The success of gene therapies for addressing inherited disorders has been demonstrated by FDA approval of AAV-based LUXTURNA and ZOLGENSMA. There is great hope that gene therapies will also be able to address more of the 10,000+ monogenic diseases that have been identified to date. Gene therapy developers face challenges in cost and access to manufacturing capacity to produce the gene therapy product. The development of treatments for ultra-rare genetic diseases, which occur in fewer than one person in a million, are especially disadvantaged as these therapies are not typically of commercial interest to biopharmaceutical manufacturers. There is an urgent need for innovation in meeting the need for developers of gene therapies for ultra-rare diseases to gain access to GMP manufacturing of small doses of drug product. We propose a flexible, cost-efficient, end-to-end manufacturing platform for production of a small number of doses of AAV gene therapy products. We use the method of triple transfection in HEK cells, which can be adapted to multiple genetic diseases by switching the disease-specific genomic insert in one of the plasmids, as well as adapted to multiple AAV serotypes by switching the AAV-specific plasmids. Our easy-to-use microbioreactor platform grows cells to high-density in perfusion. We will optimize the the triple-transfection process for high-density cell cultures to increase bioreactor productivity and generate material for treating small numbers of patients. Additionally, our platform addresses the need for innovation in downstream purification of AAV products. Empty capsids lacking the gene of interest can compose 10% - 90% of AAV produced in cell cultures. Current vector purification approaches require specialized equipment which may not be readily available in the standard laboratory. We will develop a novel downstream purification method based on electrokinetic separation which greatly simplifies elimination of empty AAV capsids. Finally, as the supply of GMP-grade plasmid needed for the triple transfection process can also be a bottleneck, as well as significant expense, we will demonstrate that our platform is capable of incorporating plasmid manufacturing, including production and purification, to provide an end-to-end solution to generation of AAV therapies. This platform, will (1) expedite the availability of AAV treatments for ultra-rare diseases by increasing the volumetric yields of upstream process with minimization of the cost; (2) reduce the time, cost and labor associated with the downstream AAV purification; and (3) incorporate plasmid production to ensure timely and low-cost supply of this critical raw material. This platform will democratize access to viral vector for developers of gene therapies for ultra-rare diseases.

Public Health Relevance

We will develop an easy-to-use, low cost platform that will enable manufacturing of small quantities (5-20 doses) of AAV gene therapy drug product. This platform will address the problem of cost and access to gene vectors which is a bottleneck for developers of gene therapies, but especially for those developing therapies for ultra-rare diseases which are not of significant commercial interest. This platform can be upgraded to GMP-readiness, which will accelerate development of gene therapies in general by providing a path to early access to material suitable for use in clinical trials.

National Institute of Health (NIH)
Food and Drug Administration (FDA)
Research Project (R01)
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Special Emphasis Panel (ZFD1)
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Osorio, Manuel
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Massachusetts Institute of Technology
Engineering (All Types)
Biomed Engr/Col Engr/Engr Sta
United States
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