Viral Vectors for Potential Gene Therapy
The primary focus of our laboratory is the development of adeno-associated virus (AAV) vectors for gene transfer. AAV vectors (“recombinant” or rAAV) offer several advantages for gene transfer because they are based upon a virus that is nonpathogenic, have a wide host range, can transduce quiescent cell populations, and have the unique capacity for site-specific integration into primate genomic DNA.
We are utilizing recombinant AAV vectors to develop tumor specific vaccines. When in our laboratory, Dr. Jiyao Sun used rAAV vectors to introduce a portion of the bcr-abl oncogene into primary human dendritic cells (DC), the major antigen presenting cells in the immune system. The bcr-abl oncogene is necessary for the development of a form of leukemia termed chronic myelogenous leukemia or CML, and is not present in normal cells. DC, in turn, play important roles in the initiation of immune responses, and are pivotal cells for generation of vaccines. Human DC genetically modified to express bcr-abl using rAAV vectors could identify and specifically lyse MHC matched target cells expressing bcr-abl in vitro. This is one step toward the development of a leukemia specific vaccine.
AAV is unique amongst all viruses in its ability to insert its genome site-specifically into a region of the human genome termed “AAVS1” on chromosome 19. This property has been mapped to an AAV encoded protein termed “Rep,” which, for a variety of reasons, has been removed from currently used rAAV vectors. Joel Conrad, a research associate in my lab, has developed fusion proteins containing Rep that may traffic between cells and mediate site-specific vector integration. If effective, these recombinant proteins will make “gene therapy” potentially safer and more effective. Research in this area has become more important following the reports of the development of T-cell leukemia in subjects participating in a clinical trial using retroviral vectors for the treatment of X-linked severe combined immunodeficiency disease (SCID).
We have initiated a study of the use of recombinant AAV vectors for expressing transgenes designed to inhibit disease-causing viruses including herpes simplex (HSV) and human papillomavirus (HPV), which is associated with cervical cancer. We have used rAAV vectors in the past to inhibit targeted viruses including human immunodeficiency virus (HIV), the cause of AIDS. Our newer approach utilizes rAAV-amplifying vectors within cells containing the targeted virus. This results in higher expression of inhibitors, and may be particularly useful against viruses such as HSV that are “latent” viruses that cause recurrent infections.
In ongoing collaborations with Dr. Saswati Chatterjee from the Department of Virology, we are continuing to assess the safety and efficacy of rAAV gene transfer to human blood and blood stem cells both in vitro and in vivo models (see material from Dr. Chatterjee). These studies are very promising, and, to date, are nontoxic.
Finally, in conjunction with Dr. Chatterjee and the Cardiology Division at Cedars-Sinai Hospital in Los Angeles, we are also utilizing rAAV vectors to express a mutant form of human apolipoprotein A-1 termed Apo A-1Milano. Individuals who naturally express Apo A-1Milano are known for longevity despite exhibiting serum lipid profiles that would normally presage the development of atherosclerosis, or “hardening of the arteries,” a major underlying cause of heart and vascular disease. In vivo expression of Apo A-1Milano has resulted in a 40-50% reduction in the development of large vessel atherosclerotic lesions. Importantly, information from other laboratories suggest that administration of recombinant Apo A-1Milano as a protein can result in actual regression of pre-existing atherosclerotic lesions, suggesting that this strategy could have important therapeutic implications.