Introduction Lung Gene Transfer Scalable Production of ... · PDF fileScalable Production of...
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rSIV.F/HN Vectors 0 100 200 300 400 500 700 800 900 Median P:I Ratio 280 Particle:Infectivity Ratio (vp/mL / TU/mL) Stephen C Hyde 1 , Eric WFW Alton 2 , A Christopher Boyd 3 , Jane C Davies 2 , Uta Griesenbach 2 , Deborah R Gill 1 , J Alastair Innes 3 & David Porteous 3 On behalf of the UK CF Gene Therapy Consortium [email protected] http://www.genemedresearch.ox.ac.uk UK Cystic Fibrosis Gene Therapy Consortium, University of Edinburgh 3 , Imperial College London 2 , University of Oxford 1 . The UK CF Gene Therapy Consortium are developing gene therapy vectors for the treatment of CF. Our non-viral platform has recently successfully completed a Phase IIb multi-dose clinical trial. In parallel, we have been developing rSIV.F/HN, a recombinant viral vector based on SIV pseudotyed with the F and HN membrane proteins from Sendai virus – the murine form of Human Parainfluenza virus I. Introduction Cellbag Scalable Production of Lentivirus Future Plans Pre-clinical and clinical studies require the production and purification of substantial quantities of rSIV.F/HN vectors. Existing cGMP viral production methodologies are based on transfection 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 animal-free suspension culture. Virus Production 293T cells adapted to animal-free growth in rocking bioreactors are transiently transfected with five-plasmid rSIV.F/HN production system. Virus is harvested 72 hours post-transfection and purified via anion exchange (AEX) and tangential flow filtration (TFF). Process yield (red dots CFTR vectors, black dots reporter vectors) typically exceeds 1x10 9 TU/L Lung Gene Transfer rSIV.F/HN vectors direct abundant gene transfer in the mouse lung Lungs harvested 14 days after delivery of 8x10 8 TU vGM107 hCEF EGFP per mouse rSIV.F/HN vectors direct long-lasting transgene expression in the mouse lung Duration of mouse lung and nose transgene expression after deliver of Integrase competent (IC) and defective (ID) vectors with a range of transgene enhancer/promoter sequences (hCEF = hybrid CpG-free CMV enhancer/elongation factor 1 alpha promoter, CMV= CMV enhancer/ promoter, EF1a = short-form elongation factor 1 alpha promoter). Transgene expression normalised to delivered dose. N=6-8 per group. rSIV.F/HN vectors direct abundant gene transfer in the sheep lung Lungs harvested 7 days after delivery of 1.7x10 9 TU vGM058 hCEF soCFTR2 or 3.4x10 9 TU vGM020 hCEF EGFPLux Additional Pre-Clinical data rSIV.F/HN administration is well-tolerated, no unusual integration patterns are observed and treated mice express transgene throughout their lifetime. rSIV.F/HN vectors direct abundant gene transfer after repeated administration to the mouse lung. rSIV.F/HN-CFTR vectors direct functional CFTR as judged by iodide efflux and intestinal organoid assays. Outsourced cGMP production of vGM058 (rSIV.F/HN hCEF soCFTR2) is planned for Q1 2016. Formal murine toxicology study assessing vector biodistribution, insertion site profiling and acute toxicity with vGM058 has been agreed with MHRA (the UK medicines regulatory authority) and is planned to start Q3 2016. Single-dose nasal clinical study protocol has been agreed with MHRA and is planned to start Q2 2017. 0 28 56 84 112 140 168 196 102 103 104 105 106 vGM020 hCEF IC vGM014 EF1a IC vGM076 hCEF ID vGM012 CMV- IC vGM074 CMV- ID Untransduced Days Post Transduction Lung Transgene Expression Avg Radiance [p/s/cm²/sr] 0 28 56 84 112 140 168 196 102 103 104 105 106 vGM020 hCEF IC vGM014 EF1a IC vGM076 hCEF ID vGM074 CMV- ID vGM012 CMV- IC Untransduced Days Post Transduction Nasal Transgene Expression Avg Radiance [p/s/cm²/sr]
Transcript of Introduction Lung Gene Transfer Scalable Production of ... · PDF fileScalable Production of...
