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546. In Vitro SFHR-Mediated Modification of Cftr Locus in Mouse Embryonic Stem Cells
LIPID MEDIATED GENE TRANSFER dialysis was less than 200nm similar to that obtained with Ca+2. Mn+2 was also tried but it yielded no trapping of plasmid and the resultant liposomes were large forming large aggregates. The trapping efficiencies were evaluated both qualitatively and quantitatively using agarose gel DNA retardation assay and fluorophore/DNA binding assay respectively. Transfection using the NLCs formed with Ca+2 was further evaluated in vitro using Human umbilical vein endothelial cells (HUVECs) for targeted gene expression and a luciferase expression plasmid to quantitate expression levels. For this purpose NLCs containing CDMRGDMFC-DOPE /DOPE/DOPC with 0.1mol% NBD-DOPE was used. CDMRGDMFC is an RGD peptide sequence in which one of the disulfide bonds have been removed. Fluorescent micrographs showed that the lipopeptide targeted NLCs were inside endosomes as evidenced by the periplasmic accumulation of punctate fluorescence. However, luciferase expression was barely above background. Thus indicating that rate-limiting steps such as escape from the endosome and nuclear uptake of the released plasmid need to be overcome. To address the first step, lipid formulations that are fusogenic at acidic pH are being evaluated with this system. The lipids being evaluated are: 1,2-Dioleoyl-sn-Glycero-3-Phophoethanolamine-N-Citraconyl, 1,2Dioleoyl-sn-Glycero-3-Posphoethanolamine-N- (succinyl) (Sodium Salt) and 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N(glutaryl) (Sodium Salt) in combination with DOPE and targeting lipopeptide. Plasmid DNA is labeled using CX-Rhodamine to follow intracellular release. This synthetic system mimics cell entry and release of genetic material similar to that of a virus but without the problematic side effects.
546. In Vitro SFHR-Mediated Modification of Cftr Locus in Mouse Embryonic Stem Cells Monica Lais,1 Federica Sangiuolo,1 Alessia Ranciaro,1 Lucia Scaldaferri,2 AnnaLucia Serafino,3 Dieter Gruenert,4 Massimo De Felici,2 Giuseppe Novelli.1 1 Biopathology and Diagnostic for Imaging, Tor Vergata University, Rome, Italy; 2Public Health and Cellular Biology, Tor Vergata University, Rome, Italy; 3Institute of Experimental Medicine, CNR, Rome, Italy; 41Human Molecular Genetics, Department of Medicine, University of Vermont, Burlington, VT. Cystic Fibrosis (CF) is a lethal genetic disease resulting in a reduced Cl- permeability caused by local misfolding of CFTR channel (Cystic Fibrosis Transmembrane Conductance Regulator). We modified Cftr locus in mouse D3 embryonic stem cells introducing a 3-bp deletion (ΔF508 mutation) by SFHR technique (Small Fragment Homologous Replacement). Transfected small DNA fragments (SDF) contain both the 3 bp deletion and an Kpn I restriction site. About 2 millions cells were transfected with fragment previously complexed with cationic liposome (Gene Porter, GTS) at different liposome to DNA charge ratio (+/-) from 16/1 to 6.1/1 (+/-). Confocal analysis confirmed the presence of DNA fragments, labelled by the fluorochrome Cy5, into the nucleus of transfected cells, 72 hours after transfection. Modification at the appropriate genomic locus by DNA fragment introduction and expression of the modified CFTR mRNA was detected by allele-specific polymerase chain reaction (PCR) amplification and subsequent restriction analysis. Positive results were obtained only from cells transfected at 7.4/1 and 6.1/1 (+/-) charge ratio. Results were confirmed by cloning and sequencing amplicons from RT-PCR of transfected cells. Quantitative PCR (Applied Biosystem 7000) and Western Blot analysis are in progress to estimate the percentage of gene modification and the defective protein respectively. Our results encourage in the application of SFHR technique for treating genetic diseases by ex-vivo gene targeting in pluripotent stem cells. Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
