Description of Project:

My project entails designing new viral vectors to mediate gene transfer , ultimately for the treatment of inherited or acquired genetic diseases (e.g. hemophilia) and/or cancer. In pursuit of this goal, I discovered that homologous elements inserted into first-generation adenoviral vectors can be used to create new vectors by recombination in 293 cells. These new recombined vectors have a number of advantages over the traditional ones including: the new recombined vectors are devoid of all viral genes rendering them non-toxic and less immunogenic, they have approximately twice the insert capacity of first-generation vectors, and they can be created to express toxic or adenoviral replication inhibiting genes. However, these new vectors still possess one disadvantage in common with their first-generation adenoviral parental vectors in that they remain episomal after therapeutic gene transfer resulting in only transient expression. In order to combat this problem, I have now incorporated two genetic elements from adeno-associated virus (AAV). First, I have added AAV ITRs flanking my therapeutic gene, which allows for random integration. Second, I have inserted the AAV rep 78 gene, which is known to confer site-specific integration into a transcriptionally active region of chromosome 19. I am currently testing this new vector system.

Publications:

1. Ahrens, C. H., C. Carlson, and G. F. Rohrmann. 1995. Identification, sequence, and transcriptional analysis of lef-3, a gene essential for Orgyia pseudotsugata baculovirus DNA replication. Virology. 210:372-82.
2. Lieber, A., D. S. Steinwaerder, C. A. Carlson, and M. A. Kay. 1999. Integrating adenovirus-adeno-associated virus hybrid vectors devoid of all viral genes. J Virol. 73:9314-24.
3. Steinwaerder*, D. S., C. A. Carlson*, and A. Lieber. 2000. DNA replication of first-generation adenovirus vectors in tumor cells [In Process Citation]. Hum Gene Ther. 11:1933-48.
4. Steinwaerder, D. S., C. A. Carlson, and A. Lieber. 1999. Generation of adenovirus vectors devoid of all viral genes by recombination between inverted repeats. J Virol. 73:9303-13.
5. Steinwaerder*, D. S., C. A. Carlson*, and A. Lieber. 2001. Human papilloma virus e6 and e7 proteins support dna replication of adenoviruses deleted for the e1a and e1b genes. Mol Ther. 4:211-6.
6. Steinwaerder, D. S., C. A. Carlson, D. L. Otto, Z. Y. Li, S. Ni, and A. Lieber. 2001. Tumor-specific gene expression in hepatic metastases by a replication-activated adenovirus vector. Nat Med. 7:240-243.
7. Carlson, C. A., D. S. Steinwaerder, H. Stecher, D. M. Shayakmetov, and A. Lieber. 2002. Rearrangements in adenoviral genomes mediated by inverted repeats. Method Enzymol. 346: in press.
8. Carlson. C. A., D. M. Shayakmetov, and A. Lieber. 2001. Reconstitution of functional transgenes by recombination between two adenoviral vectors. J Virol. submitted.