have shown the potential of gene therapy in correcting the SCD phenotype. The LVs used in these studies express either the fetal γ-globin or mutant β-globins that interfere with axial and lateral contacts in the HbS polymer, thereby reducing HbS polymerization and sickling. The anti-sickling genes are expressed under the control of a β-globin promoter and combinations of elements from the β-globin locus control region (LCR) for maximal gene expression.11Ferrari G. Cavazzana M. Mavilio F. Gene therapy approaches to hemoglobinopathies.Hematol. Oncol. Clin. North Am. 2017; 31: 835-852Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar An LV expressing one such mutant, the βAT87Q, is currently used in clinical trials for both β-thalassemia and SCD.12Ribeil J.A. Hacein-Bey-Abina S. Payen E. Magnani A. Semeraro M. Magrin E. Caccavelli L. Neven B. Bourget P. El Nemer W. et al.Gene therapy in a patient with sickle cell disease.N. Engl. J. Med. 2017; 376: 848-855Crossref PubMed Scopus (468) Google Scholar, 13Thompson A.A. Walters M.C. Kwiatkowski J. Rasko J.E.J. Ribeil J.A. Hongeng S. Magrin E. Schiller G.J. Payen E. Semeraro M. et al.Gene therapy in patients with transfusion-dependent β-thalassemia.N. Engl. J. Med. 2018; 378: 1479-1493Crossref PubMed Scopus (397) Google Scholar A second mutant, βAS3 globin, carries the T87Q and two additional mutations, G16D and E22A, which contribute to the anti-sickling activity and increase the affinity of the mutant for the α chain.14Levasseur D.N. Ryan T.M. Reilly M.P. McCune S.L. Asakura T. Townes T.M. A recombinant human hemoglobin with anti-sickling properties greater than fetal hemoglobin.J. Biol. Chem. 2004; 279: 27518-27524Crossref PubMed Scopus (54) Google Scholar βAS3 globin, expressed in the context of an LV, corrects a murine model of SCD15Levasseur D.N. Ryan T.M. Pawlik K.M. Townes T.M. Correction of a mouse model of sickle cell disease: lentiviral/antisickling beta-globin gene transduction of unmobilized, purified hematopoietic stem cells.Blood. 2003; 102: 4312-4319Crossref PubMed Scopus (155) Google Scholar and reduces the level of HbS and RBC sickling at potentially therapeutic levels when transferred in bone marrow (BM)-derived human CD34+ HSPCs from SCD patients.16Romero Z. Urbinati F. Geiger S. Cooper A.R. Wherley J. Kaufman M.L. Hollis R.P. de Assin R.R. Senadheera S. Sahagian A. et al.β-Globin gene transfer to human bone marrow for sickle cell disease.J. Clin. Invest. 2013; 123: 3317-3330Crossref Scopus (86) Google Scholar, 17Urbinati F. Hargrove P.W. Geiger S. Romero Z. Wherley J. Kaufman M.L. Hollis R.P. Chambers C.B. Persons D.A. Kohn D.B. Wilber A. Potentially therapeutic levels of anti-sickling globin gene expression following lentivirus-mediated gene transfer in sickle cell disease bone marrow CD34+ cells.Exp. Hematol. 2015; 43: 346-351Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar Here we present the characterization of an optimized vector (GLOBE-AS3) expressing the human β-AS3 gene under the control of a short human β-globin promoter and a reduced version (HS2+HS3) of the β-globin LCR. This vector design allows high transgene expression from a minimal size transgene, essential for high-titer vector production. As part of the pre-clinical validation of the vector, we analyzed the in vitro correction of the sickle phenotype in SCD patients’ cells, as well as engraftment, biodistribution, and in vivo genotoxicity of transduced human HSPCs from healthy donors after xenotransplantation in an NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse model. A vector integration analysis was carried out before and after transplantation, to analyze the clonal dynamics of transduced cells in vivo, and positive or negative selection of cells harboring integration events in specific genes or classes of genes. This study has been designed to enable a multicenter phase I/II clinical trial aimed at establishing the safety and efficacy of HSPCs transduced with GLOBE-AS3 for gene therapy for SCD. A self-inactivating (SIN) LV (GLOBE-AS3; Figure 1A) was built by cloning the human βAS3-globin gene under the control of a short (264-bp) β-globin promoter and a HS2+HS3 mini LCR in anti-sense orientation in the CCL-SIN-18 LV backbone. The vector was pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) and produced by transient transfection in 293T cells with an average infectious titer of 3.5 × 109 transducing units (TU)/mL. Transduction efficiency was tested on CD34+ HSPCs mobilized with granulocyte-colony stimulating factor (G-CSF) in the peripheral blood (PB) of five healthy donors. HSPCs were transduced at increasing MOI (50, 100, and 200) and maintained for 2 weeks in liquid culture supporting myeloid or erythroid differentiation, or cultured as individual progenitors (colony-forming cells [CFCs]) in semi-solid medium. The average vector copy number (VCN) in the myeloid bulk cultures of transduced CD34+ cells was 0.7 ± 0.2, 0.9 ± 0.3, and 0.9 ± 0.2 (mean ± SEM), respectively, whereas it reached 1.3 ± 0.3, 1.8 ± 0.3, and 1.9 ± 0.3 in erythroid cultures (Figure 1B). The % of transduced individual CFCs (myeloid + erythroid + mixed colonies) was 49% at the highest MOI (Figure 1C). At day 14 of erythroid differentiation, ∼80% of cells were positive for the erythroid markers CD36 and CD235a (data not shown). Cells were collected, total RNA was extracted, and the expression of βAS3 mRNA determined by qRT-PCR as a ratio between βAS3 mRNA and the endogenous α-globin mRNA (HbA). Relative βAS3 expression was correlated to the VCN, achieving between 4% and 30% of the α-globin expression with VCN values ranging from 0.6 to 2.5 (Figure 1D). CD34+ HSPCs were obtained from the BM of seven different SCD patients, transduced with GLOBE-AS3 at different MOIs (25–500), and maintained in erythroid cultures for 3 weeks. The VCN, measured 2 weeks after transduction, ranged between 0.3 and 3.8, with a substantial variability in cells from different donors transduced at similar MOI (Figure 2A), indicating a significant donor-dependent variability in transduction efficiency. After 3 weeks of culture, cells were harvested and the amount of vector-driven HbAS3 protein quantified by high-pressure liquid chromatography (HPLC) in RBC lysates (Figure 2B). A high correlation was observed between VCN and HbAS3 protein production (R = 0.8305; Figure 2C), with an estimated output of 11 ng of HbAS3 per integrated vector copy. The effect of the synthesis of βAS3 globin on the SCD phenotype was tested by an in vitro anti-sickling assay in erythrocytes differentiated in culture from BM CD34+ cells from one SCD donor. CD34+ cells were transduced at MOIs of 45, 150, and 450 and cultured for 3 weeks in erythroid differentiation medium to obtain hemoglobinized, enucleated RBCs. Cells were harvested and incubated in sealed chambers with sodium metabisulfite to induce sickling as previously described.16Romero Z. Urbinati F. Geiger S. Cooper A.R. Wherley J. Kaufman M.L. Hollis R.P. de Assin R.R. Senadheera S. Sahagian A. et al.β-Globin gene transfer to human bone marrow for sickle cell disease.J. Clin. Invest. 2013; 123: 3317-3330Crossref Scopus (86) Google Scholar Cell morphology was then examined under a phase-contrast microscope. RBCs derived from cells transduced with GLOBE-AS3 showed a higher percentage of phenotypically corrected, non-sickled forms compared with RBCs derived from mock-transduced cells from the same donor (Figure 2D). The percentage of phenotypically corrected cells correlated with VCN, reaching a maximum of 34.0% at a VCN of 1.7 (Figure 2D). CD34+ HSPCs were mobilized by G-CSF from three healthy donors, pre-activated overnight with a cytokine cocktail, and either mock-transduced or transduced by two rounds of infection at MOI 100 with GLOBE-AS3 or with a control vector expressing GFP from the human phosphoglycerate kinase promoter (PGK-GFP). We performed two independent transductions, the first with CD34+ cells from one donor (TD1) and the second with cells pooled from two different donors (TD2). An aliquot of cells transduced with GLOBE-AS3 was maintained in liquid culture for a week for VCN evaluation and vector integration analysis or cultured as individual progenitors in semi-solid medium for 2 weeks. A VCN of 2.8 ± 0.2 and 4.7 ± 0.8 was obtained with GLOBE-AS3 and PGK-GFP, respectively, with 51% and 75% of transduced individual progenitors. Cells transduced with PGK-GFP were also analyzed for GFP expression by flow cytometry, resulting in 60.0% ± 