Polybrene improves transfection efficacy of recombinant replication-deficient adenovirus in cutaneous cells and burned skin.
BACKGROUND
The hostile environment found in acute and chronic wounds decreases the physiological half-life of purified synthetic or recombinant peptides dramatically. Gene therapy, on the other hand, may be a viable option since it relies on the cellular machinery of the host to locally manufacture the proteins of interest. The aim of this study was to evaluate and optimize the local administration of transient cutaneous adenoviral gene delivery in wounds.


METHODS
Primary human keratinocytes (HKC) and HaCaT cells were transfected with replication-deficient adenovirus (Ad5) containing the reporter gene for beta-galactosidase (LacZ). The vector was used alone or precoated with either (1) Lipofectamine 2000, (2) FuGENE 6, or (3) Polybrene. For in vivo testing a rat burn model was used. Animals were randomized into three groups: (1) Ad5-LacZ alone; (2) Ad5-LacZ precoated with Polybrene, or (3) carrier control (phosphate-buffered saline (PBS)). Samples were harvested from burned and unburned tissue sections after either 48 h or 7 days. Transgene expression was quantified by bioluminometric assay and localized using immunohistochemistry. A BrdU assay was performed to determine the influence of the used transfection reagents on cell proliferation.


RESULTS
Transfection efficacy was significantly improved in vitro (p < 0.001) as well as in partial thickness burned (p = 0.015) and unburned skin (p > 0.001) after precoating Ad5 with Polybrene compared to Ad5 alone. Transgene expression was 10-fold higher in burned skin (9305 pg/mg protein) compared to unburned skin (859 pg/mg protein).


CONCLUSIONS
It is feasible to improve transfection efficacy in vitro and in vivo by precoating the adenovirus with Polybrene.
Jacobsen
F
F
Department for Plastic Surgery, Burn Center, BG University Hospital Bergmannsheil, Ruhr University Bochum, Bochum, Germany.
Hirsch
T
T
Mittler
D
D
Schulte
M
M
Lehnhardt
M
M
Druecke
D
D
Homann
H H
HH
Steinau
H U
HU
Steinstraesser
L
L
D000256:Adenoviridae; D000818:Animals; D002056:Burns / Q000378:metabolism* / Q000473:pathology; D002460:Cell Line; D017930:Genes, Reporter; D015316:Genetic Therapy; D005822:Genetic Vectors; D006583:Hexadimethrine Bromide; D006801:Humans; D008055:Lipids; D051381:Rats; D012867:Skin / Q000166:cytology / Q000378:metabolism / Q000473:pathology; D013997:Time Factors; D014161:Transduction, Genetic
D008055:Lipids; D006583:Hexadimethrine Bromide