Optimization of adenoviral vector-mediated transgene expression in the canine brain in vivo, and in canine glioma cells in vitro.
Expression of the immune-stimulatory molecule Fms-like tyrosine kinase 3 ligand (Flt3L) and the conditional cytotoxic enzyme herpes simplex virus type 1 thymidine kinase (HSV1-TK) provides long-term immune-mediated survival of large glioblastoma multiforme (GBM) models in rodents. A limitation for predictive testing of novel antiglioma therapies has been the lack of a glioma model in a large animal. Dogs bearing spontaneous GBM may constitute an attractive large-animal model for GBM, which so far has remained underappreciated. In preparation for a clinical trial in dogs bearing spontaneous GBMs, we tested and optimized adenovirus-mediated transgene expression with negligible toxicity in the dog brain in vivo and in canine J3T glioma cells. Expression of the marker gene beta-galactosidase (beta-Gal) was higher when driven by the murine (m) than the human (h) cytomegalovirus (CMV) promoter in the dog brain in vivo, without enhanced inflammation. In the canine brain, beta-Gal was expressed mostly in astrocytes. beta-Gal activity in J3T cells was also higher with the mCMV than the hCMV promoter driving tetracycline-dependent (TetON) transgene expression within high-capacity adenovirus vectors (HC-Ads). Dog glioma cells were efficiently transduced by HC-Ads expressing mCMV-driven HSV1-TK, which induced 90% reduction in cell viability in the presence of ganciclovir. J3T cells were also effectively transduced with HC-Ads expressing Flt3L under the control of the regulatable TetON promoter system, and as predicted, Flt3L release was stringently inducer dependent. HC-Ads encoding therapeutic transgenes under the control of regulatory sequences driven by the mCMV promoter are excellent vectors for the treatment of spontaneous GBM in dogs, which constitute an ideal preclinical animal model.
Candolfi
Marianela
M
Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90069, USA.
Pluhar
G Elizabeth
GE
Kroeger
Kurt
K
Puntel
Mariana
M
Curtin
James
J
Barcia
Carlos
C
Muhammad
A K M Ghulam
AK
Xiong
Weidong
W
Liu
Chunyan
C
Mondkar
Sonali
S
Kuoy
William
W
Kang
Terry
T
McNeil
Elizabeth A
EA
Freese
Andrew B
AB
Ohlfest
John R
JR
Moore
Peter
P
Palmer
Donna
D
Ng
Phillip
P
Young
John D
JD
Lowenstein
Pedro R
PR
Castro
Maria G
MG
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D001921:Brain / Q000502:physiology*; D001932:Brain Neoplasms / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D003587:Cytomegalovirus / Q000235:genetics; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay; D005818:Genetic Engineering / Q000379:methods; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D005910:Glioma / Q000235:genetics* / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D011401:Promoter Regions, Genetic; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic; D019076:Transgenes / Q000502:physiology*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D008565:Membrane Proteins; D014764:Viral Proteins; C084881:flt3 ligand protein; D011505:Protein-Tyrosine Kinases; D001616:beta-Galactosidase