Optimization of in vitro vascular cell transfection with non-viral vectors for in vivo applications.
BACKGROUND
Syngeneic vascular cells are interesting tools for indirect gene therapy in the cardiovascular system. This study aims to optimize transfection conditions of primary cultures of vascular smooth muscle cells (VSMCs) using different non-viral vectors and zinc as an adjuvant and to implant these transfected cells in vivo.


METHODS
Non-liposomal cationic vectors (FuGene 6), polyethylenimines (ExGen 500), and histidylated polylysine (HPL) were used as non-viral vectors in vitro with secreted alkaline phosphatase (SEAP) as reporter gene. Transfection efficiency was compared in cultured rat, rabbit and human VSMCs and fibroblasts. Zinc chloride (ZnCl2) was added to optimize transfection of rat VSMCs in vitro which were then seeded in vivo.


RESULTS
Much higher SEAP levels were obtained in rabbit cells with FuGene 6 (p <0.0001) at day 2 than in equivalent rat and human cells. Rat VSMCs transfected in vitro with FuGene 6 and ExGen 500 expressed higher SEAP levels than with HPL. In rat VSMCs, SEAP secretion was more than doubled by addition of 250 microM ZnCl2 (p <0.0001) for all vectors. Seeding of syngeneic VSMCs transfected under optimized conditions (FuGene 6/pcDNA3-SEAP +250 microM ZnCl2) into healthy Lewis rats using various routes or into post-infarct myocardial scar resulted in a peak of SEAP expression at day 2 and detectable activity in the plasma for at least 8 days.


CONCLUSIONS
FuGene 6 is an efficient non-viral transfection reagent for gene transfer in somatic smooth muscle cells in vitro and ZnCl2 enhances its efficiency. This increased expression of the transgene product is maintained after seeding in vivo.
Elmadbouh
Ibrahim
I
INSERM Unit 460, Cardiovascular Remodeling, CHU Xavier Bichat, Paris, France.
Rossignol
Patrick
P
Meilhac
Olivier
O
Vranckx
Roger
R
Pichon
Chantal
C
Pouzet
Bruno
B
Midoux
Patrick
P
Michel
Jean-Baptiste
JB
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001011:Aorta / Q000378:metabolism; D002412:Cations; D002470:Cell Survival; D002478:Cells, Cultured; D002712:Chlorides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000378:metabolism; D017930:Genes, Reporter; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D032389:Myocytes, Smooth Muscle / Q000166:cytology; D010957:Plasmids / Q000378:metabolism; D011094:Polyethyleneimine / Q000378:metabolism; D011107:Polylysine / Q000737:chemistry; D011817:Rabbits; D051381:Rats; D011917:Rats, Inbred Lew; D013997:Time Factors; D014162:Transfection; D015032:Zinc / Q000737:chemistry; D017967:Zinc Compounds / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
D002412:Cations; D002712:Chlorides; D017967:Zinc Compounds; D011107:Polylysine; C016837:zinc chloride; D011094:Polyethyleneimine; D000469:Alkaline Phosphatase; D015032:Zinc