Biomaterials 32 (2011) 9854e9865
Contents lists available at SciVerse ScienceDirect
Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials
The significance of plasmid DNA preparations contaminated with bacterial
genomic DNA on inflammatory responses following delivery of lipoplexes to
the murine lung
Reto P. Bazzania, c, Ying Caib, Henry L. Hebelb, Stephen C. Hydea, c, Deborah R. Gilla, c, *
a
b
c
Gene Medicine Research Group, NDCLS, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK
VGXI, Inc. 2700 Research Forest Dr., #180 The Woodlands, TX 77381, USA
United Kingdom Cystic Fibrosis Gene Therapy Consortium, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 August 2011
Accepted 31 August 2011
Available online 23 September 2011
Non-viral gene transfer using plasmid DNA (pDNA) is generally acknowledged as safe and nonimmunogenic compared with the use of viral vectors. However, pre-clinical and clinical studies have
shown that non-viral (lipoplex) gene transfer to the lung can provoke a mild, acute inflammatory
response, which is thought to be, partly, due to unmethylated CG dinucleotides (CpGs) present in the
pDNA sequence. Using a murine model of lung gene transfer, bronchoalveolar lavage fluid was collected
following plasmid delivery and a range of inflammatory markers was analysed. The results showed that
a Th1-related inflammatory cytokine response was present that was substantially reduced, though not
abolished, by using CpG-free pDNA. The remaining minor level of inflammation was dependent on the
quality of the pDNA preparation, specifically the quantity of contaminating bacterial genomic DNA, also
a source of CpGs. Successful modification of a scalable plasmid manufacturing process, suitable for the
production of clinical grade pDNA, produced highly pure plasmid preparations with reduced genomic
DNA contamination. These studies help define the acceptable limit of genomic DNA contamination that
will impact FDA/EMEA regulatory guidelines defining clinical grade purity of plasmid DNA for human use
in gene therapy and vaccination studies.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Cytokine
Bacterial genomic DNA
Gene therapy
Lung inflammation
Lipoplex
Plasmid
1. Introduction
Plasmid DNA (pDNA) is being investigated for therapeutic use,
including the development of strategies for gene replacement and
vaccination in humans and animals. Plasmid DNA can be administered alone, often termed ’naked’ DNA delivery, or complexed
with a cationic lipid (lipoplex), or polymer (polyplex) [1]. Non-viral
gene transfer vectors are generally regarded as being safer and less
toxic compared with viral vectors, and the absence of viral proteins
predicts that non-viral vectors should be less immunogenic and
thus can be administered repeatedly without generating an
immune response.
Gene replacement in the lung is being developed for treatment
of Cystic Fibrosis (CF), a monogenic disease affecting greater than
70,000 patients worldwide [2], where the key factor affecting
* Corresponding author. Gene Medicine Research Group, NDCLS, John Radcliffe
Hospital, University of Oxford, Oxford OX3 9DU, UK. Tel.: þ44 1865 221845;
fax: þ44 1865 221834.
E-mail address: [email protected] (D.R. Gill).
0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2011.08.092
morbidity and mortality is the chronic destructive infection of the
conducting airways, eventually leading to lung failure and premature death [3]. Plasmid DNA complexed with the cationic liposome
GL67A, which was specifically formulated for aerosol delivery, has
been delivered to the nose and lungs of CF patients. In one early
trial, pDNA/GL67A was aerosolised to the lungs of CF patients and
resulted in partial electrophysiological correction of the CF ion
transport defect in the lung after a single aerosol dose [4]. However,
lung delivery of the pDNA/GL67A lipoplex also resulted in an acute
inflammatory response [4,5], similar to that observed in early preclinical studies in mouse models [6e9]. This inflammatory
response was thought to be due in part to the plasmid containing
unmethylated CG dinucleotides (CpGs) [10,11], the ligand for the
Toll-like receptor (TLR)9 [12]. Following lipoplex delivery to the
murine lung, inflammatory markers, such as IFNg and IL-12, can be
detected in bronchoalveolar lavage (BAL) fluid [13], but are
decreased if CpGs in the pDNA are methylated [6,14], reduced [11]
or eliminated [10]. The UK CF Gene Therapy Consortium (UKCFGTC)
[15] has developed a non-viral, pDNA/GL67A formulation, where all
CpGs have been removed from the plasmid DNA, such that delivery
R.P. Bazzani et al. / Biomaterials 32 (2011) 9854e9865
of the new CpG-free plasmid (pGM169) to the murine lung was
inflammation-free [10].
Another observation from early lung gene therapy trials was
that transgene expression was transient, lasting no more than 1
week [4,16e20]. Investigation of this phenomenon in animal
models showed that loss of transgene expression did not correlate
with a loss of plasmid DNA [21], but instead, was linked to the
specific promoter used to control expression of the transgene
[10,22,23]. Replacement of commonly used viral promoters with
those from constitutively expressed ’housekeeping’ genes can
increase the duration of expression. For example, the use of the
human polyubiquitinC (UbC) promoter can lead to persistent
transgene expression in the mouse lung when delivered in the
form of naked DNA via electroporation [24] and following aerosol
lung delivery [10]. Unfortunately, this rather successful promoter
could not be incorporated into CpG-free plasmids, since the
removal of CpGs from the UbC promoter sequences abolished
promoter activity (unpublished data). However after testing
a variety of CpG-free promoter/enhancer combinations, high-level
persistent expression in the lung was achieved from a novel, CpGfree promoter based on the human cytomegalovirus enhancer and
elongation factor 1 alpha promoter (hCEFI promoter) [10]. Thus the
modification of plasmid DNA is an important area for the development of novel non-viral formulations. Plasmid sequences can be
easily manipulated to customise the level and duration of transgene expression and exploited for treatment of different disease
targets.
Plasmids can vary in size, but the majority of vectors are too
large for chemical synthesis that is economically feasible. When
produced in non-pathogenic E. coli, plasmid DNA only constitutes
1e2% of the total bacterial cell mass, which means its separation
from abundant impurities is challenging. Guidance from regulatory
bodies such as the Food & Drug Administration [25] and the
European Medicines Agency [26] recommends that in plasmid
preparations for human use, bacterial host protein, RNA, and
genomic DNA levels constitute preferably < 1% each and endotoxin
levels < 40 endotoxin units (EU)/mg, although more stringent
criteria may be required depending on the specific application.
