The fact that the blood-brain barrier poses a challenge for in vivo gene therapy is no surprise. The leading vehicles for delivery of gene therapies are adeno-associated virus (AAV) vectors, yet keeping viruses and other circulating pathogens out of the brain is why the blood-brain barrier evolved.
Taking a page out of nature’s playbook, scientists now employ directed evolution of AAVs to escort gene therapies across the highly selective barrier to treat neurological diseases. Without the need for prior understanding of molecular mechanisms governing viral tropism, directed evolution fast tracks the design of AAV vectors that can be delivered through the vasculature to target specific cell types, including those within the central nervous system (CNS).
Capsida Biotherapeutics is one company using directed evolution to advance gene therapy for both CNS and non-CNS indications. Their bioengineering platform benefits from directed evolution, from transduction optimization all the way to vector manufacturing.
“We don't just take a capsid engineering approach towards tropism or specificity,” said Nick Goeden, vice president of technology at Capsida Biotherapeutics. “Part of our screening criteria is for capsids that can be manufactured more efficiently.”
Systemic delivery of AAV-based therapies
A mere 26 nanometers in diameter with the capacity to hold a little less than five kilobases of genomic material, AAVs naturally infect humans but exhibit low immunogenicity and are not known to cause disease (1). These traits contribute to AAV vectors’ success in delivering gene therapies.
“There's almost a symbiotic relationship between the AAV and the human in that they're constantly present in our body,” said Nick Flytzanis, vice president of research at Capsida Biotherapeutics.
For neurological disorders, researchers have relied on direct administration of gene therapies into the CNS, either surgically or by injection into the cerebrospinal fluid, to bypass the blood-brain barrier. More recently, there has been a flurry of interest in the potential for gene therapies to cross the blood-brain barrier after scientists showed that a single intravenous infusion of an AAV9-based therapy was safe and effective for treating spinal muscular atrophy in pediatric patients (2). The FDA approved the treatment in 2019.
The ability to intravenously administer gene therapy for CNS disorders is important not just because it is more convenient for patients, but also because it ensures broad distribution throughout the brain. A “microfluidic delivery system” is how Viviana Gradinaru, a neuroscientist at the California Institute of Technology, likes to think about blood vessels in the body.
“Delivery through the bloodstream can access not only very broad areas, but also areas that are difficult to access by surgery, such as the cerebellum, for example, or deep brain nuclei that are relevant for Parkinson's [disease],” said Gradinaru.
While FDA-approval of the AAV9-based therapy for spinal muscular atrophy is certainly exciting, Gradinaru stressed the significance of its indication for children less than two years of age. “What we learned from our research is that the blood-brain barrier in young mammals is much more permeant to vectors such as AAV9,” said Gradinaru. “However, as the organism matures, this becomes a much more difficult problem.”
Applying directed evolution to gene therapy
Recognizing both the challenges and benefits of systemic delivery of gene therapy for neurological disorders, Gradinaru and her research group took up the goal of crossing the blood-brain barrier almost a decade ago. The directed evolution platform employed at Capsida Biotherapeutics originated from pioneering research completed in her laboratory. At its core, the method is directed evolution of AAVs paired with advanced computation.
The first step in the path to engineering AAVs for gene therapy involved generating molecular diversity around the AAV9 serotype to create AAV variants that had the potential to cross the blood-brain barrier.
“We had to implement a very stringent selection process that allowed us to sort basically the good capsids from the overwhelming noise,” explained Gradinaru. “That process gave us — from a billion plus theoretical capsids — a handful of interesting possibilities that when we put them to the test, they were able to cross the blood-brain barrier in laboratory mice.”
More recently, Gradinaru’s research group developed a method of directed evolution that identifies variants of interest through both positive and negative selection3. For example, the method allows for selecting capsids that not only cross the blood-brain barrier to reach the brain but that also avoid organs known to cause complications, such as the liver.
“That method allows us to generate capsids that can deliver on multiple asks, whether it’s organ specificity or cell type specificity to systemic delivery, while at the same time, maintaining their ability to cross the blood brain barrier,” said Gradinaru.
From capsid design to vector manufacturing
In building out the platform at Capsida Biotherapeutics, Goeden focused on creating a fully automated, scalable capsid engineering platform that incorporates high throughput screening and validation in both human cell lines and non-human primates.
“We can do an entire round of screening from data to data in 12 weeks,” said Goeden.
Flytzanis added that the speed with which capsid engineering can be completed varies based on the therapeutic goal. In general, it takes less time to engineer capsids for a disease that requires broad gene therapy distribution and high efficacy than for a disease that requires organ targeting. Of course, both of those therapeutic goals involve even less engineering time than a disease that requires cell type-specific transduction.
Goeden and Flytzanis both emphasized that directed evolution of AAV capsids can also help optimize the manufacturing process. “We can actually engineer the capsid itself, either to package with a more full-to-empty ratio or to package more specifically,” said Goeden.
“We're able to apply that selective pressure to what's going to end up being a manufacturable drug product at the end, from the earliest stages all the way through to the end,” added Flytzanis.
A major challenge still ahead for systemic delivery of AAV gene therapy is to minimize the immune response to the AAV vectors. According to Flytzanis, directed evolution provides solutions to this problem as well. For example, capsids can be engineered to steer clear of immune cells and to avoid targets in the body that engender large immune responses.
“I don't think we should compromise in obtaining vectors that have the best safety profiles and the best efficacy because even with systemic delivery, through careful de-targeting and through careful optimization of the vector, that dose can be brought down,” said Gradinaru.
“The power of directed evolution is in numbers and versatility and the unprecedented ability to look for what one needs in this collection of highly evolved capsids,” said Gradinaru.
- Wang, D., Tai, P. W. L. & Gao, G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov 18, 358-378, doi:10.1038/s41573-019-0012-9 (2019).
- Mendell, J. R. et al. Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy. N Engl J Med 377, 1713-1722, doi:10.1056/NEJMoa1706198 (2017).
- Ravindra Kumar, S. et al. Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types. Nat Methods 17, 541-550, doi:10.1038/s41592-020-0799-7 (2020).