There are 7,000 different rare genetic disorders that impact approximately 450,000 people in Wisconsin. Gene therapy has the potential to treat these diseases if two major limitations can be addressed: better targeting of the therapy agent and affordability. This project, led by Kinjal Majumder, PhD, assistant professor, Department of Oncology, sought to address these limitations by improving the nuclear targeting of gene therapy vectors.
The team used a combination of CRISPR/Cas9 technology, Big Data and high-resolution imaging to study the molecular mechanisms of Recombinant Adeno-Associate Virus (rAAV) vectors for gene therapy delivery and provide insights into engineering better rAAV gene therapy vehicles. Their findings will inform future work in the field of gene therapy, with the potential to improve treatments for rare genetic disorders and develop cancer-targeting gene therapies.
The Challenge
There are 7,000 different rare genetic disorders that impact approximately 450,000 people in Wisconsin. Advances in genomics and precision medicine have enabled early detection of these disorders, but there are challenges to effective and equitable treatment. Recombinant Adeno-Associate Virus (rAAV) vectors have the potential to solve this problem, being utilized for gene therapy applications for the treatment of rare monogenic diseases. Despite promising clinical applications, the full potential of rAAV gene therapy has not been realized due to inadequate knowledge about how these vectors navigate the host nucleus. It is essential to understand how and where rAAV vectors localize in the nucleus.
Project Goals
The aims of this project were to:
Determine how rAAV gene therapy vectors establish latency at distinct nuclear sites to express and persist long term.
Define the host-cellular factors that drive rAAV localization to distinct cellular sites to express long-term.
Results
With this project, the investigators have established experimental platforms and molecular tools necessary to make critical contributions in rAAV gene therapy over years to come. The researchers have identified an AAV binding site associated with the host transcription factor KLF4 that regulates AAV localization to cellular sites of DNA damage. They have also identified binding factors of CTCF, an insulator element, as critically responsible for the formation of rAAV chromatin and efficient transgene expression.
The researchers discovered that cellular sites that are enriched in KLF4 binding and CTCF binding regulate AAV localization, but not rAAV localization. They also made the novel discovery that rather than associating with cellular chromatin regions, rAAV genomes localize to the cellular nucleoli. These findings advance the overall goals of the project because they identify the host nuclear reservoirs where the rAAV vector genomes persist long-term.
Lasting Impact
The largest cost barrier to the application of rAAV gene therapy is the high doses that are required for them to be effective. As a result of the findings, scientists can now engineer rAAV gene therapy vectors that are effective at much lower dosage. This will reduce the dose for each patient substantially, making it safer and much more affordable. This would enable widespread application of rAAV gene therapy for all people.
