Secreted Gaussia luciferase as a sensitive reporter gene for in vivo and ex vivo studies of airway gene transfer.
The cationic lipid GL67A is one of the more efficient non-viral gene transfer agents (GTAs) for the lungs, and is currently being evaluated in an extensive clinical trial programme for cystic fibrosis gene therapy. Despite conferring significant expression of vector-specific mRNA following transfection of differentiated human airway cells cultured on air liquid interfaces (ALI) cultures and nebulisation into sheep lung in vivo we were unable to detect robust levels of the standard reporter gene Firefly luciferase (FLuc). Recently a novel secreted luciferase isolated from Gaussia princeps (GLuc) has been described. Here, we show that (1) GLuc is a more sensitive reporter gene and offers significant advantages over the traditionally used FLuc in pre-clinical models for lung gene transfer that are difficult to transfect, (2) GL67A-mediated gene transfection leads to significant production of recombinant protein in these models, (3) promoter activity in ALI cultures mimics published in vivo data and these cultures may, therefore, be suitable to characterise promoter activity in a human ex vivo airway model and (4) detection of GLuc in large animal broncho-alveolar lavage fluid and serum facilitates assessment of duration of gene expression after gene transfer to the lungs. In summary, we have shown here that GLuc is a sensitive reporter gene and is particularly useful for monitoring gene transfer in difficult to transfect models of the airway and lung. This has allowed us to validate that GL67A, which is currently in clinical use, can generate significant amounts of recombinant protein in fully differentiated human air liquid interface cultures and the ovine lung in vivo.
Department of Gene Therapy, Imperial College at the National Heart and Lung Institute, Manresa Road, London SW3 6LR, UK. u.griesenbach@imperial.ac.uk
Uta
U
Griesenbach
Catarina C
CC
Vicente
Megan J
MJ
Roberts
Cuixiang
C
Meng
Samia
S
Soussi
Stefania
S
Xenariou
Peter
P
Tennant
Alison
A
Baker
Eilidh
E
Baker
Catherine
C
Gordon
Christina
C
Vrettou
Dominique
D
McCormick
Rebecca
R
Coles
Anne-Marie
AM
Green
Anna E
AE
Lawton
Stephanie G
SG
Sumner-Jones
Seng H
SH
Cheng
Ronald K
RK
Scheule
Stephen C
SC
Hyde
Deborah R
DR
Gill
David D
DD
Collie
Gerry
G
McLachlan
Eric W F W
EW
Alton
D000818:Animals; D001992:Bronchoalveolar Lavage Fluid; D002478:Cells, Cultured; D004560:Electricity; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D008055:Lipids / Q000737:chemistry; D008156:Luciferases / Q000097:blood / Q000235:genetics* / Q000378:metabolism*; D008168:Lung / Q000378:metabolism*; D051379:Mice; D011094:Polyethyleneimine / Q000737:chemistry; D011401:Promoter Regions, Genetic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012756:Sheep; D013997:Time Factors; D014162:Transfection; D014780:Viruses / Q000235:genetics; D051598:Whole Body Imaging
D008055:Lipids; D011094:Polyethyleneimine; D008156:Luciferases