rSIV.F/HN Vectors0
100
200
300
400
500700
800
900
Median P:I Ratio 280
Part
icle
:Infe
ctiv
ity R
atio
(vp/
mL
/ TU
/mL)
Stephen C Hyde1, Eric WFW Alton2, A Christopher Boyd3, Jane C Davies2, Uta Griesenbach2, Deborah R Gill1, J Alastair Innes3 & David Porteous3
On behalf of the UK CF Gene Therapy Consortium
[email protected] http://www.genemedresearch.ox.ac.uk
UK Cystic Fibrosis Gene Therapy Consortium, University of Edinburgh3, Imperial College London2, University of Oxford1.
The UK CF Gene Therapy Consortium are developing gene therapy vectors for the treatment of CF. Our non-viral platform has recently successfully completed a Phase IIb multi-dose clinical trial. In parallel, we have been developing rSIV.F/HN, a recombinant viral vector based on SIV pseudotyed with the F and HN membrane proteins from Sendai virus – the murine form of Human Parainfluenza virus I.
Introduction
Cellbag
Scalable Production of Lentivirus
Future Plans
Pre-clinical and clinical studies require the production and purification of substantial quantities of rSIV.F/HN vectors. Existing cGMP viral production methodologies are based on transfection 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 animal-free suspension culture. Virus Production
293T cells adapted to animal-free growth in rocking bioreactors are transiently transfected with five-plasmid rSIV.F/HN production system. Virus is harvested 72 hours post-transfection and purified via anion exchange (AEX) and tangential flow filtration (TFF).
Process yield (red dots CFTR vectors, black dots reporter vectors) typically exceeds 1x109 TU/L
Lung Gene Transfer rSIV.F/HN vectors direct abundant gene transfer in the mouse lung
Lungs harvested 14 days after delivery of 8x108 TU vGM107 hCEF EGFP per mouse
rSIV.F/HN vectors direct long-lasting transgene expression in the mouse lung
Duration of mouse lung and nose transgene expression after deliver of Integrase competent (IC) and defective (ID) vectors with a range of transgene enhancer/promoter sequences (hCEF = hybrid CpG-free CMV enhancer/elongation factor 1 alpha promoter, CMV= CMV enhancer/promoter, EF1a = short-form elongation factor 1 alpha promoter). Transgene expression normalised to delivered dose. N=6-8 per group.
rSIV.F/HN vectors direct abundant gene transfer in the sheep lung
Lungs harvested 7 days after delivery of 1.7x109 TU vGM058 hCEF soCFTR2 or 3.4x109 TU vGM020 hCEF EGFPLux
Additional Pre-Clinical data rSIV.F/HN administration is well-tolerated, no unusual integration patterns are observed and treated mice express transgene throughout their lifetime. rSIV.F/HN vectors direct abundant gene transfer after repeated administration to the mouse lung. rSIV.F/HN-CFTR vectors direct functional CFTR as judged by iodide efflux and intestinal organoid assays.
Outsourced cGMP production of vGM058 (rSIV.F/HN hCEF soCFTR2) is planned for Q1 2016. Formal murine toxicology study assessing vector biodistribution, insertion site profiling and acute toxicity with vGM058 has been agreed with MHRA (the UK medicines regulatory authority) and is planned to start Q3 2016. Single-dose nasal clinical study protocol has been agreed with MHRA and is planned to start Q2 2017.
0 28 56 84 112 140 168 196102
103
104
105
106
vGM020 hCEF ICvGM014 EF1a IC
vGM076 hCEF IDvGM012 CMV- IC
vGM074 CMV- IDUntransduced
Days Post TransductionL
un
g T
ran
sgen
e E
xpre
ssio
nAv
g R
adia
nce
[p/s
/cm²/s
r]
0 28 56 84 112 140 168 196102
103
104
105
106
vGM020 hCEF ICvGM014 EF1a IC
vGM076 hCEF IDvGM074 CMV- ID
vGM012 CMV- IC
Untransduced
Days Post Transduction
Nas
al T
ran
sgen
e E
xpre
ssio
nAv
g R
adia
nce
[p/s
/cm²/s
r]
