Clinical Development of an Optimal F/HN Pseudotyped SIV Vector for Cystic Fibrosis Lung Gene Therapy

Animal models require considerable amounts of virus and clinical studies in the human lung will require production of substantial quantities of cGMP compliant virus

Existing cGMP viral production methodologies are based on transfecton of adherent cells and cannot be scaled efficiently

We have developed a scalable cGMP compliant rSIV.F/HN vector production and purification platform based on cells grown in serum-free suspension culture

Stephen C Hyde, Eric WFW Alton, A Christopher Boyd, Mary M Connolly, Mario Chan, Jane C Davies, Lee A Davies, Sabrina Gea-Sorli, Uta Griesenbach, Mamoru Hasegawa, J Alastair Innes, Makoto Inoue, Gerry McLachlan, Cuixiang Meng, Ian A Pringle, Stephanie G Sumner-Jones and Deborah R Gill

UK Cystic Fibrosis Gene Therapy Consortium, Oxford, London & Edinburgh United Kingdom and DNAVEC Corporation, Tsukuba, Japan.

Introduction Delivery of rSIV.F/HN to the Sheep LungFunctional Assessment of rSIV.F/HN in Mouse Models

The Need for Scalable Production of Lentivirus

The UK CFGTC are developing gene therapy for Cystic Fibrosis lung disease

Our non-viral platform is currently in a multi dose IIb clinical trial due to report Q1/2015 (Alton et al., 2013)

In parallel, we have been developing rSIV.F/HN, a recombinant viral vector based on SIV pseudotyped with the F and HN membrane proteins from Sendai virus - the murine form of Human Parainfluenza virus I

Overview of SIV Production Process in Serum-free Suspension Culture

Materials, Methods and References

Purification & Concentration of SIVVirus is captured on Mustand Q anion exchange membrane and eluted in high salt

Tangential flow filtration (TFF) used to concentrate rSIV.F/HN ~ x1000 fold

TFF in diafiltration mode used to exchange media for final buffer

Median vector recovery from initial harvest ~ 44 %

Process Evaluation ResultsFinal product 1 ml of vector at ≥ 1x109 TTU/ml per L cell culture

Acceptable particle : infectious particle ratio for rSIV.F/HN vectors (median 280 : 1)

Similar titres and processing characteristics for CFTR and reporter gene expression vectors

Outcomes Successful development of a scalable cGMP-compliant rSIV.F/HN production process•Methodology approved by MHRA for pre-clinical toxicology vector production•Routine weekly production of high titre rSIV.F/HN at 1-5 L production scale for development studies•

293T cells adapted to serum-free Freestyle media and maintained in suspension

Day 0Cells seeded into disposable bioreactors and grown to ~ 2 x 106 cells/ml using Wave 20/50 rocking bioreactor (GE Healthcare)

Day 2SIV production plasmids introduced into cells via transient transfection

Day 6SIV vector harvested by filtration from cell supernatant

Freeze at -80 for purification

SelectionofaLeadrSIV.F/HNVectorConfigurationinMouseModelsrSIV.F/HN vectors expressing luciferase were generated in our production system with 3 different transgene promoters:

CMV The standard hCMV IE promoter enhancer EF1a The human elongation factor 1a promoter hCEF CpG-Free human CMV enhancer and a CpG-Free version of EF1a (Hyde et al. 2008)

The viruses were delivered into the mouse lung (C57BL/6J n=10 1x108 TTU per mouse) and the duration of Luciferase activity was assessed by bioluminescent imaging over 6 months

Outcomes Identifiedleadcandidateexpressionconstructforfurtherstudies•rSIV.F.HN can be repeat administered (3 times) to the mouse without loss of activity•DemonstratedhighlevelsoftransductionefficiencyfromrSIV.F/HNpseudotypeinthemouselung•

mRNA, DNA and Luciferase Activity following SIV delivery to the sheep lungn=3 sheep received either 1.7x109 TTU or hCEF-CFTR or 3.4x109 TTU hCEF-Lux

