Molecular Therapy, Vol 23, S1, Abstract 82
The American Society of Gene and Cell Therapy Annual Conference, New Orleans, 2015
We are developing lung gene transfer vectors to direct abundant, sustained transgene expression in three therapeutic compartments:
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.
Keywords: Vector Production; Lentivirus Vectors; Respiratory/Lung Disorders
Session: Simultaneous Oral Abstract Sessions: Cell Manufacturing, Vector Production, and Biodistribution for Clinical Translation (3:15 PM-5:15 PM)
Date/Time: Wednesday, May 13, 2015 - 4:30 pm
Room: Empire D