Large scale manufacture of clinical grade pDNA still faces several
obstacles [27]. A low shear cell lysis process is critical, as this
determines the achievable concentration and purity of the final
product. Although non-chromatographic techniques such as
selective precipitation or aqueous two-phase partitioning have
been studied, anion exchange (AEX), hydrophobic interaction
chromatography (HIC), and size exclusion chromatography are
three major workhorses in plasmid purification. These allow for the
exploitation of plasmid specific characteristics such as charge
density, hydrophobicity and size. High purity is an ultimate goal,
but other factors such as cost, flexibility, broad utility and
9855
robustness are just as valuable for establishing a feasible
manufacturing process.
In this study, we investigated the inflammatory consequences of
delivering lipoplexes to the lungs of wild type (WT) and TLR9deficient (TLR9/) mice. This involved measuring a broad spectrum of inflammatory markers induced in response to delivery of
first generation plasmid DNA containing large numbers of CpGs
(CpG-rich) and comparing the results with those obtained
following delivery of lipoplexes prepared using CpG-free plasmid
DNA. In addition we investigated how scalable processes suitable
for manufacturing plasmid DNA for human use could be improved
to produce high purity plasmid and reduce inflammation following
in vivo delivery.
2. Materials and methods
2.1. Animals
Wild type female BALB/c mice aged 6e12 weeks (at point of procedure performance) were purchased from Harlan Laboratories UK (Loughborough, UK) or
Biomedical Service Unit (University of Oxford, Oxford, UK). Two breeding pairs of
TLR9/ mice were purchased from Oriental BioService Inc. (Kyoto, Japan). Mice
were housed in accordance with UK Home Office ethical and welfare guidelines and
fed on standard chow and water ad libitum. All procedures were carried out under
UK Home Office approved project and personal licenses for performing experiments
on animals under the terms of the Animals (Scientific Procedures) Act 1986.
2.2. Delivery of pDNA by intranasal instillation
Mice were anaesthetised with methoxyfluorane (Mallinckrodt Veterinary Inc.,
Mundelein, IL, USA) or isoflurane (University of Oxford Vet Services, Oxford, UK).
Lipoplexes were formed using pDNA and GL67A (1:2:0.05 M ratio of GL67:DOPE:DMPE-PEG5000); GL67 was a gift from Genzyme Corporation (Framingham, MA,
USA), DOPE:DMPE-PEG5000 was purchased from Avanti Polar Lipids Inc. (Alabaster,
AL, USA) and formulated by OctoPlus (Leiden, NL). A volume of 100 ml pDNA/GL67A
complexes, containing 80 mg pDNA, at a pDNA:GL67A mM ratio of 2.4:0.6, was
delivered to the lung via intranasal instillation as described previously [28].
2.3. Harvesting of bronchoalveolar lavage fluid
Mice were euthanised by cervical dislocation at 24 h following treatment. For
analysis of BAL fluid cytokines, the trachea was exposed by blunt dissection. A small
tear was made in the trachea and a cannula was inserted. Lungs were washed three
times with 1 ml BAL fluid solution (1 PBS, 0.1% w/v BSA, 0.05 mM EDTA). Cells from
BAL fluid were concentrated by centrifugation. The supernatant was collected to
determine cytokine levels.
2.4. Plasmid DNA
Plasmid descriptions are in Table 1. Plasmid pCIKLux [22], contains the firefly
luciferase transgene driven by the human CMV immediate-early promoter and
enhancer and contains 317 CpGs, thus also referred to as CpG-rich (Table 1). Plasmid
pCIKLux was prepared using an EndoFree Plasmid Mega Kit (Qiagen, Crawley, UK).
The CpG-free plasmid pGM169 contains the synthetic optimised cystic fibrosis
transmembrane conductance regulator (CFTR) transgene (called soCFTR2 [10])
driven by the human CMV enhancer, human elongation factor 1a promoter and
Table 1
Characteristics of plasmid preparations. Summary of plasmid DNA preparations used in this study and the quantity of bacterial gDNA measured, pre-DNase treatment and postDNase treatment where applicable. The CpG-rich plasmid was made using an EndoFree Plasmid Mega Kit (Qiagen), whereas CpG-free plasmid preparations were manufactured by commercial suppliers. Endotoxin levels were <5 endotoxin units (EU)/mg for all preparations used. CMV, cytomegalovirus; Lux, firefly luciferase; soCFTR2,
synthetic optimised cystic fibrosis transmembrane conductance regulator [10]; N/A, not applicable; hCEFI, human CMV enhancer human elongation factor 1a [10], CpG, CG
dinucleotide; SEM, standard error of the mean.
Name
Plasmid CpG-content
pCIKL ux-LB
pGM169-A2
pGM169-P1
pGM169-P2
pGM169-V15
CpGCpGCpGCpGCpG-
a
b
c
richb
freec
freec
freec
freec
Promoter
Transgene
% bacterial gDNA SEMa
CMV
hCEFI
hCEFI
hCEFI
hCEFI
Lux
soCFTR2
soCFTR2
soCFTR2
soCFTR2
1.680
4.468
4.5
0.306
0.077
The quantity of bacterial gDNA per pDNA preparation is shown as a percentage of the total DNA.
CpG-rich: pDNA construct containing 317 CpGs.
CpG-free: pDNA construct containing no CpGs.
0.058
0.164
0.146
0.008
0.003
% bacterial gDNA SEMa
after DNase treatment
N/A
N/A
0.425 0.019
0.009 0.0005
N/A
9856
R.P. Bazzani et al. / Biomaterials 32 (2011) 9854e9865
R.P. Bazzani et al. / Biomaterials 32 (2011) 9854e9865
enhancer (hCEFI [10]). Preparations of pGM169 were supplied by Aldevron (Fargo,
ND, USA), PlasmidFactory (Bielefeld, Germany) or VGXI (The Woodlands, TX, USA) as
indicated in the text. Plasmid DNA preparation pGM169-V15 was manufactured to
clinical grade standard compared with others that resulted from research-grade
processes. All plasmid preparations contained < 5 EU/mg endotoxin.
