Adeno-associated virus (AAV) is a small, non-enveloped DNA virus that can infect both dividing and non-dividing cells. AAV has been widely used as a vector for gene therapy because of its low immunogenicity, broad tissue tropism, long-term expression and safety profile. However, one of the significant challenges for AAV gene therapy is the presence of pre-existing or induced antibodies against AAV in humans, which can reduce the transduction efficiency and limit the therapeutic efficacy.
There are two types of antibodies that can affect AAV gene therapy: neutralizing antibodies (NAbs) and binding antibodies (BAbs). NAbs are able to block the binding or entry of AAV into target cells, thus preventing gene transfer. BAbs are able to bind to AAV capsid proteins but do not interfere with a viral infection. However, BAbs may trigger immune responses such as complement activation, phagocytosis or antibody-dependent cellular cytotoxicity (ADCC), which can lead to the clearance or destruction of AAV vectors. Therefore, it is essential to have reliable methods to detect and quantify anti-AAV antibodies and to develop strategies to overcome their inhibitory effects.
There are Three Categories of Antibodies for the Detection of AAV:
1) AAV capsid protein antibodies that specifically react with denatured AAV capsid proteins and thereby recognize the linear epitopes of the viral capsids. These antibodies can detect all three capsid proteins (VP1, VP2 and VP3) across different serotypes, as they share high sequence similarity. These antibodies are useful for studying the expression levels, localization and interactions of capsid proteins by western blotting, immunofluorescence, immunoprecipitation or affinity chromatography.
2) AAV particle antibodies that specifically react with intact AAV particles, i.e. empty and full capsids and thereby recognize a conformational epitope of the assembled capsids of certain serotypes. These antibodies can distinguish between fully assembled and partially assembled capsids, as they only bind to conformational epitopes that are formed by distant amino acids on the same or different proteins. These antibodies are useful for quantifying intact viral particles by ELISA or dot blotting, detecting neutralizing antibodies in patient serum, determining protein-protein interactions or purifying virus preparations by affinity chromatography or immunoprecipitation.
3) Anti-AAV replicase protein antibodies that react with selected Rep (replicase) proteins in human AAV-infected cells. Rep proteins are essential for AAV replication and integration into host genome. They are encoded by four overlapping genes (Rep78, Rep68, Rep52 and Rep40) that share a common N-terminal region but differ at their C-terminal ends. These antibodies can be used to study the expression levels, localization and functions of Rep proteins by western blotting or immunofluorescence.
Strategies to Overcome Barriers to Anti-AAV Antibodies in Gene Therapy
Several strategies have been proposed to overcome or circumvent the barriers posed by anti-AAV antibodies in gene therapy, such as:
- Selecting AAV serotypes with low seroprevalence or cross-reactivity among different populations.
- Modifying AAV capsid proteins to evade antibody recognition or enhance transduction efficiency.
- Administering immunosuppressive drugs or plasmapheresis to reduce antibody levels or activity prior to vector infusion.
- Delivering vectors locally to target tissues or organs to avoid systemic exposure and immune response.
- Co-administering decoy vectors or nanoparticles to saturate or divert anti-AAV antibodies from therapeutic vectors.
These strategies are still under investigation and require further optimization and validation before clinical application. The development of standardized and predictive assays to measure anti-AAV antibodies is also essential for patient screening and monitoring. Anti-AAV antibodies are a significant obstacle for AAV gene therapy, but not an insurmountable one. With continued research and innovation, it may be possible to achieve safe and effective gene delivery for various diseases using AAV vectors.
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