title: Functional delivery of large genomic DNA to human cells with a peptide-lipid vector.
abstract: BACKGROUND
  Nonviral gene transfer vectors have the potential to deliver much larger DNA constructs than current viral vectors but suffer from a low transfection efficiency. The LID vector, composed of Lipofectin (L), an integrin-targeting peptide (I) and DNA (D), is a highly efficient synthetic vector, both in vitro and in vivo, which may allow the transfer of genomic loci for gene therapy.
  
  
  METHODS
  Transfection efficiencies were quantitated using the green fluorescent protein (GFP) reporter. Expression of a large genomic locus (NBS1 [Nijmegen breakage syndrome], encoding nibrin) was assessed by immunofluorescence.
  
  
  RESULTS
  We report a systematic study of the parameters influencing delivery of BAC-based plasmids ranging in size from 12 to 242 kb using the LID vector. We showed 60% of cells were transfected with the smaller plasmids while plasmids up to 242 kb were consistently delivered to over 10% of cells. The number of transfected cells was related to number of plasmids in the transfection complex independent of plasmid size. Atomic force microscopy showed that LID particle size increased with plasmid size consistent with one plasmid molecule per particle. When LID vectors were used to deliver the NBS1 gene as a 143 kb construct to primary NBS cells, at least 57% of cells expressing GFP also expressed functional nibrin.
  
  
  CONCLUSIONS
  We show that LID vectors represent a promising tool for the transfer of complete genomic loci.
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
authors.forename: Robert E
authors.initials: RE
authors.lastname: White
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
authors.forename: Richard
authors.initials: R
authors.lastname: Wade-Martins
authors.affiliation: Molecular Immunology Unit, Institute of Child Health, University College London, 30 Guildford St., London WC1N 1EH, UK.
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Hart
authors.affiliation: Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, UK.
authors.forename: Jon
authors.initials: J
authors.lastname: Frampton
authors.affiliation: Department of Materials, Parks Road, Oxford, UK.
authors.forename: Bryan
authors.initials: B
authors.lastname: Huey
authors.affiliation: Molecular Genetics Program, Virginia Mason Research Centre, and Department of Immunology, University of Washington School of Medicine, Seattle, Washington, USA.
authors.forename: Ami
authors.initials: A
authors.lastname: Desai-Mehta
authors.affiliation: Molecular Genetics Program, Virginia Mason Research Centre, and Department of Immunology, University of Washington School of Medicine, Seattle, Washington, USA.
authors.forename: Karen M
authors.initials: KM
authors.lastname: Cerosaletti
authors.affiliation: Molecular Genetics Program, Virginia Mason Research Centre, and Department of Immunology, University of Washington School of Medicine, Seattle, Washington, USA.
authors.forename: Patrick
authors.initials: P
authors.lastname: Concannon
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
authors.forename: Michael R
authors.initials: MR
authors.lastname: James
mesh_terms: D018797:Cell Cycle Proteins / Q000235:genetics; D002478:Cells, Cultured; D022202:Chromosomes, Artificial, Bacterial; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D016023:Integrins / Q000235:genetics; D008164:Luminescent Proteins; D009687:Nuclear Proteins / Q000235:genetics; D010455:Peptides / Q000235:genetics; D010714:Phosphatidylethanolamines / Q000235:genetics; D010957:Plasmids; D014162:Transfection / Q000379:methods*
chemical_list: D018797:Cell Cycle Proteins; D016023:Integrins; D008164:Luminescent Proteins; C112156:NBN protein, human; D009687:Nuclear Proteins; D010455:Peptides; D010714:Phosphatidylethanolamines; D049452:Green Fluorescent Proteins; C020888:1,2-dielaidoylphosphatidylethanolamine