2.5. DNase treatment of pDNA
Plasmid-SafeTM ATP-Dependent DNase (Epicentre Biotechnologies, Madison,
WI, USA) was used according to the manufacturer’s instructions to remove residual
bacterial gDNA from plasmid preparations. Following DNase treatment, plasmid
preparations were re-purified using the EndoFree Plasmid Mega Kit (Qiagen). In
brief, 1/12 volume of endotoxin removal buffer was added to the reaction mix, followed by dilution with 10 volumes of equilibration buffer. The mixture was incubated on ice for 30 min, before transferred to a Qiagen-tip 2500 plasmid purification
column. The remainder of the purification process was performed according to the
manufacturer’s instructions.
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2.9. Bio-Plex
The levels of 23 cytokines (CCL2 (MCP-1), CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5
(RANTES), CCL11 (Eotaxin), CXCL1 (KC), G-CSF, GM-CSF, IL-1a, IL-1b, IL-2, IL-3, IL-4,
IL-5, IL-6, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-17, IFNg and TNFa) in BAL fluid
were determined using a Bio-Plex multiplex cytokine assay (Bio-Rad Ltd., Hemel
Hempstead, UK) according to manufacturer’s instructions. Samples were analysed
using a Bio-Plex 100 or a Bio-Plex 200 system with Bio-Plex Manager software v5.0
(Bio-Rad).
2.10. Statistics
Multiple groups were analysed using one-way ANOVA followed by Dunnett’s
multiple comparison test. Pairs of interest determined in advance were analysed
using one-way ANOVA followed by Bonferroni post-test correction. Data were
considered to be statistically significant different when p < 0.05 (*p < 0.05,
**p < 0.01 and ***p < 0.001). GraphPad Prism software v5 (GraphPad, San Diego, CA)
was used for the analyses.
2.6. Agarose gel electrophoresis
3. Results
Agarose gels consisted of 1.0% agarose (Invitrogen, Paisley, UK), 500 ng/ml
ethidium bromide in 1 Tris-acetate-EDTA (TAE) buffer. Samples were mixed with
10 loading dye in a final volume of 10 ml and electrophoresed at 80e120 V for
1e2 h. For DNA size estimation, 500 ng of a 12 kb DNA ladder (Stratagene, Cambridge, UK) and 500 ng of a supercoiled DNA ladder (range 2e10 kb) (Promega,
Southampton UK) were electrophoresed alongside. Plasmid DNA preparations in
Fig. 6 were analysed with ready-to-use 1% SeaKemÒ Gold Reliant agarose gels
(Lonza, Rockland, ME, USA). Prior to loading, high-salt samples were dialysed against
water on 0.025 mm MF-Millipore membrane filters (Billerica, MA, USA) for
40e50 min and mixed with 5 loading dye (Hercules, CA, USA) to a final volume of
10 ml. Electrophoresis was conducted in TAE buffer, at 100 V for 80 min. Supercoiled
DNA ladder (Invitrogen, Carlsbad, CA, USA) was used at 0.25 mg/lane. Gels were
stained by SYBR green I nucleic acid gel stain (Invitrogen, USA) for 40 min and
visualised with a Storm 840 imaging system (GE Healthcare, Piscataway, NJ, USA) at
a resolution of 200 mm pixel size and using the fluorescence scanning mode.
2.7. Quantification of bacterial gDNA by TaqMan
Absolute levels of bacterial gDNA were quantified by a GT115-specific quantitative PCR (qPCR) assay (GT115 sequence gift of Dr. Eric Perouzel, InvivoGen).The
forward (F), reverse (R) primer and probe (P) sequences being: F: 50 -TGT GAG CGT
CGC AGA ACA TT-30 ; R: 50 -TTT TAG CAA CGT ACT ATC CAC TCA-30 and P: 50 VIC-CAT
TGA CGC AGG TGA ATC GGA CGC-TAMRA-30 . Each 10 ml PCR reaction consisted of 4 ml
DNA sample (40 ng total DNA), 1 TaqMan Universal Mastermix (Applied Biosystems, Warrington, UK), 300 nM forward primer, 300 nM reverse primer and
100 nM probe. PCR plates were heat-sealed with a clear seal and placed in an 7900
HT Fast Real-Time PCR System (Applied Biosystems). Cycling conditions were: 2 min
at 50 C and then 10 min at 95 C followed by 40 cycles of 15 s at 95 C and 1 min at
60 C. Nuclease-free water was included as a no template control. The number of
bacterial gDNA copies was calculated from standard curves prepared from E. coli
GT115 gDNA and expressed as percentage gDNA of total amount of DNA.
2.8. Scalable manufacturing of highly pure plasmid DNA
The general plasmid DNA manufacturing process [29] was modified for
production of pGM169 with very low levels of gDNA. A two-step purification
process, involving anion exchange (AEX) followed by hydrophobic interaction
chromatography (HIC), is utilised to make products for human applications. AEX
employed Mustang Q (Pall, Pensacola, FL, USA) membrane capsules. Butyl 650-M
resin (Tosoh Bioscience LLC, Montgomeryville, PA, USA) was packed in BPGÔ
columns (GE Healthcare) for the HIC step (process A). For this study, a new technique
of thiophilic interaction chromatography (TIC) was explored to replace (process B),
or supplement HIC (process C). A XK50/20 column (GE healthcare) was packed with
resin PlasmidSelect Xtra (GE healthercare) to a height of 14 cm, operating at a linear
flow rate of 100 cm/h. The load of AEX product was pre-conditioned with 3 M
ammonium sulphate to 245 mS/cm before loading to the TIC column (process B), but
the pre-conditioning was omitted when TIC was added in succession to HIC (process
C). Solutions used for column equilibration, wash and elution were 2.0 M, 1.9 M and
1.7 M ammonium sulphate, respectively. Water for injection (WFI) and 0.5 M NaOH
were employed to strip and sanitise column/resin prior to and after each use.
3.1. Inflammatory cytokine levels in BAL fluid
To investigate the inflammatory consequences of delivering
lipoplexes to the lung, mice were intranasally instilled with CpGrich pCIKLux/GL67A lipoplexes, CpG-free pGM169/GL67A lipoplexes, the gene transfer agent (GTA) lipid GL67A alone, or water
for injection (WFI). The BAL fluid was harvested 24 h post treatment
and analysed using a multiplex cytokine assay (Bio-Plex) for a broad
spectrum of 23 inflammatory markers.
