AAV vectors: selection permutations, favorable fates (2023)

Adeno-associated virus (AAV) vectors may be the best option for delivering genetic material to cells in the body. But they are far from perfect. They require large doses, carry small amounts of cargo, and can be recognized by the immune system, a safety concern. Furthermore, they behave differently in animal models such as mice and non-human primates than in humans. These complications require constant innovation and development of new strategies.

LogicBio Therapeutics was recently affected by discrepancies between AAV-associated immune responses in animals and humans. The well-known company had to stop the clinical trial when two participants experienced side effects. The Akouos company has just started and hopes to avoid someAvoid the pitfalls by working in one area of ​​the body, the inner ear, where the company's AAV technology has shown promise..

learn along the way

Mark A. Kay, MD, Ph.D., a professor of genetics at Stanford University, began working with gene therapy in 1990. At the time, he recalled, people gave estimates that certain diseases would be cured in five years. .

Kay was part of the team that filed the IND application in 2000, leading to the first attempt to routinely administer AAV2 to humans. The work was done by gene therapy pioneer Katherine High, MD, who was at the University of Pennsylvania at the time. Her (Today, she is a visiting professor at Rockefeller University and professor of therapeutics at AskBio).

Over the years, Kay has learned that AAV doses and transduction efficiencies in animal studies do not necessarily correlate with humans. In short, animal tests make predictions that have nothing to do with humans.

For example, in previous experiments from 22 years ago, the team found that AAV vectors were not toxic when administered to dogs and rodents. However, humans have elevated liver enzymes. Furthermore, in humans, but not in animals, there is a T cell response against capsids that have been taken up by cells and degraded.

This experience led Kay to find a better model for AAV vector selection. The model is a mouse model containing primary human hepatocytes developed in the laboratory of Markus Grompe, MD, director of the Papé Family Pediatric Research Institute at Oregon Health & Science University. Using this model, Kay's group became the first to use "humanized" mice to compare the transduction efficiency of different AAV vectors.

Kay's group was also the first to use an AAV vector development process in which capsid variants were selected from a capsid library. In this process, scientists disassemble capsids in nature, mix the resulting capsid fragments together, and assemble new capsids at random. Eventually, the capsid becomes part of a capsid library, which may contain millions of capsid variants.

Capsids were injected into humanized mice and capsids successfully transduced into human hepatocytes were recovered. One of the most successful capsids discovered by Kay's group was the AAV-LK03 capsid. It performs 10 times better than AAV2 or AAV8. It is used in LogicBio trials as well as Spark Therapeutics trials. Spark calls the capsid Spark200 and uses it to deliver SPK-8011, a gene therapy for hemophilia A.

Kay screens for AAV vectors with different properties. For example, she examined AAV vectors that can package large amounts of DNA. Detection of AAV vectors that do not elicit an immune response is more difficult, she said. Detecting them is more difficult unless she knows more molecular details about what stimulates these responses.

First up: in vivo genome editing in children

In 2014, Kay developed work in his lab with two postdocs to form LogicBio. In 2016, LogicBio welcomed a new President and CEO, Fred Chereau, an executive with extensive experience building biotech companies.

In October 2021, LogicBio announced the results of a clinical trial that demonstrated the first use of nuclease-free genome-editing technology in children. The technology was also developed in Kay's lab.

LogicBio is focused on developing gene therapy solutions for pediatric patients with rare diseases. The company's first drug in development (LB-001) is designed to treat methylmalonic acidemia (MMA), a life-threatening condition that affects approximately 1 in 50,000 newborns in the United States.

Early data from a phase I/II trial look promising. Based on the safety data from the first two patients, an independent data safety monitoring committee recommended that the trial continue with the inclusion of younger children and the use of higher doses.

But on February 2, the company held a conference call to reveal that the FDA had suspended the MMA trial. At that time, four patients had already taken the drug. The first two patients, the eldest, are fine. But the third and fourth patients experienced thrombotic microangiopathy (TMA), a previously reported side effect with other AAV gene therapies.

Chereau said both patients are doing well and have returned home. At LogicBio, the researchers are working on implementing changes to get the experiment working again. Chereau noted that although the TMA phenomenon is not well understood, complement activation is known to be involved. While prophylactic administration of steroids and high-dose AAV vector-delivered therapies are now the standard, more is needed to prevent reactions. To do this, the LogicBio researchers can modify the protocol to add a product (a C5 inhibitor) that inhibits complement activation.

Gene editing and gene therapy are still in their infancy, and companies in the field are still learning, Chereau said. It takes time for the field to learn how to introduce steroids. Now people in the field know that steroids are good, but probably not good enough. So these guys are considering making other changes to their protocols. For example, an IND application filed by Regenxbio for its DMD program describes a protocol in which AAV8-based gene therapy is combined with strategies to reduce complement activation.

Some researchers focus on other solutions. Some do "stealth AAV" or try to engineer epitopes that tag the AAV to the immune system. Kay noted that it's not clear if they work. She advocates the strategy of finding AAVs that can be given in low doses.

