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• W2054127079 abstract "Comparative integrome analysis has revealed that the most neutral integration pattern among retroviruses is attributed to alpharetroviruses. We chose X-linked chronic granulomatous disease (X-CGD) as model to evaluate the potential of self-inactivating (SIN) alpharetroviral vectors for gene therapy of monogenic diseases. Therefore, we combined the alpharetroviral vector backbone with the elongation factor-1α short promoter, both considered to possess a low genotoxic profile, to drive transgene (gp91phox) expression. Following efficient transduction transgene expression was sustained and provided functional correction of the CGD phenotype in a cell line model at low vector copy number. Further analysis in a murine X-CGD transplantation model revealed gene-marking of bone marrow cells and oxidase positive granulocytes in peripheral blood. Transduction of human X-CGD CD34+ cells provided functional correction up to wild-type levels and long-term expression upon transplantation into a humanized mouse model. In contrast to lentiviral vectors, no aberrantly spliced transcripts containing cellular exons fused to alpharetroviral sequences were found in transduced cells, implying that the safety profile of alpharetroviral vectors may extend beyond their neutral integration profile. Taken together, this highlights the potential of this SIN alpharetroviral system as a platform for new candidate vectors for future gene therapy of hematopoietic disorders. Comparative integrome analysis has revealed that the most neutral integration pattern among retroviruses is attributed to alpharetroviruses. We chose X-linked chronic granulomatous disease (X-CGD) as model to evaluate the potential of self-inactivating (SIN) alpharetroviral vectors for gene therapy of monogenic diseases. Therefore, we combined the alpharetroviral vector backbone with the elongation factor-1α short promoter, both considered to possess a low genotoxic profile, to drive transgene (gp91phox) expression. Following efficient transduction transgene expression was sustained and provided functional correction of the CGD phenotype in a cell line model at low vector copy number. Further analysis in a murine X-CGD transplantation model revealed gene-marking of bone marrow cells and oxidase positive granulocytes in peripheral blood. Transduction of human X-CGD CD34+ cells provided functional correction up to wild-type levels and long-term expression upon transplantation into a humanized mouse model. In contrast to lentiviral vectors, no aberrantly spliced transcripts containing cellular exons fused to alpharetroviral sequences were found in transduced cells, implying that the safety profile of alpharetroviral vectors may extend beyond their neutral integration profile. Taken together, this highlights the potential of this SIN alpharetroviral system as a platform for new candidate vectors for future gene therapy of hematopoietic disorders. Chronic granulomatous disease (CGD) is a rare congenital immunodeficiency disorder characterized by the inability of phagocytes to eliminate ingested pathogens. The underlying cause of the disease is any of several mutations in the NADPH oxidase enzyme complex resulting in deficient antimicrobial activity of phagocytes. This multiprotein complex plays a pivotal role in microbial killing by reducing molecular oxygen to superoxide, which subsequently reacts to form reactive oxygen species (ROS). The genetic defect renders the patient susceptible to severe, recurrent, and life threatening bacterial and fungal infections, eventually leading to therapy-refractory granuloma and abscess formation even under prophylaxis treatment with antibiotics and antimycotics.1Segal BH Veys P Malech H Cowan MJ Chronic granulomatous disease: lessons from a rare disorder.Biol Blood Marrow Transplant. 2011; 17: S123-S131Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar Allogeneic hematopoietic stem cell transplantation is to date the only curative option for patients with an human leukocyte antigen-matched donor, when conventional care and therapy fail.2Seger RA Modern management of chronic granulomatous disease.Br J Haematol. 