Six of the 23 analytes (IL-2, IL-3, IL-4, IL-9, IL-13 and CCL11)
showed no significant differences between any treatment
(p > 0.05; Suppl. Fig. 1). For one analyte (CXCL1) all treatment
groups that contained GL67A were significantly higher than WFI
treatment (p < 0.001; Fig. 1). The remaining sixteen analytes (TNFa,
IFNg, IL-12p40, IL-12p70, IL-1a, IL-1b, IL-6, IL-5, IL-10, IL-17, CCL2,
CCL3, CCL4, CCL5, G-CSF and GM-CSF) showed significantly
(p < 0.05; Fig. 1) reduced levels in BAL fluid following treatment
with CpG-free lipoplexes compared with CpG-rich lipoplexes,
confirming our initial observations [10] that depletion of CpGs from
pDNA results in a reduced host inflammatory reaction. However, for
ten of these 16 analytes (IFNg, IL-12p40, IL-12p70, IL-1a, IL-5, IL-6,
CCL2, CCL4, CCL5 and G-CSF), a modest residual response to CpGfree lipoplexes was noted such that levels in response to CpG-free
lipolpex treatment were greater (p < 0.05) than for WFI treatment. For six of these ten analytes (IFNg, IL-12p40, IL-6, CCL2, CCL4
and CCL5) CpG-free lipoplex effects were also marginally greater
than for GL67A treatment alone.
Importantly, all plasmid preparations used contained endotoxin
levels of <5 EU/mg (Table 1), 8 times lower than the level approved
by the FDA for a drug used for intratracheal administration [25],
thus the effect of endotoxins was likely to be negligible in these
experiments. Taken together, these data suggest that in this model
of lung gene transfer pDNA per se, independent of its CpG content,
may be inflammatory, or that additional components of the
plasmid preparation may need to be considered. Interestingly, the
difference between CpG-associated cytokines IFNg, IL-12p40 and
IL-6 after delivery of CpG-free lipoplexes and delivery of lipid alone
suggested that the plasmid preparations contained a source of
unmethylated CpGs leading to the observed residual inflammatory
response.
Fig. 1. Levels of 17 inflammatory response markers in BAL fluid following nasal instillation of lipoplexes. Female BALB/c mice (n ¼ 6 per group) were treated with 100 ml CpG-rich or
CpG-free GL67A lipoplexes, lipid GL67A alone (Lipid only) or WFI via nasal instillation. BAL fluid was collected 24 h post-delivery and cytokine levels of TNFa (A), IFNg (B), IL-12p40
(C), IL-12p70 (D), IL-1a (E), IL-1b (F), IL-5 (G), IL-6 (H), IL-10 (I), IL-17 (J), CCL2 (K), CCL3 (L), CCL4 (M), CCL5 (N), CXCL1 (O), G-CSF (P) and GM-CSF (Q) were determined by Bio-Plex.
Data shown are mean SEM. Assay sensitivity limits (- - -) were 297.22 pg/ml (A), 1.61 pg/ml (B), 4.73 pg/ml (C), 3.06 pg/ml (D), 1.46 pg/ml (E), 14.12 pg/ml (F), 4.89 pg/ml (G),
3.06 pg/ml (H), 5.18 pg/ml (I), 6.39 pg/ml (J), 102.02 pg/ml (K), 578.24 pg/ml (L), 31.72 pg/ml (M) 35.45 pg/ml (N), 1.94 pg/ml (O), 7.56 (P) and 22.38 pg/ml (Q). Statistical differences
were analysed by one-way ANOVA/Dunnett to unlabelled comparator: ***p < 0.001, **p < 0.01, *p < 0.05 and ns: non-significant.
Fig. 2. Residual inflammatory response in TLR9/ mice following instillation of CpG-free lipoplexes. Female BALB/c wild type (wt) mice (light grey) (n ¼ 5 to 6 per group), or
female BALB/c TLR9-deficient (TLR9/) mice (dark grey) (n ¼ 5 to 6 per group) were treated with 100 ml CpG-free lipoplexes or WFI via nasal instillation. BAL fluid was collected
24 h post-delivery and levels of TNFa (A), IFNg (B), IL-12p40 (C), IL-6 (D), CCL2 (E), CCL3 (F), CCL4 (G), CCL5 (H), CXCL1 (I) and G-CSF (J) were determined by Bio-plex. Data shown
are mean SEM. Assay sensitivity limits (- - -) were 12.7 pg/ml (A), 6.06 pg/ml (B), 5.67 pg/ml (C), 1.16 pg/ml (D), 35.56 pg/ml (E), 9.89 pg/ml (F), 5.65 pg/ml (G), 0.91 pg/ml (H),
3.47 pg/ml (I) and 2.93 pg/ml (J). No graphical representation of assay sensitivity limits < 1 pg/ml. Statistical differences between treatments within a genotype were analysed by
one-way ANOVA/Dunnett to unlabelled comparator: ***p < 0.001; **p < 0.01; *p < 0.05 and ns: non-significant. A two-way ANOVA/Bonferroni statistical test was used to analyse
differences between the same treatment in two different genotypes:]*p < 0.05. WT: wild type; TLR9/: TLR9-deficient.
Fig. 3. Inflammatory markers in BAL fluid vary depending on CpG-free plasmid preparation. Female BALB/c mice (n ¼ 5e6 per group) were treated with 100 ml CpG-rich or CpG-free
GL67A lipoplexes, or WFI via nasal instillation. CpG-free plasmid from three commercial suppliers was used. One supplier provided a clinical grade preparation (pGM169-V15). One
supplier provided two differently manufactured plasmid preparations (pGM169-P1 and pGM169-P2). BAL fluid was collected 24 h post-delivery and levels of TNFa (A), IFNg (B),
IL-12p40 (C), IL-6 (D), CCL2 (E), CCL3 (F), CCL4 (G), CCL5 (H), CXCL1 (I) and G-CSF (J) were determined by Bio-Plex. Data shown are mean SEM. Assay sensitivity limits (- - -) were
3.46 pg/ml (A), 1.56 pg/ml (B), 0.69 pg/ml (C), 1.32 pg/ml (D), 22.26 pg/ml (E), 42.37 pg/ml (F), 5.79 pg/ml (G) 2.53 pg/ml (H), 1.6 pg/ml (I) and 1.62 (J). No graphical representation of
assay sensitivity limits < 1 pg/ml. Statistical differences between any treatment group and WFI control group were analysed by one-way ANOVA/Dunnett, whereas one-way
ANOVA/Bonferroni was used to analyse the selected preparations from the same supplier: *** and p < 0.001, and ns: non-significant.
