Semirational bioengineering of AAV vectors with increased potency and specificity for systemic gene therapy of muscle disorders.

Bioengineering of viral vectors for therapeutic gene delivery is a pivotal strategy to reduce doses, facilitate manufacturing, and improve efficacy and patient safety. Here, we engineered myotropic adeno-associated viral (AAV) vectors via a semirational, combinatorial approach that merges AAV capsid and peptide library screens. We first identified shuffled AAVs with increased specificity in the murine skeletal muscle, diaphragm, and heart, concurrent with liver detargeting. Next, we boosted muscle specificity by displaying a myotropic peptide on the capsid surface. In a mouse model of X-linked myotubular myopathy, the best vectors-AAVMYO2 and AAVMYO3-prolonged survival, corrected growth, restored strength, and ameliorated muscle fiber size and centronucleation. In a mouse model of Duchenne muscular dystrophy, our lead capsid induced robust microdystrophin expression and improved muscle function. Our pipeline is compatible with complementary AAV genome bioengineering strategies, as demonstrated here with two promoters, and could benefit many clinical applications beyond muscle gene therapy.

Preclinical Development of an AAV8-hUGT1A1 Vector for the Treatment of Crigler-Najjar Syndrome.

Adeno-associated viruses (AAVs) are among the most efficient vectors for liver gene therapy. Results obtained in the first hemophilia clinical trials demonstrated the long-term efficacy of this approach in humans, showing efficient targeting of hepatocytes with both self-complementary (sc) and single-stranded (ss) AAV vectors. However, to support clinical development of AAV-based gene therapies, efficient and scalable production processes are needed. In an effort to translate to the clinic an approach of AAV-mediated liver gene transfer to treat Crigler-Najjar (CN) syndrome, we developed an (ss)AAV8 vector carrying the human UDP-glucuronosyltransferase family 1-member A1 (hUGT1A1) transgene under the control of a liver-specific promoter. We compared our construct with similar (sc)AAV8 vectors expressing hUGT1A1, showing comparable potency in vitro and in vivo. Conversely, (ss)AAV8-hUGT1A1 vectors showed superior yields and product homogeneity compared with their (sc) counterpart. We then focused our efforts in the scale-up of a manufacturing process of the clinical product (ss)AAV8-hUGT1A1 based on the triple transfection of HEK293 cells grown in suspension. Large-scale production of this vector had characteristics identical to those of small-scale vectors produced in adherent cells. Preclinical studies in animal models of the disease and a good laboratory practice (GLP) toxicology-biodistribution study were also conducted using large-scale preparations of vectors. These studies demonstrated long-term safety and efficacy of gene transfer with (ss)AAV8-hUGT1A1 in relevant animal models of the disease, thus supporting the clinical translation of this gene therapy approach for the treatment of CN syndrome.

The EB66® cell line as a valuable cell substrate for MVA-based vaccines production.

The selection of a cell substrate is a critical step for the development and manufacturing of a viral vaccine candidate. Several parameters such as cell susceptibility and permissiveness to the viral pathogens but also performance in terms of viral antigens quality and production yields are important considerations when identifying the ideal match between a viral vaccine and cell substrate. The modified vaccinia virus Ankara (MVA) is a replication-deficient viral vector that holds great promise as a vaccine platform, however only limited cell substrates have been tested or are available for industrialization. Here we evaluate the duck embryo-derived EB66® cell line as potential cell substrate for MVA production. To this end, we used two recombinant MVA constructs and demonstrated that EB66® cells are propagating the tested MVA viruses very efficiently, while preserving viral attenuation and transgene expression for up to 20 serial passages. Furthermore we developed upstream and downstream processes that enable industrialization of the virus production. In conclusion, we showed that EB66® cells can be used as potent cell substrate for MVA-based vaccines and represent therefore an attractive alternative for vaccine production.