2008; 140: 255-266Crossref PubMed Scopus (198) Google Scholar In most cases (~65%) the X-chromosomal CYBB gene encoding for gp91phox, a subunit of the NADPH oxidase, is affected (X-CGD).3Johnston Jr, RB Clinical aspects of chronic granulomatous disease.Curr Opin Hematol. 2001; 8: 17-22Crossref PubMed Scopus (140) Google Scholar Therefore, reconstitution of oxidase activity by gene delivery of an intact gp91phox to autologous hematopoietic stem cells (HSCs) is a reasonable approach for the treatment of gp91phox-deficient patients lacking a suitable matched stem cell donor. In 2004, our group initiated a phase I/II clinical trial using a gammaretroviral, long-terminal repeat (LTR) driven vector encoding for gp91phox. Autologous CD34+ cells were transduced ex vivo and the genetically modified cells were re-infused into two patients.4Ott MG Schmidt M Schwarzwaelder K Stein S Siler U Koehl U et al.Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1.Nat Med. 2006; 12: 401-409Crossref PubMed Scopus (984) Google Scholar At first, a clear clinical benefit was observed as revealed by the resolution of therapy-resistant lesions in lung and liver. However, long-term observations revealed the occurrence of clonal dominance and a steady decline in transgene expression due to vector silencing. Furthermore, insertional activation of EVI1 led to genomic instability and to myelodysplasia with monosomy 7 in both patients.5Stein S Ott MG Schultze-Strasser S Jauch A Burwinkel B Kinner A 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 Taken together with the outcomes of other clinical trials for primary immunodeficiencies using LTR-driven gammaretroviral vectors (as reviewed elsewhere),6Rivat C Santilli G Gaspar HB Thrasher AJ Gene therapy for primary immunodeficiencies.Hum Gene Ther. 2012; 23: 668-675Crossref PubMed Scopus (51) Google Scholar these serious adverse events emphasize the need for new approaches for therapeutic gene transfer with enhanced safety profile. We report here on the evaluation of a recently developed replication-deficient alpharetroviral self-inactivating (SIN) vector with a split packaging system7Suerth JD Maetzig T Galla M Baum C Schambach A Self-inactivating alpharetroviral vectors with a split-packaging design.J Virol. 2010; 84: 6626-6635Crossref PubMed Scopus (54) Google Scholar,8Suerth JD Maetzig T Brugman MH Heinz N Appelt JU Kaufmann KB et al.Alpharetroviral self-inactivating vectors: long-term transgene expression in murine hematopoietic cells and low genotoxicity.Mol Ther. 2012; 20: 1022-1032Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar in the context of X-CGD as a paradigmatic approach in hematopoietic gene therapy. Integration site analysis had revealed that infection with avian sarcoma and leukosis virus (ASLV), an alpharetroviral subfamily, results in a relatively neutral integration pattern as compared with lenti- and gammaretroviral vectors which show a high preference to either integrate into the proximity of transcription start sites or into active genes, respectively.9Mitchell RS Beitzel BF Schroder AR Shinn P Chen H Berry CC et al.Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences.PLoS Biol. 2004; 2: E234Crossref PubMed Scopus (775) Google Scholar,10Narezkina A Taganov KD Litwin S Stoyanova R Hayashi J Seeger C et al.Genome-wide analyses of avian sarcoma virus integration sites.J Virol. 2004; 78: 11656-11663Crossref PubMed Scopus (136) Google Scholar,11Derse D Crise B Li Y Princler G Lum N Stewart C et al.Human T-cell leukemia virus type 1 integration target sites in the human genome: comparison with those of other retroviruses.J Virol. 2007; 81: 6731-6741Crossref PubMed Scopus (137) Google Scholar Combined with the SIN design, the alpharetroviral vectors are expected to have a reduced risk of insertional mutagenesis. In addition, alpharetroviruses are per se replication-deficient in mammalian cells,12Barsov EV Hughes SH Gene transfer into mammalian cells by a Rous sarcoma virus-based retroviral vector with the host range of the amphotropic murine leukemia virus.J Virol. 1996; 70: 3922-3929PubMed Google Scholar high titers can be obtained and even transduction of nondividing cells has been reported.13Greger JG Katz RA Taganov K Rall GF Skalka AM Transduction of terminally differentiated neurons by avian sarcoma virus.J Virol. 