Moving Beyond AAV: The Next Generation Of Vectors In CGT

By Erin Harris, Editor-In-Chief, Cell & Gene
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Adeno-associated virus (AAV) vectors are a proven, valuable tool in both research and clinical applications, but improving upon other existing vectors and developing new ones could drastically change the cell and gene therapy (CGT) landscape in 2024 and beyond.

Vectors are a significant and active area of ongoing R&D in various fields, including biotechnology, and more specifically, gene therapy and cancer immunotherapy. Almost 500 vectors are in development, and AAVs are currently the most widely used viral vector due to their relatively low immunogenicity compared to adenoviral vectors. But AAVs’ largest hurdle continues to be scalable manufacturing that is free of impurities, making the production of high-quality AAVs a challenge for many gene therapy manufacturers. And so, some CGT developers are relying on other vectors, such as lentiviral vectors, to deliver genetic material to target cells and tissues for therapeutic purposes while other developers are creating an entirely new class of vector that delivers their therapeutic cargo to their target cell without damaging tissue or activating the immune system.

Regarding cancer immunotherapy, lentiviral vectors can be used to engineer immune cells, such as T cells, for chimeric antigen receptor (CAR) T-cell therapy. Researchers are exploring novel CAR designs and modifications to enhance the specificity and effectiveness of these immunotherapies against cancer. And they are working on improving the safety and efficiency of all vectors, as well as expanding their applications to a wider range of diseases. Umoja Biopharma, for example, is focused on immunotherapy reprogramming T cells in vivo to target cancer cells and relies on lentivirus for delivery. Ring Therapeutics, on the other hand, is a genetic medicines company taking a novel approach to creating commensal virus-based medicines that can safely and effectively deliver therapies to target cells and tissues via anellovirus vectors.

I caught up with Konstantin Konstantinov, Ph.D., CTO at Ring Therapeutics, and Ryan Crisman, Ph.D., cofounder and CTO at Umoja Biopharma, to get their thoughts on the future of new anellovirus vectors and existing lentiviral vector technology for in vivo gene delivery, respectively.

WHAT IS AN ANELLOVIRUS?
Attend any cell and gene therapy conference, and you will, without question or hesitation, hear industry and academic leaders alike state that continuous innovation is the main ingredient to bring lifesaving therapies to patients. Developing new viral vectors that have immune stealth and can be produced outside of traditional cell-based systems is a key example of continuous innovation. One such viral vector innovation is the anellovirus. “Anelloviruses are the most abundant commensal virus in humans, making up greater than 97% of the human virome, as they have evolved and lived in harmony with us for millions of years,” explains Konstantinov. “Anelloviruses are small, circular single-stranded DNA viruses.” Konstantinov shares that, historically, virologists have largely ignored anelloviruses because they have not been connected to any diseases. However, Ring’s recent publications have shown that anelloviruses’ diversity is approximately six times greater than other families of viruses, including AAVs. “This commensalism and diversity are the key to their potential in gene therapy,” says Konstantinov. “Harnessing the unique characteristics of anelloviruses to make a new viral vector may open a wide array of possibilities for genetic medicines to reach target tissues safely and repeatedly.”

Konstantinov states that development of a new viral vector hasn’t been successful in decades, likely due to the many challenges that come with harnessing a natural virus. “We have been overcoming many of these challenges by generating proof-of-concept data for our AnelloVectors in animal models, including non-human primates. We are progressing toward IND and developing a portfolio across an array of therapeutic applications.”

Adenoviral vectors, AAVs, and retroviruses are all pathogenic viruses, eliciting an immune response that can preclude redosing when necessary. Adenoviral vectors are highly immunogenic, and much of the population has preexisting immunity that would preclude initial therapeutic dosing. AnelloVectors can be produced in a cell-free system at scale, producing high-quality vectors loaded with highly potent therapeutic cargo. Unlike AnelloVectors, lentiviruses integrate into the genome, although with reduced genotoxicity than other retroviral vectors. Some lentiviruses have been engineered to be non-integrating, greatly reducing insertional mutagenesis while maintaining transient expression in actively dividing cells. However, their current utility remains largely in ex vivo gene therapy where insertional mutagenesis can be avoided.

INNOVATION IS KEY TO IMPROVED DRUG DELIVERY
Two of gene therapy’s biggest limitations are delivery and manufacturing. Indeed, the best therapy isn’t helpful if you don’t have a way to get it to the right target or if you can’t safely scale up its production. “We’re in an exciting time for innovation, so I see both of these challenges improving with continued innovation from across the industry,” explains Konstantinov.