As development programs progress, the field of gene therapy is informed and reacts to the good and the bad. Chereau noted that other gene therapy companies have approached LogicBio considering their own IND applications. And LogicBio discovered that its problems were being shared by other companies, and those companies were willing to share information. When it comes to patient safety, the industry is willing to cooperate. “We all learn from each other,” Chereau declared.

Gene therapy for inner ear diseases

An ear for music can come with a mind for science. Check out the Longwood Symphony Orchestra (LSO), a group of Boston scientists, doctors, and other health professionals from the Longwood Medical District, home of Harvard Medical School and several hospitals.

The LSO scientist-musician is not alone. Another scientist and musician also active in the Boston area is Akouo's CEO, Dr. Manny Simons. Simmons studied music as an undergraduate at Harvard University. But his interest in neuroscience began to compete for his attention.

In fact, when Simons decided to study biomedical engineering at the Massachusetts Institute of Technology (MIT), the emphasis turned to neuroscience. In the ScD's Robert S. Langer Laboratory, Simons focuses on drug delivery to the inner ear. She received her PhD in 2008 and joined Voyager Therapeutics, an AAV gene therapy company, in 2014. But she never lost her passion for music. For Simmons, this passion has enhanced her appreciation of hearing.

In 2016, Simons co-founded Akouos, which focuses on disorders of the inner ear.Since then, Simmons has been ableCombine an interest in science with a commitment to developing treatments to restore hearing. His company has two outstanding projects. Both can appear on IND applications at the end of the year. If continued, these two programs will be the first two inner ear AAV programs to enter clinical development.

The first program, AK-OTOF, is an AAV gene therapy to restore hearing in patients with hereditary sensorineural hearing loss due to mutations in the otoferlin gene. The otoferlin gene is expressed in sensory cells (hair cells) found in the inner ear and converts sound-driven fluid waves into neural signals. The second program, AK-antiVEGF, is an AAV gene therapy for vestibular schwannoma. AK-antiVEGF is designed to induce cells in the inner ear to secrete antitumor proteins.

To date, no drug or biologic has ever been approved for direct administration into the inner ear. The inner ear is not easily accessible. It is closer to the brain than to the middle ear, with sensory cells encased in a bony, fluid-filled structure.

AAV gene therapy is "very good for the inner ear," Simons says. AAV vectors can be administered directly without exposure to other parts of the body, allowing a high proportion of vectors to reach target cells. Furthermore, hair cells do not divide, which means that the expression of functional genes can continue in the long term. (Because AAV vectors deliver genes as episomes, DNA that doesn't integrate into the cell's genome, cleavage means dilution.)

Furthermore, AAV vectors were relatively well tolerated in the immunocompromised inner ear. In Akouos's tests with non-human primates, the AAV vectors did not elicit an immune response, even when administered in high doses. Still, Akouos used lower doses of AAV vectors than are used in traditional gene therapy. Normally, the dose of AAV vector delivered into the bloodstream is 10¹⁴o 10¹⁵virus particles per kilogram. Akouos used doses three to four orders of magnitude lower than that. Together, these factors take many worries out of Akouos' job.

accidental coincidence of factors

When Akouos was founded, Simons and the company's other co-founders, Michael McKenna, MD, and Bill Sewell, MD, focused on the surgical delivery methods required to deliver vectors into the inner ear. But they don't have vectors. In fact, at that time it was not known whether a capsid with tropism for the inner ear existed.

It didn't take long for Akuos to get lucky. He comes from the laboratory of Dr. Luk Vandenberghe, director of the Grousbeck Genn Treatment Center at Massachusetts Eye and Ear. Vandenberghe constructed an ancestral library of AAV capsids using the capsid sequences of all naturally known AAV capsids and computationally predicting ancestry from him. He then developed a synthetic AAV vector that could safely and efficiently transfer the gene into the mammalian inner ear (Landegger et al., 2017).Evening. Biotechnology.2017; 35 (3): 280-284).

Vandenberghe demonstrated that the AAV Anc80 vector has a high transduction efficiency of inner ear hair cells. Akouos licensed the entire library (38,000 different capsids) for all inner ear applications. Combining Vandenberghe's Anc80 with a surgical application method for the inner ear means the company is moving forward. These factors "come together to open up a new field in medicine," Simmons said.

So Simmons is worried about a lawsuit? He admitted that he was the first of his kind and that children were recruited. Until the trial begins, the company won't know if the initial dose will have an effect or if children will benefit.

The obstacles to gene therapy are many and difficult to overcome. Even with fewer immune system complications, such as Akouos's work with the inner ear, the question of efficacy remains.

Reflecting on her early days in the field, Kay says that even small advances are cause for celebration: "If we could turn a cell into a positive mouse, we'd be jumping." Inspiration was found in bone marrow development and transplantation. These patterns took decades to build, proving that patience can be rewarded.

Many different types of methods can help. As Kay said, "We don't know which platform is better." She added that there are right and wrong ways to do things. But there is no real way to go.