2004; 78: 4902-4906Crossref PubMed Scopus (20) Google Scholar Previous evaluation of SIN alpharetroviral vectors already met most expectations: first, enhancer activity was abolished by the SIN LTR configuration in a luciferase reporter assay.7Suerth JD Maetzig T Galla M Baum C Schambach A Self-inactivating alpharetroviral vectors with a split-packaging design.J Virol. 2010; 84: 6626-6635Crossref PubMed Scopus (54) Google Scholar Even more importantly, in an in vitro immortalization assay Suerth et al.8Suerth JD Maetzig T Brugman MH Heinz N Appelt JU Kaufmann KB et al.Alpharetroviral self-inactivating vectors: long-term transgene expression in murine hematopoietic cells and low genotoxicity.Mol Ther. 2012; 20: 1022-1032Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar observed a reduced incidence of immortalized cells upon transduction of lineage negative (Lin−) murine bone marrow cells with a SIN alpharetroviral vector expressing enhanced green fluorescent protein (eGFP). In addition, most of the immortalized cells showed a reduced fitness in comparison with immortalized cells arising from transduction with gamma- and lentiviral vectors. In vivo evaluation of a SIN alpharetroviral vector driving eGFP under the transcriptional control of the spleen focus forming virus promoter (AS.SFFV.eGFP.PRE*) demonstrated not only the long-term repopulation capacity of SIN alpharetroviral vector transduced cells in a mouse transplantation model but also revealed a favorable integration pattern as compared with lenti- and gammaretroviral vectors, with long-term maintenance of multilineage transgene expression.8Suerth JD Maetzig T Brugman MH Heinz N Appelt JU Kaufmann KB et al.Alpharetroviral self-inactivating vectors: long-term transgene expression in murine hematopoietic cells and low genotoxicity.Mol Ther. 2012; 20: 1022-1032Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Besides the pivotal role of the integration profile in determining the genotoxicity potential of integrating vectors, aberrant splicing of cellular transcripts into the integrated vector backbone has recently emerged as an additional cause of adverse effects. In a β-thalassemia clinical gene therapy trial as well as in a murine model aberrantly spliced products of endogenous transcripts fused to vector sequences led to either proto-oncogene upregulation or haploinsufficiency of a tumor suppressor gene, respectively.14Moiani A Paleari Y Sartori D Mezzadra R Miccio A Cattoglio C et al.Lentiviral vector integration in the human genome induces alternative splicing and generates aberrant transcripts.J Clin Invest. 2012; 122: 1653-1666Crossref PubMed Scopus (115) Google Scholar,15Cesana D Sgualdino J Rudilosso L Merella S Naldini L Montini E Whole transcriptome characterization of aberrant splicing events induced by lentiviral vector integrations.J Clin Invest. 2012; 122: 1667-1676Crossref PubMed Scopus (94) Google Scholar,16Cavazzana-Calvo M Payen E Negre O Wang G Hehir K Fusil F et al.Transfusion independence and HMGA2 activation after gene therapy of human ß-thalassaemia.Nature. 2010; 467: 318-322Crossref PubMed Scopus (1037) Google Scholar Noteworthy, in most cases engagement of splice sites within the leader sequence contributed to abnormally spliced transcripts.14Moiani A Paleari Y Sartori D Mezzadra R Miccio A Cattoglio C et al.Lentiviral vector integration in the human genome induces alternative splicing and generates aberrant transcripts.J Clin Invest. 2012; 122: 1653-1666Crossref PubMed Scopus (115) Google Scholar,15Cesana D Sgualdino J Rudilosso L Merella S Naldini L Montini E Whole transcriptome characterization of aberrant splicing events induced by lentiviral vector integrations.J Clin Invest. 2012; 122: 1667-1676Crossref PubMed Scopus (94) Google Scholar The lack of canonical splice sites in the SIN alpharetroviral vectors' leader sequence may further contribute to the safety profile of this type of vectors.7Suerth JD Maetzig T Galla M Baum C Schambach A Self-inactivating alpharetroviral vectors with a split-packaging design.J Virol. 