Production of Adeno-Associated Virus in a Plant-Based System for the Treatment of Myotonic Dystrophy
Abstract
Groundbreaking work in the field of gene therapies has brought us from dreaming of curing genetic diseases to the edge of that reality. Gene therapies replace the missing or damaged patient’s gene with a functional copy, treating previously incurable diseases. One of the primary methods for introducing the new functional gene into the patient’s cells uses adeno-associated virus (AAV). However, using current methodologies, AAV treatments are incredibly costly to manufacture. For example, an AAV-based therapy for spinal muscular atrophy, a muscle-wasting disease, costs 1 million U.S. dollars a dose, making it the world’s most expensive drug. While the therapeutic may eventually cost less than years of treating the disease, these high prices will limit or prevent the use of AAV-based therapies. The current expense is in producing enough AAV to treat a patient. Limitations exist in the size of the AAV batch that can be made, the expense of the manufacturing raw materials, and the difficulty in timing the expression of all the critical components for AAV production. As AAV-based gene therapy treatments have become increasingly effective, the manufacturing of AAV has become the bottleneck. AAV dose requirements have increased, but batch yields have not risen to meet demand. An ideal solution would be to produce the AAV inexpensively, using a process without scaling limitations. This process would use cheap raw materials and innovative technology to express critical elements simultaneously within the same cell. To this end, in this application, we propose to replace the current expensive AAV production in mammalian cell culture systems with inexpensive production of AAV in whole plants. Myotonic dystrophy is an adult muscle wasting disease that affects approximately 1 in 8,000 people and is caused by a defective gene. Individuals who have myotonic dystrophy also have problems with muscle control, digestion, sleepiness, and thought processes that are related to the gene malfunction. Several approaches using AAV as gene carriers have shown therapeutic promise in animal models and are in preclinical development for myotonic dystrophy. Our goal is to produce a cost-effective therapy for myotonic dystrophy by reducing the cost of making AAV. The first aim will focus on building the individual components required to make functional AAV in a plant system. The second aim will focus on the empty AAV capsid’s initial formation without any genetic material. The third aim will introduce DNA into the plant-produced AAV, adding a cargo gene that encodes for an easily detectable fluorescent signal. We will determine AAV infectivity by adding it to mammalian cells and looking for the cargo gene’s production. Finally, in the fourth aim, we will make an AAV-based myotonic dystrophy gene therapy treatment in the plant production system. We will demonstrate the AAV’s therapeutic activity by testing its ability to deliver its genetic cargo functionally to laboratory-grown muscle cells. Targeting of the myotonic dystrophy disease-causing gene by AAV gene therapies has been shown to improve the outcome for muscle cells. We will compare the functionality of mammalian cell-produced AAV to the plant-produced AAV, demonstrating the plant production system’s capabilities. The production of inexpensive AAV has the potential to revolutionize gene therapies. This technology could be vital in treating myotonic dystrophy and all conditions amenable to gene therapy treatments. This proposal aligns with FY21 PRMRP Discovery Award Topic Area Myotonic Dystrophy and subtopic Development and/or testing of novel and/or innovative treatments, including those utilizing gene editing or silencing.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Dec 28, 2022
- Source ID
- W81XWH2210183
Entities
People
- Stephen Streatfield
Organizations
- Fraunhofer Center for Molecular Biotechnology
- United States Army