Fig. 4. Decreased levels of inflammatory markers in BAL fluid following reduction of bacterial genomic DNA in plasmid DNA preparations. Female BALB/c mice (n ¼ 5e6 per group)
were treated with 100 ml CpG-free GL67A lipoplexes, or WFI via nasal instillation. The CpG-free plasmid DNA preparation was pGM169-P1 and was treated with either PlasmidSafeÔ ATP-dependent DNase (Epicentre Biotechnologies) (0.43% gDNA), or a mock treatment without enzyme (4.5% gDNA). BAL fluid was collected 24 h post-delivery and levels of
TNFa (A), IFNg (B), IL-12p40 (C), IL-6 (D), CCL2 (E), CCL3 (F), CCL4 (G), CCL5 (H), CXCL1 (I) and G-CSF (J) were determined by Bio-Plex. Data shown are mean SEM. Assay sensitivity
limits (- - -) were 1.35 pg/ml (A), 12.51 pg/ml (B), 2.1 pg/ml (C), 0.48 pg/ml (D), 6.47 pg/ml (E), 13.73 pg/ml (F), 2.43 pg/ml (G) 0.09 pg/ml (H), 0.31 pg/ml (I) and 0.31 (J). No graphical
representation of assay sensitivity limits < 1 pg/ml. Statistical differences were analysed by one-way ANOVA/Dunnett to unlabelled comparator: ***p < 0.001; *p < 0.05 and ns: nonsignificant.
R.P. Bazzani et al. / Biomaterials 32 (2011) 9854e9865
9861
Fig. 5. Overview of plasmid manufacture process. Plasmid DNA production begins with batch mode fermentation to high cell density and high plasmid yields, followed by
continuous centrifugation to harvest cells. Cells are frozen prior to downstream processing. Alkaline lysis allows low shear mixing of the viscous lysate at a large scale. Removal of
solid flocculent from plasmid solution is realised with a series of filtration steps. Clarified lysate goes through the purification process (step I, II and optional III), desalting/
concentration with ultrafiltration/diafiltration and final aseptic filtration and fill; M: Motor.
To determine if the residual response was due to the presence
of unmethylated CpGs, lipoplexes were delivered to the lungs of
TLR9/ mice, deficient in TLR9 required for detection of
unmethylated CpGs. A subset of the 23 analytes, representative of
the inflammatory response, is presented throughout Figs. 2e4. As
shown in 1. 2, in WT mice, six of the ten analytes (IL-12p40, IL-6,
CCL2, CCL5, CXCL1 and G-CSF) showed increased levels in BAL
fluid after treatment with CpG-free lipoplexes compared with
WFI. Similarly in TLR9/ mice, levels of IL-12p40, IL-6, CCL2,
CCL4, CCL5 and G-CSF were significantly increased following
treatment with CpG-free lipoplexes compared with WFI. Of
the three CpG-associated cytokines IFNg, IL12-p40 and IL-6, only
IL-12p40 showed a significant (p < 0.05; 2.6-fold) reduction in
the TLR9/ mice following intranasal instillation of CpG-free
lipoplexes compared with WT mice (Fig. 2C). IFNg and IL-6
appeared reduced in TLR9/ mice compared with WT mice
(3.9-fold and 2.2- fold, respectively), but did not reach significance (p > 0.05; Fig. 2B and D). These results suggest that the
residual inflammatory cytokine response observed following
intranasal instillation of CpG-free lipoplexes may in part be due to
a CpG-dependent response. We hypothesised that bacterial gDNA
present in plasmid preparations was considered a possible source
of unmethylated CG dinucleotides.
3.2. Inflammatory cytokines associated with CpG-free plasmid
Four research or clinical grade preparations of the CpG-free
plasmid pGM169 (termed pGM169-A2, -P1, -P2 and -V15; Table 1)
were obtained from three different suppliers and their bacterial
gDNA content was evaluated. As expected, agarose gel electrophoresis was not sufficiently sensitive to reveal the presence of
sheared fragments of bacterial gDNA in pDNA preparations (data
not shown), therefore a quantitative PCR (qPCR) assay for gDNA was
developed. The qPCR assay used was highly sensitive with
a detection limit of 4 copies per reaction and was reproducible such
that results obtained for a specific sample were comparable when
analysed in separate laboratories using independent tests (data not
shown). The qPCR assay was used to measure the percentage
bacterial gDNA present and showed that this varied widely
between 0.077% 0.003e4.5% 0.15 in the preparations tested;
the clinical grade preparation (pGM169-V15) showing the lowest
levels of %gDNA measured (Table 1). Interestingly, although
plasmid preparations pGM169-P1 and pGM169-P2 were manufactured by the same supplier, production of pGM169-P2 included
a step for specific removal of bacterial chromosomal DNA, and this
appeared to reduce bacterial gDNA contamination by w15-fold
(Table 1). Next, the potential for each of these plasmid preparations to elicit an inflammatory response was investigated. Results
showed that lower levels of contaminating bacterial gDNA
appeared to correlate with lower levels of CpG-associated cytokines
IFNg and IL-12p40 (Fig. 3B and C). Furthermore, the additional
gDNA removal step for manufacturing pGM169-P2 compared with
pGM169-P1 correlated with significantly reduced cytokine levels
(p < 0.001) (Fig. 3B and C).
3.3. Bacterial gDNA contamination in pDNA
We hypothesised that bacterial gDNA contaminating plasmid
preparations, was responsible, at least in part, for the residual
inflammatory response observed in the mouse lung following
intranasal instillation of CpG-free lipoplexes. To confirm this, two
plasmid preparations (pGM169-P1 and pGM169-P2) were treated
with Plasmid-SafeTM ATP-Dependent DNase (Epicentre Biotechnologies), an exonuclease that specifically targets linear double
stranded DNA such as bacterial gDNA, but with no observed
enzymatic activity on closed circular dsDNA or supercoiled (SC)
DNA comprising the majority of pDNA. Agarose gel electrophoresis
was used to confirm that enzymatic treatment did not grossly affect
the predominantly supercoiled conformation of the plasmid (Suppl.
Fig. 2), and qPCR was used to measure gDNA. Table 1 shows that
enzymatic treatment reduced gDNA by at least 10-fold for each
plasmid. Next, the enzymatically-treated and mock-treated plasmids were complexed with GL67A lipid and intranasally instilled
into mice, so that the inflammatory response could be determined.