2010; 84: 6626-6635Crossref PubMed Scopus (54) Google Scholar Here, we demonstrate that SIN alpharetroviral vectors generate high titers and stable gp91phox expression, sufficient to functionally rescue NADPH oxidase activity in the human X-CGD PLB-985 cell line as well as in primary murine and human X-CGD HSCs, even at low vector copy numbers (VCN). In addition, no induction of aberrantly spliced transcripts upon intronic integration was observed in PLB-XCGD cells. Transplantation of transduced Lin− bone marrow HSCs in a murine model of X-CGD resulted in reconstitution of ROS-producing neutrophils in peripheral blood. Myeloid differentiation of transduced primary human X-CGD CD34+ cells either in vitro or after transplantation into a humanized mouse model led to the reconstitution of gp91phox expression and NADPH oxidase activity in the range of wild-type cells. Taken together, this report supports the application of SIN alpharetroviral vectors to rescue a monogenetic disease phenotype upon transplantation of ex vivo modified HSC. To obtain an optimal SIN alpharetroviral construct providing stable and high expression of gp91phox, we combined three previously described elements known to enhance transgene expression in vitro and in vivo: first, we chose a codon-optimized transgene (gp91s), which was shown to improve viral titers and expression levels,17Moreno-Carranza B Gentsch M Stein S Schambach A Santilli G Rudolf E et al.Transgene optimization significantly improves SIN vector titers, gp91phox expression and reconstitution of superoxide production in X-CGD cells.Gene Ther. 2009; 16: 111-118Crossref PubMed Scopus (52) Google Scholar thus reducing the number of vector copies required for an effective functional rescue of CGD. For the same reasons, an optimized posttranslational regulatory element (oPRE) devoid of any open reading frames and X protein promoter activity was placed downstream of the gp91phox coding region.18Schambach A Bohne J Baum C Hermann FG Egerer L von Laer D et al.Woodchuck hepatitis virus post-transcriptional regulatory element deleted from X protein and promoter sequences enhances retroviral vector titer and expression.Gene Ther. 2006; 13: 641-645Crossref PubMed Scopus (120) Google Scholar Finally, we chose the human elongation factor-1α promoter in its intronless, 240 bp short version (EFS)19Schambach A Bohne J Chandra S Will E Margison GP Williams DA et al.Equal potency of gammaretroviral and lentiviral SIN vectors for expression of O6-methylguanine-DNA methyltransferase in hematopoietic cells.Mol Ther. 2006; 13: 391-400Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar to drive transgene expression. This regulatory element drives physiological expression levels without perturbing the expression of genes proximal to the vector integration site.20Zychlinski D Schambach A Modlich U Maetzig T Meyer J Grassman E et al.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,21Zhou S Mody D DeRavin SS Hauer J Lu T Ma Z et al.A self-inactivating lentiviral vector for SCID-X1 gene therapy that does not activate LMO2 expression in human T cells.Blood. 2010; 116: 900-908Crossref PubMed Scopus (97) Google Scholar,22Lombardo A Cesana D Genovese P Di Stefano B Provasi E Colombo DF et al.Site-specific integration and tailoring of cassette design for sustainable gene transfer.Nat Methods. 2011; 8: 861-869Crossref PubMed Scopus (263) Google Scholar Furthermore, the EFS promoter was shown to be less prone to downregulation than other promoters within the context of lentiviral vectors.23Zhang F Frost AR Blundell MP Bales O Antoniou MN Thrasher AJ A ubiquitous chromatin opening element (UCOE) confers resistance to DNA methylation-mediated silencing of lentiviral vectors.Mol Ther. 2010; 18: 1640-1649Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar We evaluated the performance of this SIN alpharetroviral vector (AS.EFS.gp91s, Figure 1a) in comparison with its corresponding SIN lentiviral vector analogue LV.EFS.gp91s. Transduction efficiencies as well as vector titers were comparable between both constructs reaching 2.56 ± 0.88 × 108 T.U./ml (n = 5) for AS.EFS.gp91s (LV.EFS.gp91s: 2.53 ± 0.49 × 108 T.U./ml) as estimated after transduction of the human myeloid PLB-XCGD cell line24Zhen L King AA Xiao Y Chanock SJ Orkin SH Dinauer MC Gene targeting of