Gene Therapy and Re-dosing Strategy- Removal of Preexisitng anti-AAV Antibodies Using Capsid Specific Immunoadsorption

Adeno-Associated Virus, Preexisting Antibodies, & Challenges in Gene Therapy

Adeno-associated viruses (AAVs) have been widely used as a vector for the in vivo gene transfer (gene therapy) because of the following reasons
- lack of pathogenicity of the AAVs,
- the ability to establish long term transgene expression in post-mitotic cells, and
- successful gene transfer in the broad tissue and hosts
- recombinant AAVs have been used in clinical trials and approved by FDA

The presence of highly prevalent pre-existing antibodies against AAV has been observed in the population (Refer to Seroprevalence). These antibodies may have been produced by the prior exposure of wild type AAVs. These preexisting antibodies are believed to decrease bioavailability inhibit the transduction of viral gene therapy products into the target cells and reduce efficacy.  To overcome this, the screening and exclusion of the population consisting of high anti-AAV titers are excluded from the clinical studies. This approach severely limits the availability of life-saving drugs to the disease populations. Recent studies have shown that the durable expression level of the transgene may decline over time, therefore, the redosing of the gene therapy product may be essential for long term benefits. 

Plasmapheresis and Removal of Antibodies 
Plasmapheresis has been used for the removal of the autoantibodies in patients with auto-immune diseases. In this technique, the plasma is passed through the protein A column that binds and removes immunoglobulin. The immunoglobulin depleted plasma and blood cells are combined and reinfused into the patients (shown in figure). 

Although plasmapheresis has been beneficial in immune disorders,  it may not be suitable for the removal of anti-AAV antibodies because 
- Anti-AAV antibodies are present in higher titers and require repeated cycles of removal and infusion
- Removal of large quantities of antibodies with all specificities may cause hypogammaglobulinemia
- Increases the risk of infection 

AAV-capsid Specific Antibody Removal
In this immunoadsorption approach, the Sepharose Bead is covalently conjugated with AAV capsid using NHS thioester chemistry. This AAV capsid conjugated Sepharose Beads may be used similar manner as the protein A column and plasma sample may be passed through the column for plasmapheresis. The use of AAV capsid-conjugated Beads adsorps antibodies and inhibitory factors specific to the AAV-capsid, therefore, allowing removal or specific antibodies and binding proteins to the AAV capsid. 

This immunoadsorption approach has been used to deplete plasma and test in vitro viral transduction in cell culture and in vivo animal plasmapheresis in animal models. The results from the study have shown (Ref- Orlowski et al, 2020, Bertin et al. 2020)
- The successful removal of AAV capsid specific antibodies,
- Empty capsid retains IgG more efficiently than full capsids
- Improved in vitro transduction and expression
- No leakage of the AAVs from the column

Promising Immunoadsorption Approach in Clinic
These proof of concept studies have shown the successful removal of AAV capsid specific antibodies in vitro and in-vivo animal models. The translation of this method into the clinic would provide will make the AAV based in vivo gene transfer more accessible for patients with high antibody titers and pave a path for redosing the gene therapy products.