662 In Vitro and In Vivo Gene Expression Mediated by the Human Artemis Promoter Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S252 GENE REGULATIO[.]
GENE REGULATION: ORGANOTROPIC APPROACHES - HEART, MUSCLE, LIVER, NEURO, BLOOD 660 Effects of Culture Conditions and Viral Envelope on Gene Expression Profile and Retroviral Vector Integration in Human Hematopoietic CD34+ Cells Amrita Ghosh, Chenwei Wang, Abdel G Elkahloun, Chris Silvin,1 Fabio Candotti.1 NHGRI, NIH, Bethesda, MD 1 Gammaretrovirus-based vectors are known to target actively transcribing genes, indicating that the transcriptional activity of genes in target cells can be predictive of the pattern of vector integrations Therefore, culture conditions that affect gene expression may, in turn, affect vector integration profile In addition, cell-surface receptors for viral envelope proteins are differentially expressed in different cell types, as well as in distinct stages of lineage differentiation and maturation Therefore, vectors carrying different envelopes may target biologically distinct cells, characterized by different gene expression profiles Accordingly, the integration pattern in cells transduced with vectors pseudotyped with different envelopes may be significantly dissimilar The integration patterns in X-SCID patients treated with gene therapy in France and UK have shown differences in the frequency of common integration sites (CIS) that may have resulted from minor differences in (a) culture conditions (60U/ml IL3 and 4% FCS vs 20 U/ml IL3 and 1% HSA) and (b) vector envelope proteins (amphotropic vs GALV) To address this issue, we cultured human bone marrow CD34+ cells from three donors under the French (S1) and UK (S2) stimulation conditions Both groups of cells were transduced with amphotropic and GALV pseudotyped EGFP retroviral vectors after 48, 72, and 96 hours of culture After each transduction EGFP-expressing cells were FACS-sorted Microarray analysis of gene expression in S1 and S2 cells identified culture-specific sets of 1.5-fold upregulated genes Pathway and network analyses of both sets showed enrichment of genes involved in hematopoietic differentiation and cancer However, these functional similarities were seen at different culture time points, suggesting that both S1 and S2 conditions expose similar sets of genes to vector integration but at different transduction times Of note, oncogenes, except for CCND2, activated in human trials (e.g LMO2, BMI1, JUND, and LYL1) were not upregulated compared to unstimulated cells However, all oncogene transcripts were among those expressed at high levels (>75th percentile) on the microarray Interestingly, human cancer genes were significantly enriched among genes with high constitutive expression (p=4.62 × 10-16), suggesting that the risk for oncogene insertional activation could be independent of culture conditions To confirm that gene expression correlates with vector integration, we mapped retroviral integration sites (RIS) in transduced cells Preliminary results showed that 17 out of 18 RIS were within 20 kb of genes that were either upregulated during culture or belonged to the highest quartile of expression These results may help reconciling the apparent discordance between the differences in frequency of CIS in the French and UK trials and the observed similar risk for insertional oncogenesis To address if envelope selection may affect the vector integration profile in target cells, we are performing similar bioinformatic comparisons of genes activated in S1 and S2 transduced cells These studies may have implications in further understanding factors involved in safety for clinical gene therapy S252 661 Engineering XID (Btk) Site Specific Homing Endonucleases for Gene Repair in Hematopoietic Stem Cells Yupeng Wang,1 Jordan Jarjour,1,2 Jim Havranek,3 David Baker,3 Andrew M Scharenberg,1,2 David J Rawlings.1,2 Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA; 2Pediatrics and Immunology, Universtiy of Washington, Seattle, WA; 3Biochemistry, University of Washington, Seattle, WA X-linked agammaglobulinemia (XLA), a human immunodeficiency caused by mutations in Bruton tyrosine kinase (Btk), represents an ideal candidate target for hematopoietic stem cell (HSC) gene therapy However, current gene replacement methods including viral vectors carry risks of insertional mutagenesis as well as gene silencing In contrast, gene repair induced by site specific DNA double strand breaks offers a novel means to treat genetic disease with potentially less risk and the potential to utilize endogenous control elments for gene expression LAGLIDADG Homing Endonucleases (LHEs) have been shown to efficiently induce site specific double strand breaks and markerless modification of genes without toxicity However, the limited number of site specific LHEs currently available limits their application in gene therapy Thus, engineering site specific LHEs represents a critical step for enabling technology for double strand break-induced gene modification In the current study