9.0% GFP+ cells. After transduction, CD34+ cells were transplanted in sub-lethally irradiated, female NSG recipient mice (10 mice per group) by retro-orbital injection (∼2 × 106 cells/mouse) (Figure 3A). Transplanted mice were maintained for 3 months and monitored weekly for health and body weight. One mouse that received untransduced cells and two mice that received cells transduced with GLOBE-AS3 were sacrificed at an early time point because of loss of weight due to the irradiation, with no significant difference among the three groups (χ2 test, p = 0.3). White blood cell (WBC), RBC, and platelet counts were determined at sacrifice and showed no apparent difference among the three groups (Figure S1A). At the end of the study, human cell chimerism was evaluated in the BM by flow cytometry after labeling with a human-specific anti-CD45 antibody. Engraftment of human cells was comparable in mice receiving cells transduced by the GLOBE-AS3 or by the control vector (Figure 3B). The average VCN/donor cell of the test group was ∼0.4, showing efficient transduction of repopulating stem cells by the GLOBE-AS3 vector (Figure 3B). The average VCN was significantly lower than that obtained in cells transduced with the PGK-GFP vector, reflecting the lower transduction efficiency measured in pre-transplant cells. The presence of human T, B, and NK cells was evaluated by flow cytometry after immunostaining for human CD45+/CD3+, CD45+/CD19+, and CD45+/CD56+ cells, respectively. No significant difference in engraftment of immune cells was detected in the three groups of mice, with animals showing either predominant B cell or predominant T cell engraftment (Figure 3C; mice in the GLOBE-AS3 group are individually identified), a pattern reproduced also in PB and spleen (Figure S1B, top and middle panels). In animals where a thymus could be analyzed, this showed a high proportion of CD4+/CD8+ double-positive T cells, with no difference among the three groups (Figure S1B, bottom panel). As expected for this mouse model, engraftment of CD45+/CD33+ myelomonocytic cells was <10% in all hematopoietic organs (Figures 3C and S1B, top and middle panels), with no significant difference among the three groups. Overall, histopathological analysis of hematopoietic and non-hematopoietic organs revealed comparable size, cellularity, and organ architecture in mice receiving untransduced cells or cells transduced with either vector. No malignancy was observed in any of the groups (data not shown). We tested the insertional mutagenesis potential of the GLOBE-AS3 vector in three independent in vitro immortalization (IVIM) assays.18Modlich U. Bohne J. Schmidt M. von Kalle C. Knöss S. Schambach A. Baum C. Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity.Blood. 2006; 108: 2545-2553Crossref PubMed Scopus (276) Google Scholar, 19Zychlinski D. Schambach A. Modlich U. Maetzig T. Meyer J. Grassman E. Mishra A. Baum C. Physiological promoters reduce the genotoxic risk of integrating gene vectors.Mol. Ther. 2008; 16: 718-725Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar As a positive control for vector-induced immortalization, we included a mutagenic gammaretroviral vector with intact LTRs. Non-transduced cells (Mock) were the negative control. Mutagenic, LTR-driven vectors like RSF91 contain strong enhancer-promoter sequences and can activate proto-oncogenes near the insertion sites.20Stein S. Ott M.G. Schultze-Strasser S. Jauch A. Burwinkel B. Kinner A. Schmidt M. Krämer A. Schwäble J. Glimm H. et al.Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease.Nat. Med. 2010; 16: 198-204Crossref PubMed Scopus (633) Google Scholar In the IVIM assay, these mutants show a proliferation advantage when cells are seeded at very low density. While non-immortalized cells stop growing, insertional mutants can be quantified by their replating phenotype on a 96-well plate. We used a cumulative MOI of 400 in two rounds of transduction for GLOBE-AS3 and achieved a VCN of 6.5 ± 0.5 in 10 out of 11 transductions. One sample had a very low VCN of 0.12 for unknown reason. The positive control RSF91 reached VCN values of 8.8 ± 2.1. After transduction of 1 × 105 starting cells, cultures were expanded for 2 weeks and then re-plated at 100 cells/96-well