An adenoviral vector containing an arg-gly-asp (RGD) motif in the fiber knob enhances protein product levels from transgenes refractory to expression.
Genetic manipulation of the adenovirus type 5 represents one strategy to modify viral transduction properties in vitro and in vivo. In the majority of studies to date, reporter gene activity has been monitored to assess transduction efficiency. BRCA1 is a gene whose protein product is clinically important, biologically toxic, difficult to overexpress, and difficult to detect as an untagged protein species. Thus, it represents an attractive candidate from which to evaluate the efficacy of a gene delivery system. In the present study, transgene expression was assessed employing otherwise isogenic viruses, which differed only in the presence or absence of an RGD integrin-binding motif in the HI loop of the Ad fiber knob. We utilized a combination of BRCA1 expression level comparisons among several human BRCA1/mutant BRCA1/murine Brca1 constructs and reporter gene activity following transduction of a panel of human breast and ovarian tumor cell lines representative of both sporadic and hereditary cases. A general overall concordance in efficiency was observed, whether the biological readout measured was reporter gene activity or steady-state level of ectopic BRCA1 protein produced. Importantly, the expression of full-length wild-type BRCA1 protein, clinically relevant mutant BRCA1 proteins or murine Brca1 was superior when the gene was delivered via the RGD-modified Ad. The ectopic BRCA1 stabilized endogenous BARD1 and this functional effect was evident at lower input viral doses when BRCA1 was delivered via the RGD-modified Ad. Quantitative, noninvasive, real-time image analysis of reporter gene function in nude mice harboring human ovarian tumor xenographs demonstrated a similar enhancement of expression in vivo by the RGD fiber modification, with low levels of transduction of normal mouse mesothelium. These results provide additional evidence supporting the concept that rational modification of viral vectors can result in the delivery of functionally active therapeutic proteins such as BRCA1 that present with technical difficulties with regard to their expression.
Campbell
Mel
M
Department of Pathology, Vanderbilt-Ingram Comprehensive Cancer Center, Nashville, Tennessee 37232, USA. mel.campbell@vanderbilt.edu
Qu
Shimian
S
Wells
Sam
S
Sugandha
Herlina
H
Jensen
Roy A
RA
D000256:Adenoviridae / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D005260:Female; D018014:Gene Transfer Techniques; D019398:Genes, BRCA1; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D016023:Integrins / Q000032:analysis; D051379:Mice; D009842:Oligopeptides; D014162:Transfection / Q000379:methods*; D014407:Tumor Cells, Cultured
D016023:Integrins; D009842:Oligopeptides; C047981:arginyl-glycyl-aspartic acid