Sheep were culled 7 days post dosing and samples prepared for Lux activity assay or Taqman analysis of mRNA/DNA

Naive segments in the same sheep were used as controls

Sheep ResultsrSIV.F/HN derived transgene mRNA was present on average at 23 % of endogenous ovine CFTR levels

1 copy of virus DNA was detected for every 10 sheep diploid genomes - therefore transduction efficiency was estimated ~ 10 % of total cells

Luciferase activity was consisently high in all three sheep and throughout the dosed segments

This dataset demonstrates the highest levels of activity we have every observed in the sheep lung with these assays for any gene transfer agent

Prior to commencing clinical studies with rSIV.F/HN CFTR vector we intend to complete a formal toxicology study assessing vector biodistribution, acute and chronic toxicity and vector insertion site profiling

Future Plans

Harvest Setup Primary Harvest Supernatant

Mouse Promoter Selection ResultsAll viruses demonstrated long lasting Lux activity (≥ 6 months) in the lung and nose

The highest level Lux activity in both tissues was observed when hCEF was used

hCEF resulted in mean Lux activity ~ 1 log higher than CMV

We have previously used the sheep lung as a large animal model for lung gene transfer (McLachlan et al., 2011)

For initial rSIV.F/HN sheep studies a segmental instillation approach was used (Emerson et al., 2003)

Determination of Taqman Transducing Units (TTU) DNA Titre

1. Serial dilutions of purified virus were added to 293F cells (0.9 ml @ 5 x 105 cells/ml, Freestyle media, 8 µg/ml Polybrene) in suspension culture2. DNA was extracted at Day 3 and Day 6 post transduction3. TTU was determined by Taqman analysis on integrated copies of the WPRE element in the integrated virus DNA

Luciferase assessment by bioluminescent imaging (BLI) in mice

1. Mice (virus dosed and naive controls) were anaesthetised with isoflurane and instilled with 100 µl Luciferin (Caliper Life Sciences, 15 mg/ml)2. Imaging was performed in an IVIS Imager (Perkin Elmer) over 4 minutes exposure3. Analysis was conducted using Ivis Imager (Caliper) and average radiance/cm2 was calculated for the mouse and lungs

Alton, E. W. et al. A randomised, double-blind, placebo-controlled phase IIB clinical trial of repeated application of gene therapy in patients with cystic fibrosis. Thorax, 2013, 68, 1075-1077

McLachlan, G. et al. Pre-clinical evaluation of three non-viral gene transfer agents for cystic fibrosis after aerosol delivery to the ovine lung. Gene Ther, 2011, 18, 996-1005

Mitomo, K. et al. Toward gene therapy for cystic fibrosis using a lentivirus pseudotyped with Sendai virus envelopes. Mol Ther, 2010, 18, 1173-1182

Emerson, M. et al. Transfection efficiency and toxicity following delivery of naked plasmid DNA and cationic lipid-DNA complexes to ovine lung segments. Mol Ther, 2003, 8, 646-653

Outcomes Successful delivery of rSIV.F/HN to the sheep lung•High levels of rSIV.F.HN mediated transgene expression in the sheep lung•

Bronchoscope Mediated Segmental Delivery of SIV to the Sheep Lung

Dissected into multiple samples for processing Delivery by bronchoscope to lung segments Segments identified at PM

Other Studies (data not shown)rSIV.F/HN directs functional expression of CFTR (hCEF-CFTR) in cell culture models

At least 6 months duration of expression from integrase deficient (ID) rSIV.F.HN in the mouse lung and nose

Lux activity from ID vectors was 5-10 fold lower than from integrase competent IC vectors

rSIV.F/HN can be repeat administered (3 times) to the mouse lung and nose without loss of activity

Lung Activity Nose Activity

TransductionEfficiencyofrSIV.F/HNinMouseLungA GFP expressing virus driven by hCEF was generated and dosed to 3 mice (8x108 TTU/mouse)

Mice were harvested at 14 days and lung sections were prepared for direct visualisation of GFP