plate. For RSF91, we observed insertional mutants in seven out of nine transductions with a mean replating frequency (RF) of 4.1 × 10−3. In contrast, the GLOBE-AS3 vector showed a significantly lower incidence of positive plates (2 out of 11; p = 0.022, Fisher’s exact test) and an over 10-fold lower RF in the IVIM assay, which was statistically not different from non-transduced Mock cells (RF = 1.08 × 10−4; p = 0.05, Kruskal-Wallis test with Dunn’s correction) (Figure 4). Hence, GLOBE-AS3 showed a low risk for induction of insertional mutagenesis, even with multiple integrated proviral copies. The global integration characteristics of the GLOBE-AS3 vector were determined on an aliquot of transduced human HSPCs before transplantation. Integration sites (ISs) were recovered by linker-mediated PCR (LM-PCR) followed by high-throughput Illumina sequencing and mapped to the human genome (hg19 assembly) by a previously described, custom-designed bioinformatic pipeline21Poletti V. Charrier S. Corre G. Gjata B. Vignaud A. Zhang F. Rothe M. Schambach A. Gaspar H.B. Thrasher A.J. Mavilio F. Preclinical development of a lentiviral vector for gene therapy of X-linked severe combined immunodeficiency.Mol. Ther. Methods Clin. Dev. 2018; 9: 257-269Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar (Figure 5A). The aim of the analysis was to compare pre- and post-transplantation integration profiles to uncover differences in integration characteristics or frequency and function of the targeted gene pool that may suggest altered dynamics in the hematopoiesis of transplanted mice caused by positive or negative selection of cells harboring specific integration events. The integration profile of GLOBE-AS3 in pre-transplant CD34+ cells was analyzed in each transduction and in the merged TD1+TD2 dataset. Sequencing of the LM-PCR libraries generated 2.6–2.8 million reads/sample, corresponding to about 5.5 million reads in the merged TD1+TD2 dataset. Overall, a total of 41,979 ISs were retrieved from pre-transplant CD34+ cells, with 13,459 and 28,721 unique ISs retrieved from TD1 and TD2 CD34+ cells, respectively (Table 1). The GLOBE-AS3 vector showed the canonical LV genome-wide integration profile in CD34+ cells, with a prevalence of intragenic, mostly intronic ISs (81% in both TD1 and TD2; Figure 5B). Overall, the 41,979 ISs targeted 7,989 genes, corresponding to 28.5% of the annotated human genes. Most of the targeted genes were in common between TD1 and TD2, with the larger dataset (TD2) including the majority (77%) of the genes targeted in the smaller one (TD1) (Figure 5C).Table 1GLOBE-AS3 Integration Sites Retrieved from Pre-transplantation G-CSF-Mobilized PB CD34+ HSPCs and BM Samples from NSG Mice 3 Months after TransplantationSampleHuman Chimerism in BM (% huCD45)VCNNo. of ISsNo. of Target GenesPre-transplant CD34+ cells (merged TD1+TD2)–2.7541,9797,989TD1 CD34+ cellsNA2.613,4594,814TD1 BM26350.5615359TD1 BM27680.8677479TD1 BM28730.3810536TD1 BM29570.41,018651TD1 BM30630.3949598TD1 BM31300.21,190672TD1 BM cells (merged)540.43,8221,970TD2 CD34+ cells–2.928,7216,869TD2 BM32130.72,0361,300TD2 BM33270.81,7291,096TD2 BM34131.91,101720TD2 BM35160.71,013679TD2 BM cells (merged)171.05,3322,559BM cells (merged TD1+TD2)––8,8703,595huCD45, human CD45 antigen. Open table in a new tab huCD45, human CD45 antigen. We then analyzed the genes targeted at a statistically significant (p < 0.001) higher frequency with respect to an in silico-generated random set of integrations in the human genome, after Bonferroni correction for false discovery rate. By this analysis, 266 and 415 genes, respectively, were significantly over-targeted by GLOBE-AS3 in TD1 and TD2, respectively. Most of the genes over-targeted in TD1 (56%) were over-targeted also in TD2 (Figure 5C), and among the top-100 over-targeted genes (by IS frequency), 55 were in common between the TD1 and TD2 datasets (Table S1). The top-100 list contains most of the genes preferentially targeted by LV integration in HSPCs of patients treated by gene therapy for hematological and non-hematological disorders (in bold in Table S1). Among the targeted genes (merged TD1+TD2), several functional categories were enriched by DAVID analysis (Bonferroni corrected, p ≤ 