title: Circumventing recombination events encountered with production of a clinical-grade adenoviral vector with a double-expression cassette.
abstract: Delivery of multiple exogenous genes into target cells is important for a broad range of gene therapy applications, including combined therapeutic gene expression and noninvasive imaging. Previous studies ( Mol Ther 4: 223-231, 2001 ) have described the adenoviral vector RGDTKSSTR with a double-expression cassette that encodes herpes simplex virus thymidine kinase (HSVtk) for molecular chemotherapy and human somatostatin receptor subtype-2 (hSSTR2) for indirect imaging. In this vector, both genes are inserted in place of the E1 region of the adenoviral genome and expressed independently from two cytomegalovirus (CMV) promoters. During production of clinical-grade RGDTKSSTR, we found that the CMV promoters and simian virus 40 (SV40) poly(A) regions located in both expression cassettes provoked homologous recombination and deletion of one of the cassettes. To resolve this problem, we designed a strategy for substituting the duplicate promoters and poly(A) regions. We placed the hSSTR2 gene in the new Ad5.SSTR/TK.RGD vector under the control of a CMV promoter with a bovine growth hormone poly(A) region, whereas the SV40 promoter, enhancer, and poly(A) signal controlled HSVtk expression. This use of different regulatory sequences allowed independent expression of both transgenes from a single adenoviral vector and circumvented the recombination problem. Reconstruction of the vector with a double-expression cassette enables its use in human clinical trials.
authors.affiliation: Division of Human Gene Therapy, Gene Therapy Center, The University of Alabama at Birmingham, 901-19th Street, South, BMR2-502, 35249-2184, USA.
authors.forename: Natalya
authors.initials: N
authors.lastname: Belousova
authors.forename: Raymond
authors.initials: R
authors.lastname: Harris
authors.forename: Kurt
authors.initials: K
authors.lastname: Zinn
authors.forename: Mary Ann
authors.initials: MA
authors.lastname: Rhodes-Selser
authors.forename: Alexander
authors.initials: A
authors.lastname: Kotov
authors.forename: Olga
authors.initials: O
authors.lastname: Kotova
authors.forename: Minghui
authors.initials: M
authors.lastname: Wang
authors.forename: Rosemarie
authors.initials: R
authors.lastname: Aurigemma
authors.forename: Zeng B
authors.initials: ZB
authors.lastname: Zhu
authors.forename: David T
authors.initials: DT
authors.lastname: Curiel
authors.forename: Ronald D
authors.initials: RD
authors.lastname: Alvarez
mesh_terms: D000256:Adenoviridae / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D002478:Cells, Cultured; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D016679:Genome, Viral / Q000235:genetics; D006801:Humans; D010455:Peptides / Q000737:chemistry; D011995:Recombination, Genetic / Q000235:genetics*; D015203:Reproducibility of Results; D017384:Sequence Deletion; D017385:Sequence Homology; D019076:Transgenes
chemical_list: D010455:Peptides