82. Scalable, Animal-Free, Suspension-Based Production of Siv Lentiviral Vectors

Abstract

We are developing lung gene transfer vectors to direct abundant, sustained transgene expression in three therapeutic compartments: (i) within lung cells to treat lung disorders such as cystic fibrosis (CF), (ii) secreted from lung cells into the lung lumen to treat viral infections and airway diseases such as alpha1 anti-trypsin deficiency, (iii) secreted from nasal and lung cells into the circulatory system to treat hematologic and a range of other disorders. Such diseases are associated with a high burden of care often involving frequent antibiotic treatments, physiotherapy and supportive systemic infusions; and considerable patient morbidity and mortality. We have developed an efficient lung gene transfer platform based on a European and US Pharmacopoeial compliant, minimal recombinant SIV lentiviral vector which is specifically pseudotyped with the Sendai virus envelope proteins F and HN (rSIV.F/HN). Initially, production of rSIV.F/HN vectors was established in a serum-dependent, calcium phosphate-mediated, transient transfection process. Here we report the development of a scalable, animal-free, cGMP-compliant suspension cell culture based upstream production (USP) method. Serum-free media screening identified a suspension cell growth conditions that support efficient (>70%) multiple plasmid transient transfection of HEK293T cells with a range of cGMP-compliant liposomal and polymeric gene transfer agents. Small volume (40–400mL) studies in Erlenmeyer shake flasks and Design Of Experiments statistical approaches identified robust growth and transfection conditions that yield ≥5e6 TU/mL of unpurified rSIV. F/HN vectors. These parameters supported transition to 1L and 5L scale WAVE bioreactor cultures, where virus yield is maintained and plasmid DNA usage (a high proportion of the cGMP cost of goods) has been reduced to 0.33mg/L. F/HN pseudotype specific DNA removal and virus activation steps are incorporated into the downstream purification (DSP) method which relies on anion-exchange membrane virus capture and hollow-fibre UF/DF for vector purification and final formulation. In 56 independent, pH-controlled WAVE bioreactor cultures, purified yield exceeds 2.0e9 TU/L for a range of reporter gene constructs with a variety of promoters and 1.2e9 TU/L for a range of CFTR constructs (p>0.05; n=42/14 respectively). The final product directs abundant in vivo airway gene transfer, transducing 14.1% epithelial cells in the murine lung (p<0.001; 8e8 TU rSIV.F/HN hCEF EGFP), and directing CFTR transgene expression at ~30% of endogenous CFTR mRNA levels in the ovine lung (p<0.001; 8e8 TU rSIV.F/HN hCEF soCFTR2), exceeding presumed therapeutic levels. While these regulator friendly USP/DSP approaches have been tuned to yield highly purified and potent rSIV.F/HN vectors, the methodology is readily transferrable to VSV-G pseudotype production.