Investigators headed by a team at Brigham and Women’s Hospital have developed an adeno-associated virus (AAV) vector that studies show can far more efficiently cross the blood-brain barrier (BBB), in primate and rodent models, than previously developed delivery vehicles. The new vector construct, designated AAV.CPP.16, displays a cell-penetrating peptide (CPP) on the viral capsid. In vivo tests show that this new vector could effectively be used to deliver antitumor payloads in a mouse model of gliobalstoma.

“Our study is exciting because it shows that we are one step closer to being able to deliver gene therapy across the blood-brain barrier in humans,” said Fengfeng Bei, PhD, at Brigham’s department of neurosurgery. “Our findings demonstrate that AAVs could provide a valuable tool for developing systemic gene therapies against glioblastoma and other diseases where CNS delivery is required.”

Bei and colleagues reported their development in Nature Biomedical Engineering, in a paper titled, “Variants of the adeno-associated virus serotype 9 with enhanced penetration of the blood–brain barrier in rodents and primates.” In their paper, they concluded: “… we have engineered a potentially translatable BBB-penetrant AAV capsid that could be applied for systemic gene therapy in a broad range of CNS disorders as well as GBM … AAV capsids that can efficiently penetrate the BBB will facilitate the clinical translation of gene therapies aimed at the central nervous system.”

The BBB represents a major obstacle for gene therapy. Formed of cells wedged tightly together, the BBB keeps toxins and pathogens that may be present in the blood from entering brain tissue, but it also keeps out potential treatments for diseases that affect the central nervous system (CNS). AAVs are small, non-disease-causing viruses that can be engineered to carry and deliver DNA sequences to targeted cells. Previous studies have found them to be safe delivery vehicles for gene therapy, which aims to directly modify genes in cells to treat disease.

Scientists have developed some AAVs that can penetrate the BBB in mouse models, but most AAVs identified to date are not efficient enough to be considered for use in clinical settings. So, as the authors further commented, “Improving the efficiency of gene delivery remains a major challenge for gene therapies based on AAVs for the treatment of diseases of the CNS. The development of gene therapies for such diseases has “… been hindered by the limited availability of AAVs that efficiently traverse the BBB.”

Investigators from Brigham and Women’s Hospital, a founding member of the Mass General Brigham healthcare system, are working to optimize AAVs as gene delivery vehicles, improving their efficiency and their potential to deliver drugs to treat brain cancers such as glioblastoma and genetic diseases that affect the central nervous system.

To improve upon existing AAVs, Bei and colleagues turned to CPPs—a group of short peptides that are known to be able to cross biological membranes like the BBB. “CPPs are a group of short peptides that can cross biological membranes and that facilitate cellular uptake of otherwise membrane-impermeable molecular cargoes,” they explained. “It was reported that some CPPs could be used as conjugates to enhance the delivery of impermeable chemicals and nanoparticles across the BBB in both in vitro and in vivo models.”

The team collected about 100 of these CPPs and inserted them into the AAV capsid, testing them one by one to identify which could more efficiently deliver genes to the CNS. One of the peptides looked particularly promising. “We got lucky,” said Bei. “We got a hit right around number 16.”

The team tested out the AAV.CPP.16 finding in preclinical models, looking in both mice and nonhuman primates (NHPs). Their experiments confirmed that AAV.CPP.16 far more efficiently crossed the BBB than previously tested AAVs, and was 6- to 249-fold more efficient in different mouse strains, and approximately 5-fold more efficient in the cynomolgus monkey NHPs, than the parent AAV9 construct, in systemic gene delivery to the CNS. Experiments also confirmed that AAV.CPP.16 could deliver a secreted payload (PD-L1 antibody) and a nonsecreted payload (HSV-TK1 suicide gene) to the mouse brain, demonstrating the potential to use the system for systemic gene therapy against glioblastoma, the team noted. “When combined with other treatment modalities such as antibodies, AAV.CPP.16 could also be applied to tackle more complex CNS disorders such as GBM, a highly vascularized and infiltrative malignant brain tumor with a substantial unmet need. These findings show that AAV.CPP.16 could have broad application for multiple pathologies of the CNS.”

The newly reported data suggest the novel vector could be used to treat genetic diseases in which turning on protein production in a specified number of cells could reverse a disease. Bei’s lab is looking to make further improvements to the system. “We’d like to develop a version that is even more efficient and more restricted to the central nervous system. Our studies to date tell us we’re headed in the right direction,” he said.

Yulia Grishchuk, PhD, who leads a lab in the Center for Genomic Medicine at Massachusetts General Hospital, recently collaborated with Bei and sees potential disease applications for his research team’s laboratory-based advancements. “New treatments are urgently needed for neurometabolic diseases, lysosomal storage diseases, and other diseases that affect both CNS tissue and other tissues in the body,” said Grishchuk. “What is exciting here is that this work could represent a way to treat a broad spectrum of CNS disorders that are hard to target with current treatment approaches.”