Konstantinov shares three keys to drug delivery. The first is tropism or tissue selectivity. Current viral vectors can successfully target some key tissues, including the liver, heart, and muscle, but many tissues remain unreachable due to their limited diversity.

The second key is potency or the ability to cause significant and durable therapeutic effects. Here is where viral vectors are the most effective delivery vehicle, harnessing the millions of years of viral evolution to effectively enter target cells to deliver therapeutic cargo.

The third key to drug delivery is redosability and, in some cases, initial dosing. Delivery through viral vectors has largely relied on pathogenic viruses such as AAVs and lentiviruses that elicit a robust immune response, producing antibodies specific to these vectors. These neutralizing antibodies preclude any subsequent therapeutic dosing. Additionally, these viruses are naturally occurring, and many people have preexisting immunity against them. For example, 20%-80% of humans have preexisting antibodies across all AAV serotypes, making these people unable to receive even a first dose of AAV-delivered therapies.

Scalable manufacturing of viral vectors also poses a major barrier to clinical development and ultimately commercialization. Current viral vectors rely on cell-based systems that are costly, time intensive, and often variable. Empty capsids that result from these cell-based systems are also a major problem for developers. Empty capsids contain no therapeutic cargo but still elicit an immune response, often increasing the doses required to have a therapeutic effect and therefore the likelihood for immunotoxicity.

“In the next few years, gene therapy will evolve in several directions, driven by rapid scientific progress and economic and social forces. While traditional gene therapies utilizing natural viral vectors, such as AAV and lentiviruses, will continue to dominate pipelines, the novel synthetic or hybrid approaches will progress rapidly, reflecting a major shift in the gene therapy field to combine the strengths of the viral and non-viral vectors,” states Konstantinov.

ARTIFICIAL INTELLIGENCE AND MACHINE LEARNING TO IMPACT VECTORS
Konstantinov states the field of gene therapy will also see growing applications of AI and machine learning (ML) targeting optimal design of the capsid and the payload. The growing amount of data available on viral capsid structure and function along with the payload engineering will fuel rapid improvements to the next generation of viral vectors. He explains the field will also see the development of manufacturing technology with the potential to eliminate the major bottlenecks of traditional gene therapy, including large cost and limited productivity. “More specifically, the high cost of current gene therapies [often $1 million to $2 million per treatment] can be dramatically reduced [≥10X] with key process innovations, making these advanced therapies more accessible,” states Konstantinov.

LENTIVIRAL VECTOR – THE DELIVERY PLATFORM OF CHOICE FOR IN VIVO GENE DELIVERY
Research in the field of lentiviral vectors is dynamic and continually evolving. Scientists are dedicated to addressing safety concerns, improving vector design, expanding applications, and ensuring efficient vector production and delivery. Crisman shares that the lentiviral vector is Umoja Biopharma’s drug product delivery platform. “We have surface-engineered the lentiviral vector particles such that they can be directly injected into the patient to deliver cancer fighting genetic information to the patient’s immune cells,” Crisman says. “Our commercial manufacturing process can provide thousands of doses per manufacturing run, overcoming the supply chain bottlenecks for patient access in the current CAR-T field. Lentiviral vectors will become the delivery platform of choice for in vivo gene delivery.”

Crisman explains that lentiviral vectors have decades of proven safety data, large genetic payload capacity, and efficient transduction capabilities. Additionally, he shares that lentiviral vector gene delivery provides stable integration of the payload into the target cells, resulting in a single dose being sufficient to provide a persistent therapeutic effect. Lastly, lentiviral vector manufacturing is highly scalable, reproducible, and a commercially viable platform for in vivo gene delivery. “The safety, efficiency, and manufacturability of lentiviral vectors make them the delivery platform of choice for in vivo gene delivery,” Crisman says. “We will see the field migrate toward lentiviral vectors away from AAV, for example, as in vivo data starts to come out.”

A BRIGHT FUTURE FOR VIRAL VECTOR PRODUCTION
Konstantinov states, “I believe the future of viral vector production and manufacturing is bright and poised to tackle some of the largest hurdles facing the field today. Key innovations coupled with AI/ML-optimized processes and designs will make these promising therapies accessible for the wide array of patients in need.”

“We are in the early days of unlocking the potential of lentiviral vectors for in vivo gene delivery, and we have a great deal of opportunity to enhance the processes to continue improving the productivity that ultimately impacts patients,” Crisman states. “When we started Umoja Biopharma four years ago, the world of in vivo lentiviral vector included us and maybe one other player. Now the field is starting to grow, and there are many more players in the space. That’s exciting because it means the science is there. We have engineering issues we need to solve, which is possible now because both scientists and engineers are involved. I’m confident and very excited about where the field is headed.”