Use Of Molecularly Imprinted Polymers To Improve Efficiency Of Adeno Associated Virus Mediated Gene Therapy

Gene therapy has the possibility to change the lives of patients around the world suffering from genetic diseases. By addressing the underlying genetic cause, it may be able to provide lifelong curative treatments to currently incurable conditions.

Adeno associated virus has emerged as a leading vector in this field to deliver corrective genes to patients for the treatment of monogenic disorders. Interest in the field is reflected in the rapidly growing number of therapies in clinical trials. There are, however, some limitations in their practical applications due to challenges both in their delivery and manufacture which must be overcome if these therapies are to reach their full potential of prolonging and improving the lives of as many patients as possible.

Developing techniques for addressing these limitations will improve the overall efficiency of gene therapy. This thesis describes the first steps taken to address these issues by implementing molecularly imprinted polymers (MIPs) to identify potential peptide targets for separation and as novel biomimetic affinity chromatography solutions.

The work described seeks to overcome one clinical and one manufacturing challenge. The clinical challenge to address is neutralising of viruses by neutralising antibodies (NAbs). These are present in certain patient populations with pre-existing humoral immunity to AAVs and prevent gene transfer. The key industrial challenge is achieving separation of the therapeutically efficacious genome containing capsids from empty capsids in a manner that is robust, efficient, and scalable. Empty and Full particles are similar in size, capsid morphology and physicochemical properties and empty particles in the drug product increase the immunogenic load to the patient while providing no therapeutic benefit.

Based upon the challenges posed by NAbs and Empty/Full particle separation this thesis successfully demonstrates use of a novel molecular imprinting technique to identify novel peptide sequences on NAbs and AAV capsids as potential targets for affinity purification. The peptides were used to generate molecularly imprinted polymers which were then integrated with chromatography resin as biomimetic affinity ligands. Generating novel prototype nanoMIP columns for technically challenging biological separations and looking towards what manufacturing challenges and scales are required to achieve this.

Providing affinity solutions to these clinical and industrial challenges is critical to the successful widespread proliferation of AAV based gene therapies and thus bringing life changing curative treatments to those with monogenic disorders.