Overcoming the challenges of recombinant adenoassociated virus production to treat Muscular dystrophy
Gene therapy is defined as the treatment of a disease by transferring genetic material into cells. One of the most commonly used viral vectors in gene therapy and with the greatest potential are recombinant adeno-associated virus (rAAV). rAAVs are highly versatile and have several advantages: To date rAAV have shown to be safe and effective in preclinical and clinical settings; rAAV can be used in a wide range of clinical applications in multiple diseases due to its unique biological and biophysical properties and rAAV presents low immunogenicity comparing to other viral vectors. Presently, clinical grade rAAV are mainly generated by transient transfection method. However, this approach presents difficulties when scaling up production and with robustness. The alternative used for commercial production are stably-transfected, producer cell lines systems where rAAV production is activated by the infection with a helper virus. Nonetheless, the construction of these systems is complex and time consuming due to several challenges ranging from cytotoxicity caused by viral infection to suboptimal product
quality and low product titer. Hence, in order to accelerate cell line development process, better approaches need to be developed to fulfil the production demand.
Genetic screens are a powerful tool to discover new genotypes/mutations that may be responsible for a phenotype of interest (ie, a mutation that confers drug resistance or confers better fitness to environmental conditions) and are evolving rapidly: The discovery of the Cas9 nuclease removed many of the technical and financial barriers to perform deletion and overexpression genetic screens in mammalian cells. The central AIM of this project is to develop a state-of-the-art Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR)-Cas9 based genetic screen technology to improve the cell line development process for rAAV production and to evaluate its direct applicability on a mouse model of a human disease.
Loss of function and gain of function genetic screens will be carried out to identify genes responsible for phenotypic variants that would improve product bioprocess and, consequently, accelerate cell line development process. As a proof or concept to evaluate the improved cell line development process generated in this proposal, we will produce a rAAV vector to treat LAMA2-related muscular dystrophy (LAMA2-MD), the most common congenital muscular dystrophy and which is a life threatening and currently incurable disease. For that we will produce a unique gene therapy aiming at reversing LAMA2-MD mutation; We will design a novel strategy by producing an all-in-one rAAV vector encoding a CRISPR- Cas9 system. By using the smaller Cas9 protein from Campylobacter jejuni, we will be able to assemble into rAAV vector the gRNA and donor sequence required to heal the muscular dystrophy mutation presence in the nmf417 mouse model of LAMA2-MD. Moreover, in order to stablish a production platform at iBET, we will develop a downstream processing protocol for purification of the CRISPR-Cas9 rAAV vector produced by Hela-S3 cell line and state-of-the-art analytical tools for the accurate quantification and characterization of the final product.