X chromosome-linked chronic granulomatous disease locus in a human myeloid leukemia cell line and rescue by expression of recombinant gp91phox.Proc Natl Acad Sci USA. 1993; 90: 9832-9836Crossref PubMed Scopus (183) Google Scholar (Supplementary Figure S1c online). The levels of gp91phox expression, as estimated from the mean fluorescence intensity (MFI), were similar for both vectors at comparable mean vector copy numbers (0.39 ± 0.23 vs. 0.42 ± 0.05 for AS.EFS.gp91s and LV.EFS.gp91s, respectively) (Figure 1b) with an estimate of more than 90% of the integrated copies expressing the gp91s transgene (AS.EFS.gp91s 90.2 ± 2.0% vs. LV.EFS.gp91s 91.1 ± 5.9%; P = 0.88, Figure 1c and Supplementary Figure S1d online). As expected, the percentage of gp91phox expressing PLB-XCGD cells and vector copy numbers increased with increasing multiplicity of infection (Figure 1d and Supplementary Figure S1a,b online). Noteworthy, the mean fluorescence intensities (MFI) of the gp91phox positive populations and hence the expression levels were in the range of the parental wild-type cell line PLB-985, even at low VCNs (Figure 1d). Analysis of transduced clonal populations obtained by limiting dilution revealed expression levels independent of VCN and cell differentiation state (Figure 1e), suggesting that maximal gp91phox expression can be achieved with a single AS.EFS.gp91s integrant. Since a previous report implicated that retroviral delivery of gp91phox was prone to silencing in PLB-XCGD cells,25Zentilin L Qin G Tafuro S Dinauer MC Baum C Giacca M Variegation of retroviral vector gene expression in myeloid cells.Gene Ther. 2000; 7: 153-166Crossref PubMed Scopus (35) Google Scholar we monitored alpharetroviral gp91phox expression in this cell line over 150 days. In order to minimize the risk of masking silencing due to expression from several vector copies per cell, PLB-XCGD cells were transduced at a low multiplicity of infection of 0.1 followed by immunomagnetic sorting of gp91phox-expressing cells resulting in a cell population with an average vector copy number of 1.3. Long-term analysis of this population revealed stable expression levels (MFI) and percentages of gp91phox positive cells (Figure 2a,b), recapitulating similar observations made with an alpharetroviral vector expressing eGFP under the transcriptional control of the EFS promoter.8Suerth JD Maetzig T Brugman MH Heinz N Appelt JU Kaufmann KB et al.Alpharetroviral self-inactivating vectors: long-term transgene expression in murine hematopoietic cells and low genotoxicity.Mol Ther. 2012; 20: 1022-1032Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Upon differentiation and phorbol myristate acetate stimulation of the transduced and sorted PLB-XCGD cells restoration of NADPH oxidase activity to levels similar to that of the wild-type cell line (P = 0.498) were observed as assessed by superoxide production over time in a quantitative cytochrome C reduction assay (Figure 2c). Even after 150 day culture, no significant changes in superoxide production (P = 0.645) were observed in this population after differentiation and subsequent functional analysis (Figure 2c). Taken together, the AS.EFS.gp91s vector not only provides efficient transduction and long-term stable gp91phox expression but also complete functional rescue of the X-CGD phenotype at low VCN in a model X-CGD cell line in vitro. We also assessed the performance of the SIN alpharetroviral vector in comparison with its lentiviral equivalent in terms of functional activity. Therefore, we chose two sorted gp91phox+ cell populations with similar mean VCN (alpha: 1.27 vs. LV: 1.30; Supplementary Figure S1d online) and subjected them to differentiation and subsequent cytochrome C assay. Both cell populations revealed comparable kinetics for superoxide production close to wild-type levels (Figure 2d). Superoxide production in cells transduced with AS.EFS.gp91s was equivalent to that of LV.EFS.gp91s transduced cells (5.3 vs. 5.5 nmol/min 106 cells−1, respectively). Hence, the vectors were not only comparable in terms of titer and expression levels, but also in terms of functional activity. Since aberrantly spliced cellular transcripts have recently been detected caused by lentiviral vector integrations, we addressed the occurrence of aberrant splicing in cells transduced with SIN alpharetroviral vectors. In contrast to lentiviral vectors, SIN alpharetroviral vectors are devoid of canonical splice sites in the leader region,7Suerth JD Maetzig T Galla M Baum C Schambach A Self-inactivating alpharetroviral vectors with a split-packaging design.J Virol. 