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Novel Viral Vector Manufacturing Approach Could Improve Safety of Gene Therapy

Novel Viral Vector Manufacturing Approach Could Improve Safety of Gene Therapy 1024 683 Lauren Dembeck

New AAV proviral plasmid for vector manufacturing has potential to increase patient safety and make gene therapies more affordable, researchers say.

Gene therapy is one of the most exciting frontiers in modern medicine, offering potential cures for previously devastating or fatal disorders, such as spinal muscular atrophy and aromatic L-amino acid decarboxylase deficiency. Adeno-associated virus (AAV) vectors — non-pathogenic viruses used to deliver therapeutic genetic material to human cells — have been central to advances in the field. Many of the gene therapies approved to date by the Food and Drug Administration rely on AAVs due to their favorable properties such as providing stable transgene expression and relative safety. However, issues related to AAV vector manufacturing have become a concern for long-term safety.

“Gene therapy using AAV as a delivery system moved from science fiction to reality in 2017 with the approval of Luxturna, a treatment for a form of blindness. Today, we have several AAV-based gene therapies on the market, including ones that literally save babies’ lives and restore sight, among other things,” explains Scott Harper, PhD, principal investigator in the Jerry R. Mendell Center for Gene Therapy at the Abigail Wexner Research Institute at Nationwide Children’s Hospital. “However, nearly all AAV products made today use DNA plasmids as raw materials, and these DNA plasmids are typically synthesized for us by bacteria. While these bacterial-generated raw materials produce large quantities of AAV products, some potentially harmful bacterial sequences may remain as DNA contaminants in essentially all AAVs manufactured today.”

The potentially toxic DNA contaminants arise from AAV proviral plasmids, circular DNA required for manufacturing the genetically engineered therapeutic AAVs. These circular DNAs typically contain three essential components:

  1. A therapeutic gene cassette — all the genetic information required by the cell to replace a missing or faulty protein
  2. Natural AAV inverted terminal repeat sequences — short DNA sequences that guide proper packaging of the therapeutic gene cassette into the viral capsid
  3. A plasmid backbone — bacterial DNA sequences required for replication of the proviral plasmid in bacteria for large-scale production

During production, most AAVs package the intended therapeutic payload. However, some package the bacterial sequences comprising the proviral plasmid backbone instead. Because these bacterial sequences can be expressed, they are potentially toxic, representing a safety risk.

“Recent studies have demonstrated that even relatively small amounts of AAV contaminants can be toxic to the brains of animal models, leading to ataxia, tremor, dysmetria and brain lesions,” adds Dr. Harper.

In a new study published in the journal Molecular Therapy: Methods & Clinical Development, Dr. Harper and colleagues describe a new AAV proviral plasmid for vector manufacturing.

“We think we can improve manufacturing and safety by optimizing the DNA raw materials required to make an AAV product, and the focus of our study was to design a new plasmid system for AAV manufacturing that would decrease the amount of bacterial DNA sequences present in an AAV prep and improve the overall uniformity of the product. By increasing the percentage of correctly packaged AAV material, we also hope to reduce manufacturing costs, and hopefully lower the costs of the therapies, which are currently the most expensive drugs on the planet.”

They demonstrated that, compared with the original plasmid, their new AAV proviral plasmid significantly decreased cross-packaged plasmid backbone sequences by 46%, increased the ratio of correctly packaged AAV payloads to backbone sequences, and reduced cross-packaged potentially toxic bacterial sequences by 70%.

“We modified the AAV vector by adding insulator sequences to prevent unwanted gene activation, incorporating safe human DNA to the backbone to avoid packaging bacterial sequences, and removing start signals to eliminate unintended protein production,” says Dr. Harper. “Our new plasmid should be safer than the first-generation plasmids currently used for AAV manufacturing; however, we do not yet know in practice if this is true,” says Dr. Harper, who is also a professor of Pediatrics at The Ohio State University College of Medicine.

Dr. Harper and colleagues are now evaluating additional improvements to the system to further reduce DNA contaminants and improve the potential safety of AAV preps in general.

 

Reference:

Taylor NK, Guggenbiller MJ, Mistry PP, King OD, Harper SQ. A self-complementary AAV proviral plasmid that reduces cross-packaging and ITR promoter activity in AAV vector preparations. Mol Ther Methods Clin Dev. 2024 Jun 26;32(3):101295.

About the author

Lauren Dembeck, PhD, is a freelance science and medical writer based in New York City. She completed her BS in biology and BA in foreign languages at West Virginia University. Dr. Dembeck studied the genetic basis of natural variation in complex traits for her doctorate in genetics at North Carolina State University. She then conducted postdoctoral research on the formation and regulation of neuronal circuits at the Okinawa Institute of Science and Technology in Japan.

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