we have begun to generate a site specific variant of the LHE I-AniI, targeted to recognize a unique mutation in the mouse BTK gene in the XID model of human XLA Our approaches combine: protein computational design with random mutation and selection using yeast library surface display; or alternatively, iterative somatic hypermutation and selection within DT40 B cell library targeted to the light chain locus Using these combinatorial methods we have generated a panel of I-AniI variants with novel DNA binding and cleavage specificity towards the XID site Preliminary characterization of a subset of these I-AniI variants using cell surface binding and cleavage, structural studies, and in vitro biochemical analysis will be presented Ultimately, new I-Anil variants (for induction of site specific double strand breaks) and donor DNA repair template will be delivered into into lin- HSC (or B cell progenitors) derived from XID/Tec-/- mice using non-integrating lentiviral vectors; and evaluated for capacity to re-engraft and reconstitute B lineage development and function in vivo 662 In Vitro and In Vivo Gene Expression Mediated by the Human Artemis Promoter Megan M Multhaup,1 Sweta Gurram,1 Kelly M Podetz-Pedersen,1 Andrea D Karlen,1 Debra L Swanson,1 Nikunj V Somia,1 Perry B Hackett,1 Morton J Cowan,2 R Scott McIvor.1 Department of Genetics, Cell Biology, and Development, The University of Minnesota, Minneapolis, MN; 2Department of Pediatrics, The University of California, San Francisco, CA Artemis is an endonucleolytic hairpin opening enzyme involved in nonhomologous end joining and repair of double strand breaks formed by ionizing radiation and by cellular processes such as V(D) J recombination Deficiency of Artemis results in a radiosensitive severe combined immunodeficiency (SCID-A) characterized by the inability to arrange immunoglobulin and T cell receptor genes, ultimately resulting in a B- T- NK+ phenotype We generated several lentiviral vector constructs for transduction of the human Artemis sequence into hematopoietic stem cells of Artemis-deficient mice as a model for correction of human SCID-A Transduction by a lentiviral vector in which Artemis is regulated by a strong EF1α promoter resulted in a dose-dependent loss of cell viability and global DNA damage that was not observed in cultures exposed to identical amounts of control vector Upon further investigation, it was found Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy GENE SILENCING that toxicity was diminished when Artemis was regulated by a lower strength PGK promoter ( Molecular Therapy 16(S1) S226, 2008) These results demonstrate the necessity of establishing conditions that provide Artemis expression at a level that is non-toxic but nonetheless sufficient to complement Artemis deficiency In this study, we recovered the human Artemis promoter region and explored the possibility of employing innate regulation of human Artemis cDNA using its own endogenous promoter sequence A one kilobase DNA sequence upstream of the human Artemis gene was PCR amplified from genomic DNA to recover the Artemis promoter (APro) The one kb APro sequence conferred significant expression in vitro when luciferase expression constructs were transfected into HEK 293 cells Deletion constructs were tested to identify the minimal sequence required for human Artemis promoter activity, and the 5’ end of the Artemis message was mapped by 5’-RACE The effectiveness of the APro sequence in mediating in vivo expression was tested by transduction with a lentiviral vector containing an APro-regulated green fluorescent protein (GFP) sequence After transplantation with APro-GFP transduced donor marrow, GFP expression was observed in several hematopoietic lineages of recipient mice at reduced mean fluorescence intensity compared to control mice transplanted with EF1α-GFP transduced cells APro-regulated GFP expression was sustained in secondary transplant recipients We conclude that the human Artemis promoter provides moderate levels of expression in vitro and in vivo after lentiviral transduction into murine hematopoietic stem cells This promoter will therefore be useful for the purpose of establishing a clinical vector that provides Artemis expression at a non-toxic level that is nonetheless sufficient to correct the B-T- SCID-A phenotype Gene Silencing 663 Effect of siRNA in PEG-Coated siRNALipoplex on the Anti-PEG IgM Production as Induced by the PEG-Coated siRNA-Lipoplex Tatsuaki Tagami,1 Kazuya Nakamura,1 Taro Shimizu,1 Tatsuhiro Ishida,1 Hiroshi Kiwada.1 Department of Pharmacokinetics and Biopharmaceutics, Subdivision of Biopharmaceutical Sciences, Institute of Health Biosciences, The University of Tokushima, Tokushima, Japan Polyethylene glycol (PEG) has been used to prolong the circulation time of drug carriers such as liposome, polymer and nanoparticle The universal function of PEG in this case is the prevention of PEGcoated substances from recognition by immune system, especially by macrophages in the mononuclear phagocyte system (MPS) PEG