547. Strategies for Incorporation of NonClassical Nuclear Localization Signal Sequences into Non-Viral Delivery Systems That Promote High Transfection without Cell Division Kenneth J. Longmuir,1 Eric A. Murphy,1 Alan J. Waring,2 Sherry M. Haynes.1 1 Physiology & Biophysics, University of California, Irvine, CA, United States; 2Department of Medicine, University of California, Los Angeles, CA, United States. We have previously reported the development of multicomponent non-viral gene delivery systems composed of peptides, zwitterionic lipid, and PEG. At this meeting we report that incorporation of non-classical nuclear localization signal sequences allows non-viral complexes to achieve high transfection without cell division. In particular, we incorporate the peptide sequence NEWTLELLEELKNEAVRHF, which corresponds to amino acids 16-34 of viral protein r (“vpr”) of the pre-integration complex of HIV-1. The nuclear targeting mechanism of this non-classical NLS is fundamentally different from the classical NLS’s, such as SV40. The non-classical NLS sequence does not require importin α or β, nor does it require energy, to promote transport into the nucleus. Recent evidence also indicates that vpr binds directly to nucleoporins of the nuclear pore complex. We use a basic delivery system of plasmid DNA with EGFP reporter gene, a seven-fold charge excess of a DNA compacting peptide, and 5 nmol of dimyristoleoylphosphatidylethanolamine per μg DNA. HeLa cells were growth-arrested for 24 hours with 5 mM hydroxyurea. Addition of the vpr peptide to the delivery system (5 nmol/μg DNA) resulted in equally efficient transfection of both dividing and growth-arrested HeLa cells after 12 hours (dividing cells 44.5 ± 4.3% positive for GFP; growth-arrested cells 48.0 ± 0.9% positive for GFP; statistically the same). Incorporation of the vpr peptide into our delivery complexes is driven by hydrophobic interactions. For a different assembly strategy, we added 15 amino acids composed of the repeating sequence KKP to the n-terminal end of the vpr peptide. This “kkp-vpr” peptide allows for direct electrostatic interaction of the vpr non-classical sequence with the plasmid DNA. Using the delivery system described above, high transfection (cells positive for GFP at 12 h) was also observed in both dividing and growtharrested HeLa cells. We incorporated the kkp-vpr peptide into complexes of EGFP DNA and 25 Kd branched PEI (N/P ratio of 10 for PEI, N/P ratio of 2.5 for peptide) and incubated with both dividing and growth-arrested HeLa cells for 12 hours. Equally efficient transfection was observed in dividing HeLa cells with or without the kkp-vpr peptide (35-40%). For growth-arrested HeLa cells, the percentages were 21.9 ± 1.9% for the complexes with the peptide, and only 7.2 ± 1.5% positive for complexes without the peptide (statistically significant p<0.001). In contrast, we observed no enhancement of transfection of growth-arrested HeLa cells by the vpr sequence when it was combined with the cationic lipid system Lipofectamine. Hence, the overall chemistry of non-viral gene delivery systems significantly influences the effectiveness of a non-classical NLS. Finally, we have found that complexes containing the kkp-vpr peptide show high transfection activity after extrusion through either 200, 100, 80, or 50 nm Nuclepore filters, and do not re-aggregate upon incubation with serum. The ability of these small complexes to transfect cells without cell division makes this system a strong candidate for development for systemic gene delivery in adult animal models. President, Lipomer, Inc.