2010; 84: 6626-6635Crossref PubMed Scopus (54) Google Scholar suggesting the absence of cellular exon–vector fusion transcripts in cells transduced with these vectors. However, recent reports have revealed that cryptic splice sites are also engaged in splicing leading to aberrant cellular exon–vector fusion transcripts.14Moiani A Paleari Y Sartori D Mezzadra R Miccio A Cattoglio C et al.Lentiviral vector integration in the human genome induces alternative splicing and generates aberrant transcripts.J Clin Invest. 2012; 122: 1653-1666Crossref PubMed Scopus (115) Google Scholar,15Cesana D Sgualdino J Rudilosso L Merella S Naldini L Montini E Whole transcriptome characterization of aberrant splicing events induced by lentiviral vector integrations.J Clin Invest. 2012; 122: 1667-1676Crossref PubMed Scopus (94) Google Scholar,26Heckl D Schwarzer A Haemmerle R Steinemann D Rudolph C Skawran B et al.Lentiviral vector induced insertional haploinsufficiency of Ebf1 causes murine leukemia.Mol Ther. 2012; 20: 1187-1195Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar Therefore, we analyzed the SIN alpharetroviral leader sequence in silico for cryptic splice sites using the NetGene2 software (http://www.cbs.dtu.dk/services/NetGene2/) as this procedure led to the identification of cryptic splice sites in the leader region of lentiviral vectors involved in aberrant splicing.15Cesana D Sgualdino J Rudilosso L Merella S Naldini L Montini E Whole transcriptome characterization of aberrant splicing events induced by lentiviral vector integrations.J Clin Invest. 2012; 122: 1667-1676Crossref PubMed Scopus (94) Google Scholar In total, no splice donor and seven cryptic splice acceptors with intermediate score (0.2 < score < 0.5) were predicted within the alpharetroviral leader sequence (Table 1). To identify potential aberrantly spliced transcripts in alpharetrovirally transduced PLB-XCGD cells, a clonal population harboring five well-defined intronic integrations was selected for this analysis. A similar procedure was followed for the analysis of aberrant splicing in lentivirally transduced cells (Table 2).Table 1Internal vector splice sitesVectorNameNetGene2 score (>0. 20)aHighly confident acceptor and donor sites (H) ↑ ≥95%; nearly all true acceptor (donor) sites ≥ 20% (50%).StrandSequenceAberrant transcriptsAlphaSAa10. 31+ ACCGTCGGAQAGGAGCTCCAG-SAa20. 34+ TCGGAGGGAGACTCCAGGGCC-SAa30. 43+ GGAGCTCCAG^GGCCCGGAGC-SAa40. 45+ GGGCCCGGAG^CGACTGACCC-SAa1r0. 25- GATAGACGAG^ACGGATGGAG-SAa2r0. 34- CTCCCGATAG^ACGAGACGGA-SAa3r0. 34- CCTCCACCAG^GGTCATCGAA-LVSD10. 83+ GCGGCGACTG^GTGAGTACGC+SA2 (C)0. 28+ ATCGTTTCAG^ACCCACCTCC+SA4(SA7)-+ AAATCTCTAG^CAGTGGCGCC+Lentiviral splice site nomenclature according to Moiani et al. 14 and Cesana et al15. The strand in which the splice site is located is indicated by “+” for the positive strand and “-” for the negative strand. Splice sites are indicated by “^” in the sequence. Presence or absence of aberrant transcripts is indicated according to RT-PCR.a Highly confident acceptor and donor sites (H) ↑ ≥95%; nearly all true acceptor (donor) sites ≥ 20% (50%). Open table in a new tab Table 2Intronic vector integrations in monoclonal PLB-XCGDVectorNameGeneVector orientation to geneIntegration site (UCSC browseraBLAT by UCSC Genome Browser on Human February 2009 (GRCh37/hg19) Assembly.)Gene expression (RT-PCR)AlphaA1MYB+chr6: 135523691+A2UBAC2+chr13: 99912281-A3MAML3+chr4: 140929380+A4ZKSCAN2+chr16: 25262924+A5ADAMTS17-chr15: 100728200+LVL1PSMD14-chr2: 162, 214, 323+L2ZFAND2A-chr7: 1, 196, 636+L3HNRNPR+chr1: 23, 665, 489+L4ZNF655+chr7: 99, 164, 741+L5ZFR+chr5: 32, 406, 083+a BLAT by UCSC Genome Browser on Human February 2009 (GRCh37/hg19) Assembly. Open table in a new tab Lentiviral splice site nomenclature according to Moiani et al. 14 and Cesana et al15. The strand in which the splice site is located