The Effect of Plasmid DNA Quality on Gene Transfer Outcome ... · Non-Viral Gene Transfer to Lung...
Transcript of The Effect of Plasmid DNA Quality on Gene Transfer Outcome ... · Non-Viral Gene Transfer to Lung...
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The Effect of Plasmid DNA Quality on GeneTransfer Outcome in vivo
Reto BazzaniGene Medicine Research GroupUniversity of Oxford& United Kingdom Cystic Fibrosis Gene Therapy Consortium
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Non-Viral Gene Transfer to Lung
• Non-viral gene therapy for Cystic Fibrosis• Plasmid DNA complexed with cationic lipid• Plasmid DNA is:
• Circular, bacterial, double stranded DNA• Carrier of gene of interest• Manufactured in bacteria
• UK Cystic Fibrosis Gene Therapy Consortium• 5 clinical trials• DNA-Lipid (GL67A) complex applied to the lung• Patients showed flu-like symptoms• Unmethylated CG dinucleotides (CpGs)
PlasmidDNA
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Development of CpG-Free Plasmid DNA
Successful deletion of all CG dinucleotides on the plasmid DNA
= 1 CpG
CpG-rich pDNA317 CpGs
CpG-free pDNA0 CpGs
Hyde et al. (2008) Nat Biotech 26:549
Previous plasmid in clinical trial Current plasmid in clinical trial
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Test Plasmids in Mouse Lung Model
• Plasmid DNA/Lipid (GL67A) complexes (80 µg plasmid DNA in 100 µl)• Delivered by intranasal instillation under general anaesthesia• Female BALB/c mice (n=6 per group)• At day 1 post delivery collection of bronchoalveolar lavage fluid (BALF)• Analysis for pro-inflammatory cytokines• Water (Mock) treated group as negative control
Significant decrease in CpG-associated pro-inflammatorycytokines
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Residual levels of pro-inflammatory cytokines
***: sig different from CpG-free groupOne way ANOVA/Dunnett
• Significant increase of IFNγ and IL-12p40 after CpG-free plasmid DNA delivery
• Additional component in plasmid DNA preparations may contribute to residualinflammatory cytokines
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GMP Specifications for Clinical Plasmid DNA
• Is a component of the plasmid DNA preparation contributing to residual inflammatorycytokines?
• DNA concentration ≥5.3 mg/mL• Circular forms ≥80%• % host-cell DNA ≤2%• % host-cell RNA ≤2%• % host-cell protein ≤2%• Endotoxin levels ≤5 EU/mg• Total aerobic microbial, yeast and mold count <10 CFU/mL• Specific detection of S. aureus, P. aeruginosa,
Salmonella sp & E. coli <10 CFU/mL
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Hypothesis
• Plasmid DNA preparations contain residual host bacterial chromosomal DNA thatcan act as a source of unmethylated CpGs contributing to low-levels of pro-inflammatory cytokines in the lung
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Aims
• Plasmid forms in different preparations - capillary gel electrophoresis• Quantify chromosomal DNA in preparations - Q-PCR• Analyse plasmid performance in mouse lung model
• Quantify a range of pro-inflammatory cytokines - Multiplex assay (Bio-Plex)• Transgene expression - vector mRNA quantification by TaqMan
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Characteristics of different pDNA preparations
• 5 different batches of pGM169 plasmid DNA tested• Multiple manufacturers/processes
• Similar levels of plasmid integrity & forms
Plasmid ccc monomer[%]
ccc dimer[%]
oc forms[%]
pGM169-A 91.2 3.3 1.4pGM169-B 91.1 1.9 0.3pGM169-C 93.0 2.8 3.8pGM169-D 96.7 2.7 0.4pGM169-E 96.85 2.2 0.9
Ladder
Plasmid
ccc monomer
ccc dimer & open circular
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Characteristics of different pDNA preparations
• 5 different batches of pGM169 plasmid DNA tested• Multiple manufacturers/processes
• Similar levels of plasmid integrity & forms
• Considerable variation in level of bacterial chromosomal DNA
Plasmid ccc monomer[%]
ccc dimer[%]
oc forms[%]
chrDNA[%]
pGM169-A 91.2 3.3 1.4 4.1pGM169-B 91.1 1.9 0.3 2.6pGM169-C 93.0 2.8 3.8 0.5pGM169-D 96.7 2.7 0.4 0.2pGM169-E 96.85 2.2 0.9 0.05
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Effect of chrDNA on pro-inflammatory cytokines
R2=0.8867p=0.0015
R2=0.8913p=0.0014
• Significant correlation between levels of residual bacterial chromosomal DNA & cytokines
• Different plasmid DNA preparations were tested in the mouse lung model
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Effect of chrDNA on Vector mRNA Expression
Trend of higher vector mRNA expressionlevels with preparations containing lowlevels of chromosomal DNA
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Further Reduction of chromosomal DNA level
scDNase
5kb6kb~6.4kb
–DNase +DNase
Treatment of plasmid DNA samplewith ‘Plasmid-Safe’ DNase withoutaltering plasmid DNA forms
Reduced bacterial chromosomal DNAafter DNase treatment:
pre-treatment: 0.2%post-treatment: 0.002%
+–
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Measuring effect of reduction of chrDNA in vivo
• DNase treated sample tested in mouse lung model
• Significant decrease in total number of white blood cells after delivery of DNasetreated plasmid DNA preparations
• Preliminary results of an ongoing study
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Conclusion
• CpG-free plasmid DNA preparations contain low level bacterial chromosomal DNA• Level depends on manufacturer & process• Potential source of unmethylated CpGs
• Performance in vivo:• CpG-free plasmid DNA preparations containing low level bacterial chromosomal
DNA can result in low levels of pro-inflammatory cytokines• May lead to reduced transgene expression
• Development of manufacturing processes to further reduce or eliminate bacterialchromosomal DNA is desirable
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Acknowledgment
• Oxford UniversityDeborah Gill & Steve HydeRebecca ColesMary ConnollyLee DaviesRebekka Harding-SmithAnna LawtonDominique McCormickChela NunezIan PringleSarah SmithStephanie Sumner-Jones
Edinburgh
London
Oxford
Edinburgh UniversityImperial College
Martin SchleefMarco Schmeer