Senior Scientist
,
Regenerative Medicine
, Ottawa Hospital Research Institute
BACKGROUND AND RECENT STUDIES
BACKGROUND AND RECENT STUDIES
Gene therapy – the delivery of therapeutic DNA to affected individuals – holds great promise for the treatment of many hereditary and acquired diseases. Most gene therapy studies utilize viral vectors to deliver the therapeutic gene, since viruses are very efficient at delivering their DNA payload to the nucleus of infected cells. The most commonly used viral vector is based on adenovirus (Ad). Our research focuses on improving our understanding of basic Ad biology, and using this knowledge to improve the efficacy and safety of Ad-based vectors in gene therapy applications.
For the past 12 years, our lab has been at the forefront of Ad vector design and development, and we have pioneered several key advances in the field. Shown below are just some of the studies performed in our lab.
Development of helper-dependent Ad vectors. Most gene therapy studies utilize Ad vectors that are deleted of the essential early region 1 (E1) coding sequence to deliver therapeutic genes to target cells; these vectors can infect cells but they cannot replicate. However, the effectiveness of these vectors is limited due to low level expression of other viral genes which stimulates very strong anti-vector immune responses in the host. We were the first group to develop an efficient method for producing Ad vectors devoid of all viral coding sequences (Parks, PNAS 93:13565), termed helper-dependent Ad (hdAd), and showed these vectors provide very long term gene expression (>1 year) in mice and non-human primates. This system is now used world-wide for generating hdAd. We have also made several key discoveries which have provided insight into elements of hdAd vector design that significantly enhance the stability and efficacy of the vector (Parks, J Virol. 71:3293; Parks, J Virol. 73:8027). Our current work focuses on studying the effectiveness of hdAd-mediated delivery of therapeutic genes in a variety of animal models of human disease.
Genome size and capsid stability. We are also studying several aspects of basic Ad biology that contribute to vector stability. We determined that the Ad DNA plays an important role in physically stabilizing the Ad virion (Smith, J Virol. 83:2025; Kennedy, Mol. Ther. 17:1664). Virions containing wildtype length DNA (36 kb) are stable and lose little infectivity when heated at 47oC for 30 min. Vectors with small genomes (<33 kb) rapidly lose infectivity with a half-life of ~4 min, accompanied by a loss of capsid integrity. Thus, not only does the viral DNA encode all of the heritable information essential for virus replication, it also plays a critical role in maintaining capsid strength and integrity. We are now examining various methods to modify the Ad capsid to increase the stability of Ad vectors containing small genomes.
Ad DNA “chromatinization.” Although Ad has been studied for over 50 years, little is known about the fate and structure of Ad DNA within the nucleus of the infected cell. Considering the fundamental importance of chromatin in regulating gene expression in host cells, it is surprising that it remains unknown whether Ad DNA interacts with cellular histones or assembles into chromatin. We were the first group to conclusively show that Ad DNA is wrapped in nucleosomes shortly after infection, and that this event is crucial for efficient expression of virus-encoded genes (Ross, 2009 J. Virol. 83:8409). This observation has important implications for vector function: many Ad vectors contain “tissue-specific” promoter elements to restrict expression of the therapeutic gene to the desired tissue (for example - muscle promoters for muscle-specific expression), and we are currently examining the effect of Ad vector chromatinization on promoter function, which should lead to methods to enhance promoter fidelity.
Characterization of Ad capsid proteins. We have also completed several studies characterizing various aspects of Ad capsid protein IX (pIX) function in the virus lifecycle (Sargent, Gene Ther. 11:504; Sargent, J. Virol. 78:5032). More recently, we showed that pIX can be used to present large molecules on the surface of the virion (Meulenbroek, Mol. Ther. 9:617): a pIX-GFP (green fluorescent protein) fusion protein was efficiently incorporated into the Ad virion, and could be used to track virus infection in tissue culture and in animals using fluorescence microscopy. We have expanded upon this observation to develop pIX as a platform for presentation of large targeting ligands, which will allow us to redirect virus infection to specific cell types, such as cancer cells. This approach will lead to enhanced vector efficacy and safety, since the Ad vector will only infect the target tissue.
These and other studies have significantly improved our understanding of many aspects of basic Ad biology, and have translated into improved function, safety and stability of Ad vectors.
A more detailed summary of our previous research efforts are described here.
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