is indicated by “+” for the positive strand and “-” for the negative strand. Splice sites are indicated by “^” in the sequence. Presence or absence of aberrant transcripts is indicated according to RT-PCR. First, we asked if the genes targeted by the integration of alpharetroviral or lentiviral vectors were actively transcribed. Using RT-PCR primers spanning exons flanking the integration sites, transcripts were detected for all targeted loci with exception of A2 (Table 2; Supplementary Figures S2a,b online). For the detection of cellular exon–vector fusion transcripts in transduced cells RT-PCR primers spanning the upstream exon adjacent to the integration sites and the in silico predicted splice acceptor sites for the alpharetroviral vector were selected, whereas previously published primer sets15Cesana D Sgualdino J Rudilosso L Merella S Naldini L Montini E Whole transcriptome characterization of aberrant splicing events induced by lentiviral vector integrations.J Clin Invest. 2012; 122: 1667-1676Crossref PubMed Scopus (94) Google Scholar were used for the evaluation of aberrant splicing in lentivirally transduced cells (Figure 3a,b; Supplementary Table S1 online). In alpharetrovirally transduced cells no cellular exon–vector fusion transcripts were detectable (Supplemenatry Figure S2c online). Using the same cDNA sample, sustained expression of the targeted genes was confirmed by PCR (Supplementary Figure S2a online), indicating that the lack of exon–vector fusion transcripts was not due to inefficient cDNA synthesis. On the contrary, cellular exon–vector fusion transcripts were detected (and confirmed by sequencing) in all LV integrants orientated in sense to the targeted gene (Figure 3b and Supplementary Figure S2d online). We then extended our analysis to polyclonal PLB-XCGD populations. Therefore, we chose two transduced populations that matched in VCN of either AS.EFS.gp91s or LV.EFS.gp91s (VCN: 2.92 and 2.94, respectively) after FACS sorting of gp91phox positive cells. A 5′RACE was performed to recover aberrantly spliced transcripts originating from endogenous genes spliced to cryptic or canonical splice sites within the proviral leader sequences as described above. Transfected HEK293T cells served as positive control for this approach (Supplementary Figure S2e,f online). In line with our previous findings, no RT-PCR products containing cellular exons fused to alpharetroviral sequences were found in AS.EFS.gp91s transduced cells whereas multiple cellular exon–LV fusion transcripts were identified in LV.EFS.gp91s transduced cells (Figure 3c and Supplementary Figure S2g and Tables S2–S4 online). The abundance of lentivirally induced aberrant splice products recovered by these approaches as compared with the lack of detectable cellular exon–vector fusion transcripts in SIN alpharetroviral vector transduced cells indicate that the contribution of the alpharetroviral vector backbone to splice-mediated genotoxicity may be lower than that observed for lentiviral vectors. Next we transduced murine Lin− cells obtained from X-CGD mice with AS.EFS.gp91s at a moderate multiplicity of infection of 15. This resulted in 33% gp91phox positive cells as measured 5 days after transduction by flow cytometry (Figure 4a). Transduction of hematopoietic precursors was confirmed by superoxide positive colonies in an nitroblue tetrazolium (NBT) assay obtained after plating transduced cells in semi-solid media. The percentage of NBT-positive colonies matched with 35 ± 6% (n = 4) the initial gp91phox expression in Lin− cells (Figure 4a,b). Moreover, five out of seven (71.4%) provirus positive colonies showed functional reconstitution of superoxide activity (Figure 4c). The lack of superoxide detection in the two vector positive colonies can be explained by incomplete myeloid differentiation and/or low gp91phox expression. Please note that only 30% of the hematopoietic colonies derived from wild-type" @default.
• W2054127079 created "2016-06-24" @default.
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• W2054127079 title "Alpharetroviral Vector-mediated Gene Therapy for X-CGD: Functional Correction and Lack of Aberrant Splicing" @default.
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