Gene therapy is well versed as a promising approach for treating and preventing genetic disorders, including lymphoma, glioma, osteoarthritis, haemophilia A & B, and congenital blindness (to name a few).1 The field encompasses diverse methods such as gene replacement, addition, editing, and silencing, reflecting its rapidly evolving nature.
Typically, functional genes are introduced into defective target cells, providing the capacity to correct, replace, or supplement defective genes and modulate gene expression. This offers the potential for long-lasting therapeutic effects across a wide range of disorders.
One key avenue for gene delivery is through viral vectors, which are viruses that have been genetically engineered to deliver genetic material into target host cells efficiently.2 Viral vectors offer high transduction efficiency, stable gene expression, and the ability to target specific cell types.
Among viral vectors, adeno-associated viruses (AAVs) are widely used. What sets AAV vectors apart from other viral vectors, such as adenovirus, lentivirus and retrovirus products, is that 1) AAVs naturally have low pathogenicity, which stems from their inability to replicate without a helper virus, and 2) AAVs produce a lower immune response compared with alternative viral vectors.3,4 However, AAVs are not without their limitations.
The case for EV-AAVs
Classically considered non-enveloped viruses (i.e., lacking a biological membrane), AAVs have recently been shown to possess the ability to acquire a membrane. During production, some AAVs are naturally secreted in association with EVs – either encapsulated within or attached to the surface.5 This EV-AAV association has been shown to offer clinically desirable characteristics over solo-AAVs, including improved cellular transduction, targeted tissue delivery, and antibody neutralisation evasion.
Key benefits over solo-AAVs
For AAV vectors to mediate gene therapy effects in vivo, they must evade neutralisation by pre-existing anti-AAV antibodies in the blood stream. An estimated 50% of the population is believed to have some degree of anti-AAV antibodies, which can significantly reduce the efficacy of AAV vectors by lowering gene transfer and associated expression.6 EV-AAVs prevent this neutralisation, demonstrating up to 136-fold greater resistance to neutralisation compared to solo AAV vectors in vitro.7
Lung carcinoma is one disease where AAV-mediated gene therapy shows potential. However, solo-AAV vectors are found to exhibit low specificity to certain subtypes of lung carcinomas – perhaps due to antibody neutralisation. EV-AAVs showed improved delivery to lung carcinomas, increasing cellular transduction and evading antibody neutralisation compared with solo-AAVs.8
Moreover, evidence of EV-AAVs passing the blood-brain barrier (BBB) makes them an appealing approach for treating neurological disorders.9 This ability to cross the BBB is not dependent on the AAV serotype, but is instead (according to a recent preprint) attributed to the EV surface proteins, which mediate entry into the brain.10
Upon reaching the target cell, transduction must take place. Transduction is the process of a viral vector delivering its genomic package into a target cell, where it can elicit protein translation and therapeutic effects. EV-AAVs reportedly have higher cell transduction than AAVs alone, in addition to evading the immune system.11,12 Specifically, EV-AAVs showed improved transduction over solo-AAVs when used in treating inherited retinal degeneration disorders.12
SEC as a tool for purifying EV-AAVs
EV-AAV are promising candidates for gene therapy, offering attractive benefits over solo-AAVs – but achieving high purity is crucial to fully unlocking these advantages. In line with its established role in the EV field, size-exclusion chromatography (SEC) is emerging as a reproducible and scalable isolation method that can facilitate clinical translation of EV-AAVs.
In the investigation into EV-AAVs and their ability to cross the BBB described above,10 SEC via qEVoriginal 70 nm columns demonstrated improved yield and transgene expression efficiency over differential ultracentrifugation (UC), while reducing contamination. Although the size of EV-AAVS and the total protein amount was unchanged between the two methods, SEC yielded a 6.6-fold higher amount of EV-AAVs over UC. Importantly, this increase in isolated EV-AAV yield with SEC did not correspond with an increase in contaminating proteins, suggesting that some EV-AAVs may have been lost with UC.
Given these results, SEC not only improves yield and reduce contamination compared with UC, but it also offers scalability. A study investigating this aspect confirmed that SEC is a more scalable solution for isolating EV-AAVs compared with UC and density gradient method.13
Facilitating clinical translation of EV-AAVs via the qEV Isolation platform
In summary, EV-AAVs represent a promising advancement for gene therapy, offering enhanced transduction efficiency, targeted delivery and immune evasion compared with solo-AAVs. To fully harness these benefits, high-purity isolation is essential. Izon's SEC qEV columns are a scalable and effective method for isolating EV-AAVs.
Interested in isolating EV-AAVs with SEC? Learn more about the qEV Isolation platform here.
