Pediatric Pulmonology, Volume 50, Issue S41, Abstract 263
The North American Cystic Fibrosis Conference, Phoenix, 2015
Preparations are underway to progress a new Lentivirus-based vector into CF patients to evaluate viral-mediated gene therapy for CF lung disease. In anticipation of the need for highly efficient gene transfer to the airway epithelium, the UK CF Gene Therapy Consortium has developed a Lentivrus vector pseudotyped with the F and HN coat proteins from Sendai virus providing high affinity for airway cell transduction. A single dose has been shown to provide high-level, sustained gene expression in the murine airways for the lifetime of the animal. The new, regulator-compliant (EU & US Pharmacopoeia) Lentivirus vector (rSIV.F/HN) is approximately 2 logs more efficient than the leading non-viral vector in the mouse lung and can be repeatedly administered.
However a key challenge for progression to clinical studies is the generation of sufficient purified virus expressing CFTR. Initially, rSIV.F/HN vector production utilised standard, serum-dependent, calcium phosphate-mediated, transient transfection protocols. Here, however, we report the development of a scalable, animal-free, cGMP-compliant suspension cell culture upstream process (USP). Screening in serum-free media identified suspension cell growth conditions that support efficient (>70%) multiple plasmid transient transfection of HEK293T cells with cGMP-compliant gene transfer agents. Small volume (40 - 400mL) studies identified robust growth and transfection conditions yielding ≥5e6 Transducing Units pr mL (TU/mL) of unpurified rSIV.F/HN.
We have also scaled the process to 1L and 5L WAVE bioreactor cultures, where virus yield is maintained and plasmid DNA usage - a costly proportion of virus production - is reduced. A downstream process (DSP) specific to the F/HN pseudotyped vector incorporates anion-exchange membrane virus capture and hollow fibre ultra-filtration/dia-filtration for purification and final formulation. In 56 independent, pH-controlled WAVE bioreactor cultures, the purified yield was >2.0e9 TU/L for a range of reporter gene vectors and >1.2e9 TU/L for CFTR vectors (p>0.05; n=42/14 respectively).
This regulator-compliant USP/DSP process is transferable to other Lentiviral pseudotypes and has also been approved for planned toxicological studies in support of clinical trials.
Supported by grants from the Medical Research Council (UK) & Cystic Fibrosis Trust (UK).