PIECING TOGETHER THE PUZZLE OF TREATING HEMOPHILIA A UTILIZING AAV BASED GENE THERAPY

Abstract

Genetic disorders are a major health issue. They include any disease caused by a genetic mutation or deletion. Disorders range from Hemophilia, Cystic fibrosis, and Duchenne muscular dystrophy to diseases like Cancer and Huntington’s Disease. While these diseases may seem vastly different, they all are due to issues in the genetic makeup of the patient. Gene therapy treats or cures genetic disorders by any number of different mechanisms. Gene therapy can be used to augment the genome, inhibit detrimental genes, or kill off malignant cells. While the concept of gene therapy seems straight forward the application is not. Many hurdles need to be overcome from the host’s response to the therapy to the understanding of the disease in general. One of the biggest hurdles to overcome is the delivery of the genetic material to the host. Viruses represent a potential solution to the problem of delivery. While viruses tend to be thought of as pathogenic and deadly, they can be co-opted for use in gene therapy. Viruses have evolved to target and deliver genetic material, which makes them a strong candidate for this application. Among the viruses currently being studied for use in gene therapy is AAV. AAV is a small virus, which is relatively easily augmented to produce replication deficient therapeutic agents. While AAV looks to be a potential solution to the delivery aspect of gene therapy, there are still many issues with AAV use. AAV is a very small virus that cannot hold more than 4.7kb of DNA. Production of AAV is time and labor intensive. AAV tropism has not been documented to all tissue types and some tissues like the lungs and heart are underserved in that respect. Contamination of viral preparations is also an issue as the production of AAV creates many virions that do not contain the therapeutic cassette of interest. Some of these issues like size cannot be addressed. However, by expanding studies and looking into the life cycle and production of AAV, this delivery system can be refined. By using technology more recently developed, new viral tropisms can be identified. In the experiments conducted we utilized cloning to generate plasmids containing only one ITR. Our work shows that plasmids containing only a single ITR can be packaged into virions during normal rAAV production. This is an issue as single ITR containing plasmids are found in plasmid preparations used for AAV production. These single ITR containing plasmids have detrimental effects on the overall yield of recombinant AAV. This results in lower overall yields of AAV. Another focus of the research presented is on the tropisms of AAV. It is known that viruses target different tissues and cell types. By identifying viruses that have not yet been studied we can find viruses with specific tropisms possibly reducing the need for high viral loads. We identify AAV-Go.1 as a virus with promise targeting specifically the mouse lungs. AAV-Go.1 could be useful for the treatment of cystic fibrosis or alpha-1 trypsin deficiency. We see that viral tropism differs between humanized and wild type mouse livers. This may mean that we will need to examine different viruses for studies on different organisms. All of these findings help us to push forward in refining AAV for use in clinical applications.