The pGM169-P1 preparation, where bacterial gDNA was reduced
w10-fold from 4.5 to 0.43%, (Table 1) resulted in significantly
(p < 0.05) reduced levels of seven of the ten analytes (TNFa, IFNg,
IL-12p40, IL-6, CCL2, CCL3 and G-CSF; Fig. 4). However, for some of
the markers (IL-12p40, IL-6, CCL2, CCL4, CCL5, CXCL1, G-CSF) the
residual levels were still significantly (p < 0.05) higher than the WFI
control group, despite the reduction in bacterial gDNA. Delivery of
the enzymatically-treated plasmid preparation pGM169-P2, where
bacterial gDNA was reduced w33-fold from 0.3 to 0.009% (Table 1),
resulted in no significant (p > 0.05) decrease for any of the ten
analytes investigated (TNFa, IFNg, IL-12p40, IL-6, CCL2, CCL3, CCL4,
CCL5, CXCL1 and G-CSF) (data not shown). In addition, seven of the
ten analytes (IFNg, IL-12p40, IL-6, CCL2, CCL3, CCL4 and CCL5) were
significantly (p < 0.05) increased compared with animals treated
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R.P. Bazzani et al. / Biomaterials 32 (2011) 9854e9865
Fig. 6. Agarose gel electrophoresis of samples of pGM169 processed by AEX, HIC and/or TIC. Following purification, samples of plasmid pGM169 were analysed by agarose gel
electrophoresis. Bands represent genomic DNA (gDNA), open circular (OC) pDNA, supercoiled (SC) pDNA and RNA. (A): HIC process samples of Lot#1 (Process A, AEX þ HIC). Lane 1,
SC plasmid ladder (Invitrogen); Lane 2, Load; Lane 3, flowthrough (FT) initial; Lane 4, FT middle; Lane 5, FT end; Lane 6, wash; Lane 7, Eluate; Lane 8, WFI strip. (B): TIC process
samples of Lot#2 (Process B, AEX þ TIC). Lane 1, SC plasmid ladder; Lane 2, Load; Lane 3, FT middle; Lane 4, FT end; Lane 5, wash; Lane 6, Eluate; Lane 7, Eluate end; Lane 8, WFI
strip. (C): HIC process samples of Lot#3 (Process C, AEX þ HIC þ TIC). Lane 1, SC plasmid ladder; Lane 2, Load; Lane 3, FT initial; Lane 4, FT middle; Lane 5, FT end; Lane 6, wash; Lane
7, Eluate; Lane 8, WFI strip. (D): TIC process samples of Lot#3 (Process C, AEX þ HIC þ TIC). Lane 1, SC plasmid ladder; Lane 2, Load; Lane 3, FT initial; Lane 4, FT middle; Lane 5, FT
end; Lane 6, Eluate; Lane 7, Eluate end; Lane 8, WFI strip.
with lipid alone (Fig. 4). Together, these results suggest that the
quality of plasmid preparations, at least with respect to contaminating levels of bacterial gDNA, should be improved and might be
of importance in minimising unwanted inflammatory side effects if
used for therapeutic purpose.
3.4. Large scale pDNA production
Preparation of pDNA with total yields in the region of 2e10 mg
per column (e.g. Large-Scale Plasmid DNA Purification Kits) is
routine in the laboratory, but can be more efficiently manufactured to higher purity by commercial suppliers, particularly at
larger scales; and is essential when producing plasmid for therapeutic purposes. However, the purity of pDNA preparations
seemed to depend on the manufacturing process used (Table 1).
Therefore, we wished to develop a process for manufacturing
clinical grade pDNA, where we could reliably achieve low levels of
bacterial gDNA. Fig. 5 outlines the main steps used in the manufacture of clinical grade pGM169 plasmid by one commercial
supplier, VGXI. Following a 10 L scale batch fermentation, cells
were processed by alkaline lysis, and plasmid was isolated and
purified with either two or three chromatographic steps. Anion
exchange membrane chromatography was effective in primary
capture of pDNA and major reduction of impurities. However,
different polishing steps following AEX, HIC and/or TIC, were
explored to achieve maximum gDNA removal. Thus, three robust
purification processes were developed: (A) AEX þ HIC; (B)
AEX þ TIC; (C) AEX þ HIC þ TIC. The CpG-free plasmid, pGM169
[10], was generated using each process and the purity of each final
product was compared (Table 2).
Process A resulted in high %SC and low levels of impurities
(Table 2). In this process, the HIC step load conditions were
R.P. Bazzani et al. / Biomaterials 32 (2011) 9854e9865
9863
Table 2
Comparison of final drug product specifications following the use of different purification processes. Three different purification processes (two 2-step processes (A and B) and
one 3-step process (C)) were tested. The resulting filtered drug product was analysed using quality control release assays. The purification processes were tested following
a medium scale fermentation of 10 L, yielding 100e400 mg of final drug product. AEX: anion exchange membrane chromatography, HIC: hydrophobic interaction chromatography, TIC: thiophilic interaction chromatography, EU: endotxin units, SC: supercoiled, and OC: open circular.
Process
Process A: AEX þ HIC
Process B: AEX þ TIC
Process C: AEX þ HIC þ TIC
Concentration (mg/ml)
Purity (A260/280)
Restriction
Appearance
5.618
2.016
Conform to standards
Clear colourless solution with
no visible particulates
0.06
0.125
0.5
0.26
97.4
1.2
1.4
5.606
2.025
Conform to standards
Clear colourless solution with
no visible particulates
0.06
0.362
0.1
0.24
97.2
1.3
1.5
5.563
2.042
Conform to standards
Clear colourless solution with
no visible particulates
0.06
0.204
0.26
0.003
97.7
0.8
1.6
Host cell protein (%)
Endotoxin (EU/mg)
Host cell RNA (%)
Host cell genomic DNA (%)
Forms
SC (%)
OC (%)
Other (%)
optimised to achieve pDNA isoform separation, with SC pDNA
binding to the resin while open circular (OC) and trace multimeric
pDNA forms along with gDNA (fragmented to w 20 kb during
alkaline lysis and subsequent processing) passing through the
column in the non-absorbed flowthrough (FT) phase (Fig. 6A; lanes
3e5). Single-stranded RNA exhibited much higher hydrophobicity
than SC pDNA and was only recovered from the HIC column during
WFI column strip/regeneration step (Fig. 6A; lane 8). Host protein
and endotoxin predominantly co-eluted with RNA in the WFI
column strip/regeneration step (data not shown). A typical lot of
pGM169 produced by this process (Fig. 6A; lane 7) contained 0.26%
residual gDNA as determined by qPCR (Table 2).