is believed to be immunologically inert, but we and other researchers found that repeated injection of PEG-coated liposome with several days interval induced a reduction of blood circulation of second dose This phenomenon is called the “accelerated blood clearance (ABC) phenomenon” We recently clarified that anti-PEG IgM production induced by the first injection of PEG-coated liposome is the major cause of the phenomenon In the present study, we investigated the issue of whether siRNA in PEG-coated siRNA-lipoplex (PSCL) effects on the anti-PEG IgM production as induced by the PSCL following intravenous injection of the lipoplex We found that PSCL produced anti-PEG IgM production and consequently attenuated the blood circulation time of second dose PEG-coated naked cationic liposome (PCL) (the ABC phenomenon) Anti-PEG IgM responses to PSCL were inversely related to the PSCL dose Interestingly, anti-PEG IgM responses were significantly lower for PSCL than for PCL The studies with splenectomized mice and nude mice indicated that anti-PEG IgM response was closely related to an interaction of PSCL and PCL with the spleen in a T cell-independent manner In addition, PSCL induced apoptosis on IgM-expressing splenic cells more strongly than PCL, suggesting that siRNA in the PSCL rather Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy attenuated anti-PEG IgM production by causing apoptosis in the splenic cells including B cells In conclusion, our study indicating that PEG-coated cationic liposome (PCL) needs to be used with caution for applications involving siRNA-based therapeutics, increases the importance of evaluating the carrier-mediated effect in siRNA delivery system 664 Generation of Epstein Barr Virus Specific Cytotoxic T Lymphocytes (EBV-CTLs) Resistant to the Immunosuppressive Drug Tacrolimus (FK506) Biagio De Angelis,1 Gianpietro Dotti,1 Concetta Quintarelli,1 Leslie E Huye,1 Lan Zhang,1 Ming Zhang,1 Helen E Heslop,1 Malcolm K Brenner,1 Cliona M Rooney,1 Barbara Savoldo.1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX Adoptive transfer of autologous EBV-CTLs to hematopoietic stem cell transplant and solid organ transplant recipients is safe and effective for prevention and treatment of EBV-associated post transplant lymphoproliferative disorders (PTLD) However, CTLs expansion, persistence and efficacy can be limited by immunosuppressive drugs, which can often be tapered in patients developing PTLD, but not completely withdrawn due to the risk of graft rejection One of the most used immunosuppressive agents is FK506 whose effects are highly dependent on binding of FKBP12 proteins, since T cells generated from FKBP12 knockout mice are completely resistant to the inhibitory effects of FK506 We therefore hypothesize that EBV-CTLs can be rendered resistant to FK506 by knocking down FKBP12, using a small interfering RNA (siRNA) stably expressed from a retroviral vector After extensive screening of potential target sequences, we identified by Western blotting that siRNA4 knocked down>90% of FKBP12 in CTLs We then generated two retroviral vector encoding for siRNA4/eGFP and irrelevant siRNA/eGFP, respectively These vectors were used to transduce established CTLs generated from EBV-seropositive donors Transduction efficiency was 46%±22% for siRNA4 and 55%±27% for irrelevant-siRNA We measured the proliferation of transduced CTLs in the presence of FK506, in short term and long term cultures Using a thymidine uptake assay, we found that the inhibition of proliferation by increasing concentrations of FK506 was significantly diminished in siRNA4+ CTLs compared to control CTLs (41%±4% inhibition vs 74%±2%, respectively) To evaluate the effects of knocking down FKBP12 in long-term cultures, control and siRNA4+CTLs were stimulated weekly with autologous LCL, low dose IL-2 (20U/mL) and in the presence or absence of FK506 (5ng/ml) The proportion of siRNA4+ CTLs increased over time not only as a percentage of GFP+ cells (from 46%±22% to 89%±5% after stimulations) but also numerically (median fold expansion: 39, range 5-111) In contrast, control EBVCTLs did not show any selection in culture, since the percentage of GFP+ cells remained unchanged (from 55%±27% to 57%±23%) and CTLs ceased to proliferate (median fold expansion: 2, range 0-11) In addition, siRNA4+ CTLs kept their MHC-restricted cytotoxic activity, assessed by Cr release assay (66%±22% killing of autologous LCL S253 ... identify the minimal sequence required for human Artemis promoter activity, and the 5’ end of the Artemis message was mapped by 5’-RACE The effectiveness of the APro sequence in mediating in vivo expression. .. sustained in secondary transplant recipients We conclude that the human Artemis promoter provides moderate levels of expression in vitro and in vivo after lentiviral transduction into murine hematopoietic... region and explored the possibility of employing innate regulation of human Artemis cDNA using its own endogenous promoter sequence A one kilobase DNA sequence upstream of the human Artemis gene