0.001), and included metabolism and transport of proteins, RNA and DNA, chromatin modifications, regulation of gene expression, and mitosis (the top-20 categories are listed in the Table S2). The integration profile of GLOBE-AS3 was then analyzed in the BM of each transplanted mouse in the TD1 and TD2 groups, as well as in the merged TD1+TD2 dataset, and the post-transplantation profiles compared with the corresponding pre-transplant CD34+ datasets. Sequencing of the LM-PCR libraries generated 1–2.3 million reads/sample, corresponding to about 12 million reads in the merged TD1+TD2 dataset (Table 1). We retrieved between 615 and 2,036 ISs from individual BMs, suggesting a highly polyclonal repopulation in all transplanted mice (Table 1). On average, TD2 BMs yielded a higher number of ISs, in line with the higher average VCN observed in these animals and possibly reflecting a better transduction of the TD2 CD34+ cells (Table 1). The global post-transplant LV integration profile showed a prevalence of intragenic ISs (71% in TD1 and 78% in TD2), although their proportion was significantly decreased with respect to the corresponding pre-transplant CD34+ samples (χ2 test, p < 0.0001; Figure 5B), suggesting a negative in vivo selection of cells hosting intragenic integrations. The difference was significant also when considering only the integrations in exons (4.88% in the TD1+TD2 pre-transplant CD34+ cells versus 4.44% in the corresponding post-transplant BM; χ2 test, p < 0.0001; values rounded to 5% and 4% in Figure 5B). No selection for the orientation of the provirus with respect to the target gene was observed in the post- versus pre-transplant samples (Table S3). Most of the genes hosting a GLOBE-AS3 IS in the post-transplant BMs were targeted also in the corresponding pre-transplant CD34+ dataset (68% in TD1 and 85% in TD2; Figure 5D), and no gene was targeted at a statistically higher frequency in BM cells (merged TD1+TD2) with respect to the corresponding pre-transplant samples. Almost half of the targeted genes (Figure 5D) and 20 out of the top-50 targeted genes were in common between the TD1 and TD2 BM samples (Table 2), and 23 (46%) and 29 (58%) of the top-50 targeted genes in TD1 and TD2, respectively, were listed in the top-100 over-targeted genes in the corresponding pre-transplant samples (second and fifth columns in Table 2). These data suggest no substantial positive or negative selection for cells carrying ISs in specific genes after transplantation. Again, most of the genes described as preferentially targeted by LV integration in patients treated by gene therapy are among the 50 top-targeted genes in BM cells (footnote “a” in Table 2).Table 2Top 50 Genes Targeted by GLOBE-AS3 in the BM of NSG Mice Transplanted with the TD1 and TD2 CD34+ Cells, Ranked by Number of Independent ISs per GeneFor each gene, an X indicates that the gene was also targeted in the respective pre-transplant CD34+ cell sample. Genes targeted in both TD1 and TD2 BM samples are highlighted in matching colors. Genes found preferentially targeted by LV integration also in gene therapy patients are indicated in bold. Open table in a new tab For each gene, an X indicates that the gene was also targeted in the respective pre-transplant CD34+ cell sample. Genes targeted in both TD1 and TD2 BM samples are highlighted in matching colors. Genes found preferentially targeted by LV integration also in gene therapy patients are indicated in bold. A comprehensive functional analysis of the targeted genes in BM cells (merged TD1+TD2) performed by DAVID indicated no significant enrichment (Bonferroni-corrected p ≤ 0.01) in functional categories with respect to the pre-transplant CD34+ cells (merged TD1+TD2) except for the category “regulation of GTPase activity” (data not shown). Although no gene was targeted at a statistically higher frequency in TD1, TD2, and merged TD1+TD2 BM cells with respect to the corresponding pre-transplant samples, 34 genes were f" @default.
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• W2898845079 title "Pre-clinical Development of a Lentiviral Vector Expressing the Anti-sickling βAS3 Globin for Gene Therapy for Sickle Cell Disease" @default.
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