In an attempt to reduce residual gDNA levels, we developed
Process B e an alternative two-step chromatography process,
where HIC is replaced by TIC. Process B demonstrated a similar
impurity profile to Process A (Table 2). TIC, like HIC in Process A,
allowed flowthrough removal of OC pDNA along with gDNA
(Fig. 6B; lanes 3 and 4). A typical lot of pGM169 produced by this
process (Fig. 6B; lanes 6 and 7) was highly enriched for SC pDNA
(Table 2). However, residual gDNA levels achieved with Process B
(0.24%) were broadly similar to levels achieved with Process A
(Table 2). We hypothesised that a three-step chromatographic
process would be required to substantially reduce levels of residual
gDNA in the final product.
Consequently, we developed Process C, combining AEX, HIC and
TIC. Comparable with the two-step processes, OC pDNA and gDNA
removal was facilitated in the flowthrough of both, the HIC (Fig. 6C;
lanes 3e5) and TIC (Fig. 6D; lanes 3e5) steps. In a typical lot of
pGM169 produced by this process, this resulted in a modest
reduction in residual OC pDNA levels and a substantial w100-fold
reduction in residual gDNA levels in the final product (Table 2).
These data confirm that by process modification, clinical grade
pDNA for human use can be generated with extremely low levels
(0.003%) of contaminating bacterial gDNA.
4. Discussion
Pre-clinical and clinical studies have documented an acute
inflammatory response to lung delivery of lipoplexes [4e6,10,14],
which may limit their therapeutic use. This inflammatory response,
characterised by increased pro-inflammatory cytokine levels and
an influx of white blood cells (WBC) to the lung, can be measured in
BAL fluid from the lungs of treated mice [10]. Similar to mice,
a WBC influx, dominated by neutrophils, has been reported in
sheep following lipoplex delivery to the lung [30]. In this present
study, a highly sensitive, multiplex assay was used to further
characterise the response by measuring a broad spectrum of 23
markers of Th1 and Th2 immune responses and leukocyte trafficking and maturation (Fig. 1 and Suppl. Fig. 1). The results obtained were consistent with published reports of a Th1-biased
inflammatory cytokine response triggered by unmethylated CpGs
[6,10,11,14]. In addition, the inflammatory response was accompanied by increased levels of CC-chemokines, also associated with
a type I (Th1-related) inflammation mediated by monocytes/
macrophages [31], and increased CXCL1, the murine functional
homologue of the strong neutrophil attractant human CXCL8 (IL-8).
Furthermore, the observed Th1-biased inflammatory response is in
line with the use of unmethylated CpG oligodeoxynucleotides as an
adjuvant for immunisation [32].
In an attempt to mitigate the observed acute inflammatory
response following lung delivery of GL67A lipoplexes for CF gene
therapy [4,5], the UKCFGTC [15] developed plasmid pGM169, which
is capable of persistent CFTR expression in the mouse lung, and is
entirely devoid of CpGs such that inflammation in the mouse lung,
as judged by three cytokines analysed by ELISA, was reduced to
background (naïve) levels [10]. However, with the development of
the more sensitive and extensive assays used in these studies,
further characterisation of the response to lipoplex delivery has
revealed that even when using a CpG-free plasmid, a low-grade,
residual inflammatory response remains (Fig. 1). To investigate
this further, CpG-free lipoplexes were delivered via intranasal
instillation to the lungs of mice deficient in the TLR9 (TLR9/)
receptor for unmethylated CpGs [12]. Results indicated the presence of a TLR9-independent inflammatory response to lipoplex
delivery (Fig. 2), consistent with reports that TLR9 signalling was
only partially responsible for toxic responses following systemic
delivery of lipoplexes [33].
Possible contaminants of plasmid preparations were considered. Bacterial lipopolysaccharide fragments, also known as endotoxins, can readily contaminate plasmid preparations and cause
inflammation. However, all plasmid preparations used in this study
had endotoxin levels of <5 EU/mg, at least 8 times lower than
recommended by regulatory bodies [25]. Furthermore, studies have
shown previously that routine endotoxin contamination of plasmid
preparations is unlikely to contribute substantially towards the
inflammatory response elicited by lipoplexes [6]. Results presented
here showed that part of the inflammatory response could be due
to the lipid component of the gene transfer agent, since delivery of
GL67A alone resulted in a slight inflammatory response (Fig. 1 and
Suppl. Fig. 1). Intriguingly, levels of CpG-associated cytokines IFNg,
IL-6 and IL-12p40 were further increased when lipid was combined
with CpG-free pDNA. This, together with the observation that
these cytokines showed a > 2-fold reduction in their levels in
TLR9/ mice, indicated the presence of an additional source of
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R.P. Bazzani et al. / Biomaterials 32 (2011) 9854e9865
unmethylated CpGs in plasmid preparations, most likely contaminating bacterial gDNA. In support of this, cytokine levels, (especially
IFNg, IL-6 and IL-12p40) were found to vary between plasmid
preparations (Fig. 3), and correlated with the levels of bacterial
gDNA in the plasmid preparation used (Table 1). This hypothesis
was confirmed by enzymatic digestion of bacterial gDNA in plasmid
preparations prior to use in vivo and the concomitant reduction in
pro-inflammatory cytokines (Fig. 4).
Our observations, that plasmid preparations can induce a variable inflammatory response in vivo, are in line with a recent report
of toxicity following hydrodynamic limb vein injection of naked
plasmid DNA into rodent limb muscles [34]. Some of the pDNA
preparations caused muscle necrosis whilst others did not; the
results were dependent on the batch of pDNA used and correlated
with the presence of bacterial gDNA, such that 5% of bacterial
gDNA in plasmid preparations was sufficient to cause muscle
damage [34]. The authors recommended that the amount of
contaminating bacterial gDNA present in a plasmid preparation
should be reported along with the experimental results. Unfortunately, since sheared fragments of bacterial gDNA in a plasmid
preparation are variable in size, they are difficult to visualise and
quantify using routine agarose gel electrophoresis, thus qPCR
assays are required to accurately report residual low levels of
gDNA. When we used our qPCR assay to measure gDNA levels in
research-grade plasmid DNA prepared in our laboratory, we found
that these were generally low (pCIKLux; Table 1). The %gDNA
present in commercially prepared research-grade plasmid preparations was similar, except for pGM169-P2 where an additional
processing step was included resulting in lower levels (Table 1).
