Functional delivery of large genomic DNA to human cells with a peptide-lipid vector.
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.
White
Robert E
RE
Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
Wade-Martins
Richard
R
Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
Hart
Stephen L
SL
Molecular Immunology Unit, Institute of Child Health, University College London, 30 Guildford St., London WC1N 1EH, UK.
Frampton
Jon
J
Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, UK.
Huey
Bryan
B
Department of Materials, Parks Road, Oxford, UK.
Desai-Mehta
Ami
A
Molecular Genetics Program, Virginia Mason Research Centre, and Department of Immunology, University of Washington School of Medicine, Seattle, Washington, USA.
Cerosaletti
Karen M
KM
Molecular Genetics Program, Virginia Mason Research Centre, and Department of Immunology, University of Washington School of Medicine, Seattle, Washington, USA.
Concannon
Patrick
P
Molecular Genetics Program, Virginia Mason Research Centre, and Department of Immunology, University of Washington School of Medicine, Seattle, Washington, USA.
James
Michael R
MR
Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
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*
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