QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: mol.106.025619v1 70/5/1488    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Belousova, N. Articles by Alvarez, R. D. Search for Related Content PubMed PubMed Citation Articles by Belousova, N. Articles by Alvarez, R. D.

Accelerated Communication

Circumventing Recombination Events Encountered with Production of a Clinical-Grade Adenoviral Vector with a Double-Expression Cassette

Division of Human Gene Therapy, Departments of Medicine, Pathology, and Surgery, and the Gene Therapy Center (N.B., M.W., D.T.C., Z.B.Z.), Department of Medicine (K.Z.), Vaccine and Vector Production Facility (A.K., O.K.), and the Department of Obstetrics and Gynecology (R.D.A.), University of Alabama at Birmingham, Birmingham, Alabama; and Biopharmaceutical Development Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland (R.H., M.A.R.-S., R.A.)

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[CrossRef][Medline] ) 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.

Address correspondence to: Dr. David T. Curiel, Division of Human Gene Therapy, Gene Therapy Center, The University of Alabama at Birmingham, 901-19th Street, South, BMR2-502, Birmingham, AL 35249-2184. E-mail: curiel{at}uab.edu

This article has been cited by other articles:

				D. A. Mankoff, J. M. Link, H. M. Linden, L. Sundararajan, and K. A. Krohn Tumor Receptor Imaging J. Nucl. Med., June 1, 2008; 49(Suppl_2): 149S - 163S. [Abstract] [Full Text] [PDF]

				R. Vogels, D. Zuijdgeest, M. van Meerendonk, A. Companjen, G. Gillissen, J. Sijtsma, I. Melis, L. Holterman, K. Radosevic, J. Goudsmit, et al. High-level expression from two independent expression cassettes in replication-incompetent adenovirus type 35 vector J. Gen. Virol., November 1, 2007; 88(11): 2915 - 2924. [Abstract] [Full Text] [PDF]