Anion-exchange HPLC assay for separation and quantification of empty and full capsids in multiple adeno-associated virus serotypes.

Gene therapy has entered a new era where numerous therapies for severe and rare diseases are generating robust and compelling clinical results. The rapid improvements in gene therapies over the past few years can be attributed to better scientific understanding of the critical quality attributes that contribute to a safe and efficacious product, as well as a better understanding of the manufacturing processes that are required to yield consistent products, which routinely meet the quality standards required for clinical studies. Of particular concern is the need for an effective, quality control (QC)-compatible, and versatile test method for the quantification of empty and full capsids in recombinant adeno-associated virus (rAAV) samples from multiple serotypes. In that regard, we describe the development of a QC-compatible anion-exchange chromatography method consisting of a modular discontinuous gradient to achieve full baseline peak separation and quantification of empty and full AAV capsids. Using an rAAV6 vector, our assay was shown to be precise, linear, robust, and accurate-correlating well with orthogonal methods such as analytical ultracentrifugation (AUC) and cryogenic transmission electron microscopy (Cryo-TEM). Additionally, we demonstrate the versatility of our approach by adapting the method to separate and quantify empty/full capsids in samples from several rAAV serotypes.

A novel telomerase activator suppresses lung damage in a murine model of idiopathic pulmonary fibrosis.

The emergence of diseases associated with telomere dysfunction, including AIDS, aplastic anemia and pulmonary fibrosis, has bolstered interest in telomerase activators. We report identification of a new small molecule activator, GRN510, with activity ex vivo and in vivo. Using a novel mouse model, we tested the potential of GRN510 to limit fibrosis induced by bleomycin in mTERT heterozygous mice. Treatment with GRN510 at 10 mg/kg/day activated telomerase 2-4 fold both in hematopoietic progenitors ex vivo and in bone marrow and lung tissue in vivo, respectively. Telomerase activation was countered by co-treatment with Imetelstat (GRN163L), a potent telomerase inhibitor. In this model of bleomycin-induced fibrosis, treatment with GRN510 suppressed the development of fibrosis and accumulation of senescent cells in the lung via a mechanism dependent upon telomerase activation. Treatment of small airway epithelial cells (SAEC) or lung fibroblasts ex vivo with GRN510 revealed telomerase activating and replicative lifespan promoting effects only in the SAEC, suggesting that the mechanism accounting for the protective effects of GRN510 against induced lung fibrosis involves specific types of lung cells. Together, these results support the use of small molecule activators of telomerase in therapies to treat idiopathic pulmonary fibrosis.