New Method to Enhance AAV Gene Therapy

Adeno-Associated Viruses (AAVs) are important tools for delivering genes to treat various diseases. Several AAV Gene Therapies are currently available in the United States, including Luxturna for vision loss and Zolgensma for spinal muscular atrophy. There are many clinical trials using AAVs, and companies are working on improving their design for better performance. However, there has been a slowdown in progress due to the body's immune system recognizing these viruses as foreign. A significant portion of the population already has antibodies that target these viruses, which can prevent effective treatment for many patients. Finding ways to deal with these Neutralizing Antibodies is a key challenge in AAV gene therapy.

#Current Challenges in AAV Gene Therapy

Various strategies have been attempted to overcome the problem of neutralizing antibodies. One approach involves modifying the outer structure, or capsid, of the virus through techniques like directed evolution and machine learning. While these methods can create new virus versions that can evade some antibodies, they often do not fully solve the issue and can sometimes make the virus less effective.

Other methods have included treatments like plasmapheresis, which removes antibodies from the blood, but this process can be expensive and risky. Researchers also discovered in 2012 that AAVs can sometimes be packaged into protective carriers called exosomes, which can help them avoid destruction by antibodies. However, this method has faced challenges such as low production levels and difficulty in measuring the amount needed for treatment. Chemical modifications to the capsid have been tested as well, but these often lead to reduced effectiveness in getting the virus into target cells.

#Introduction to Vaults

Vaults are tiny structures found in most cells and are thought to have existed in early cell ancestors. They can be produced in a lab using insect or yeast cells. These vaults can be used to carry drugs and vaccines because they do not trigger an immune response. They naturally target certain immune cells, but scientists can change their targeting abilities to deliver cargo to specific cell types.

Vaults can effectively release their contents inside target cells, which is beneficial for delivering various treatments. However, researchers have faced challenges when trying to package larger molecules such as nucleic acids inside vaults. Despite these challenges, vaults have proven useful for delivering smaller proteins and drugs, especially in vaccine development.

#Developing a New Delivery System

Our team has developed a new method of delivering AAVs that can get around the issue of neutralizing antibodies while also improving the ability of the virus to enter cells. This method uses a technology called SpyTag-SpyCatcher to connect the AAVs with the INT peptide, enabling them to be packaged inside vaults, leading to what we call vaultAAV (VAAV). This is a new achievement since no group has previously packaged an entire virus into vaults.

The new VAAV has shown the ability to enter cells effectively, even when neutralizing antibodies are present. This suggests that our approach has the potential to provide a solution to the challenge faced in AAV gene therapy.

#The Process of Creating VAAV

To specifically attach the INT peptide to the AAV, we used SpyTag-SpyCatcher technology. We modified a part of the AAV capsid to include the SpyTag. This change allows us to control the virus's ability to connect with the INT peptide, which is essential for packaging it into vaults.

Once we made these changes, we mixed the modified AAV with the INT peptide to form a new compound called AAV9-INT. Following this, we combined this new compound with vaults. To our surprise, just a short incubation period allowed for the packaging of many AAVs into vaults, even though some AAVs remained unencapsulated.

#Imaging and Validation of VAAV

To confirm that our AAVs were successfully packed into vaults, we used electron microscopy techniques. These images showed that VAAVs were formed, indicating that the AAVs were enclosed within the vault structures. Some vaults even appeared to carry two AAVs at once.

Further imaging techniques provided a more detailed view of where the AAVs were located inside the vaults. This work provided strong evidence of our successful packaging.

#Testing VAAV Against Neutralizing Serum

To test how well VAAV could protect AAVs from neutralizing antibodies, we conducted experiments using serum from mice that had been immunized against our modified AAV. In a test, we found that the serum completely blocked AAV from entering target cells. However, when we introduced the VAAV, it was still able to enter the cells, even in the presence of neutralizing antibodies, which was an encouraging result.

We compared the performance of VAAV with other groups, including a mix of vaults and AAVs without INT, and a control group using only AAV. The results showed that VAAV had a significant advantage in protecting its cargo and effectively entering cells compared to the other groups.

#Implications of VAAV Technology

AAV gene therapy has struggled with the neutralizing antibody issue for a long time, and current solutions have shown limited success. Our VAAV approach represents a potential breakthrough by using the vault as a protective shield around the AAV. This technology could be useful since vaults can be modified to target specific cell types more effectively.

Our results also suggest that even when vaults are mixed with AAVs, there is a notable improvement in the ability to enter cells. This might help reduce the need for large doses of AAV, which can often be toxic and costly. Our observations indicate that as VAAV is developed further, it could be valuable in targeting different tissues and expanding treatment options for diverse patient populations.

#Conclusion

VAAV represents a potential new solution to the challenges faced in AAV gene therapy, particularly in overcoming neutralizing antibodies and improving the efficiency of cellular entry. With our use of SpyTag-SpyCatcher technology for the targeted packaging of AAVs into vaults, we have demonstrated that it's possible to protect these viruses while enhancing their performance. This research lays the groundwork for future developments that could make AAV gene therapy more effective and accessible for patients, including those who previously could not benefit from such treatments.

Overall, VAAV not only aims to address key issues in current gene therapies but also opens the door for further innovations in targeted drug delivery, potentially transforming how various diseases are treated in the future.