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546. In Vitro SFHR-Mediated Modification of Cftr Locus in Mouse Embryonic Stem Cells Monica Lais,1 Federica Sangiuolo,1 Alessia Ranciaro,1 Lucia Scaldaferri,2 AnnaLucia Serafino,3 Dieter Gruenert,4 Massimo De Felici,2 Giuseppe Novelli.1 1 Biopathology and Diagnostic for Imaging, Tor Vergata University, Rome, Italy; 2Public Health and Cellular Biology, Tor Vergata University, Rome, Italy; 3Institute of Experimental Medicine, CNR, Rome, Italy; 41Human Molecular Genetics, Department of Medicine, University of Vermont, Burlington, VT. Cystic Fibrosis (CF) is a lethal genetic disease resulting in a reduced Cl- permeability caused by local misfolding of CFTR channel (Cystic Fibrosis Transmembrane Conductance Regulator). We modified Cftr locus in mouse D3 embryonic stem cells introducing a 3-bp deletion (ΔF508 mutation) by SFHR technique (Small Fragment Homologous Replacement). Transfected small DNA fragments (SDF) contain both the 3 bp deletion and an Kpn I restriction site. About 2 millions cells were transfected with fragment previously complexed with cationic liposome (Gene Porter, GTS) at different liposome to DNA charge ratio (+/-) from 16/1 to 6.1/1 (+/-). Confocal analysis confirmed the presence of DNA fragments, labelled by the fluorochrome Cy5, into the nucleus of transfected cells, 72 hours after transfection. Modification at the appropriate genomic locus by DNA fragment introduction and expression of the modified CFTR mRNA was detected by allele-specific polymerase chain reaction (PCR) amplification and subsequent restriction analysis. Positive results were obtained only from cells transfected at 7.4/1 and 6.1/1 (+/-) charge ratio. Results were confirmed by cloning and sequencing amplicons from RT-PCR of transfected cells. Quantitative PCR (Applied Biosystem 7000) and Western Blot analysis are in progress to estimate the percentage of gene modification and the defective protein respectively. Our results encourage in the application of SFHR technique for treating genetic diseases by ex-vivo gene targeting in pluripotent stem cells. Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
547. Strategies for Incorporation of NonClassical Nuclear Localization Signal Sequences into Non-Viral Delivery Systems That Promote High Transfection without Cell Division Kenneth J. Longmuir,1 Eric A. Murphy,1 Alan J. Waring,2 Sherry M. Haynes.1 1 Physiology & Biophysics, University of California, Irvine, CA, United States; 2Department of Medicine, University of California, Los Angeles, CA, United States. We have previously reported the development of multicomponent non-viral gene delivery systems composed of peptides, zwitterionic lipid, and PEG. At this meeting we report that incorporation of non-classical nuclear localization signal sequences allows non-viral complexes to achieve high transfection without cell division. In particular, we incorporate the peptide sequence NEWTLELLEELKNEAVRHF, which corresponds to amino acids 16-34 of viral protein r (“vpr”) of the pre-integration complex of HIV-1. The nuclear targeting mechanism of this non-classical NLS is fundamentally different from the classical NLS’s, such as SV40. The non-classical NLS sequence does not require importin α or β, nor does it require energy, to promote transport into the nucleus. Recent evidence also indicates that vpr binds directly to nucleoporins of the nuclear pore complex. We use a basic delivery system of plasmid DNA with EGFP reporter gene, a seven-fold charge excess of a DNA compacting peptide, and 5 nmol of dimyristoleoylphosphatidylethanolamine per μg DNA. HeLa cells were growth-arrested for 24 hours with 5 mM hydroxyurea. Addition of the vpr peptide to the delivery system (5 nmol/μg DNA) resulted in equally efficient transfection of both dividing and growth-arrested HeLa cells after 12 hours (dividing cells 44.5 ± 4.3% positive for GFP; growth-arrested cells 48.0 ± 0.9% positive for GFP; statistically the same). Incorporation of the vpr peptide into our delivery complexes is driven by hydrophobic interactions. For a different assembly strategy, we added 15 amino acids composed of the repeating sequence KKP to the n-terminal end of the vpr peptide. This “kkp-vpr” peptide allows for direct electrostatic interaction of the vpr non-classical sequence with the plasmid DNA. Using the delivery system described above, high transfection (cells positive for GFP at 12 h) was also observed in both dividing and growtharrested HeLa cells. We incorporated the kkp-vpr peptide into complexes of EGFP DNA and 25 Kd branched PEI (N/P ratio of 10 for PEI, N/P ratio of 2.5 for peptide) and incubated with both dividing and growth-arrested HeLa cells for 12 hours. Equally efficient transfection was observed in dividing HeLa cells with or without the kkp-vpr peptide (35-40%). For growth-arrested HeLa cells, the percentages were 21.9 ± 1.9% for the complexes with the peptide, and only 7.2 ± 1.5% positive for complexes without the peptide (statistically significant p<0.001). In contrast, we observed no enhancement of transfection of growth-arrested HeLa cells by the vpr sequence when it was combined with the cationic lipid system Lipofectamine. Hence, the overall chemistry of non-viral gene delivery systems significantly influences the effectiveness of a non-classical NLS. Finally, we have found that complexes containing the kkp-vpr peptide show high transfection activity after extrusion through either 200, 100, 80, or 50 nm Nuclepore filters, and do not re-aggregate upon incubation with serum. The ability of these small complexes to transfect cells without cell division makes this system a strong candidate for development for systemic gene delivery in adult animal models. President, Lipomer, Inc.
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