Importantly, it is possible for occasional plasmid preparations to
contain > 5% gDNA (data not shown), which could contribute to
toxicity when used in vivo.
Irrespective of the procedures included to reduce bacterial
gDNA in research-grade plasmid preparations, those plasmids
ultimately intended for therapeutic use required a scalable, reproducible, production process. We investigated the feasibility of
modifying a clinical plasmid manufacturing process to produce
plasmid with minimal bacterial gDNA contamination. The two-step
plasmid purification process, using AEX þ HIC (Process A), has been
used to produce cGMP plasmid at 1e50 g scale for over 80 different
plasmids, generating high SC% (95%) product and purity
exceeding the recommendations set by the FDA [25] and other
regulatory agencies. However, the levels of impurities in the final
product can vary, typically correlating inversely with specific
plasmid yields (data not shown). A high copy number plasmid can
easily be produced with levels of RNA 0.1%, protein 0.1%,
endotoxin 0.1 EU/mg and gDNA 0.01% (where “” designates
result lower than assay limit of detection). Recent human papillomavirus and influenza DNA vaccines that used pUC derived backbone were manufactured with the two step process (AEX þ HIC)
under cGMP regulations, all showing undetectable levels of impurities at a pDNA concentration of up to 10 mg/ml [35]. However,
CpG-free plasmid pGM169, derived from R6K vectors [36] exhibited
lower copy number and reduced yields, even under optimised
fermentation conditions. Fragments of gDNA generated by shear
during processing, share similar physical-chemical characteristics
to SC pDNA and are most difficult to eliminate. The AEX step can
reduce bulk protein, RNA and endotoxin, but is less effective at
reducing fragmented gDNA that co-elutes with plasmid. A process
of AEX þ HIC or AEX þ TIC is sufficient to reduce gDNA to < 1%, or
lower with typical high yield plasmids. When initial plasmid yield
is low, the ratio of %gDNA in the AEX eluate becomes relatively high,
thus resolution with subsequent HIC or TIC is limited. In this study,
two-step processes reduced bacterial gDNA to w0.2%; whereas
combining them in a three-step process further reduced residual
gDNA levels w100 fold (Table 2). However, one must take into
consideration that such low level bacterial gDNA was obtained at
the cost of a 10e20% loss of plasmid product. The extra processing
step will reduce overall downstream yield and an increased
fermentation scale will be required to achieve the desired quantity
of final product. Eventually, the need for increased purity has to be
balanced with the increased costs involved. Whilst 5% gDNA was
toxic for delivery to rodent muscle, preparations with <1.3% gDNA
did not cause significant muscle damage [34]. In this study, gDNA at
contaminating levels of w4.5% correlated with a CpG-dependent
inflammatory response (Figs. 3 and 4 and Table 1), which was
reduced although still measureable when bacterial gDNA was
reduced to < 0.43% (Fig. 4). The authors believe that the acceptable
range of %gDNA for a plasmid preparation is reasonable at 1% for
most applications and possibly as low as 0.1% for therapeutic
purposes.
After minimising the CpG-dependent inflammatory response
by using CpG-free plasmid DNA and reducing the %gDNA
contamination, the data indicated there was an additional CpGindependent and TLR9-independent cause of low level inflammation following intranasal instillation of lipolexes to the mouse
lung. This additional mechanism could be due to the lipid
component of the complex, which could potentially mask any
auxiliary reduction in inflammatory cytokine levels that could be
achieved by further improving the purity of plasmid preparations.
In addition, TLR9-independent systems that recognise ’foreign’
DNA in the cytoplasm such as the inflammasomes [37] or the DNAdependent activator of IFN-regulatory factors (DAI) [38] might also
play a role. For example, cell culture studies using non-TLR9expressing Glomerular endothelial cells transfected with CpGfree lipoplexes showed production of cytokines and chemokines,
such as IL-6, CCL2, CCL5 and type I IFNs, in a MyD88-independent
cytosolic DNA recognition pathway [39]. A hallmark of TLR9/
MyD88-independent DNA recognition pathways is the resulting
expression of Type I IFNs [40], which were not amongst the
inflammatory analytes studied herein. It would therefore be
appropriate to investigate alternative inflammatory response
markers and/or pathways associated with cytoplasmic DNA
recognition, such as type I IFNs, and establish if their levels change
upon treatment with CpG-rich lipoplex compared with CpG-free
lipoplex or WFI.
5. Conclusion
Removal of CpGs from plasmid expression vectors minimises
the inflammatory response to plasmid/lipid complexes in the
mouse lung and may help mitigate the acute inflammatory
response in clinical studies. However, a low-level CpG-dependent
response is still detectable due to contaminating bacterial gDNA
present in plasmid preparations. Our results demonstrate that
scalable manufacturing processes can be adapted to further reduce
contaminating levels of gDNA in plasmid preparations, but the cost
of additional processing must be considered.
Acknowledgement
We would like to thank Dr. Martin Schleef from PlasmidFactory
for providing us with two different batches of pGM169, G. NuñezAlonso for helping with BAL fluid collection and Dr. I. Pringle for
critically reading the manuscript. This work was funded by
a studentship from the UK Cystic Fibrosis Trust via the UK Cystic
Fibrosis Gene Therapy Consortium (http://www.cfgenetherapy.
org.uk).
R.P. Bazzani et al. / Biomaterials 32 (2011) 9854e9865
Appendix. Supplementary material
Supplementary information associated with this article can
be found, in the online version, at doi:10.1016/j.biomaterials.2011.
08.092.
References
[1] Gao X, Kim KS, Liu D. Nonviral gene delivery: what we know and what is next.
AAPS J 2007;9:E92e104.
[2] Griesenbach U, Alton EW. Current status and future directions of gene and cell
therapy for cystic fibrosis. BioDrugs 2011;25:77e88.
[3] Welsh MJ, Ramsey BW, Accurso F, Cutting GR. Cystic fibrosis. In: Scriver CR,
Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of
Inherited Diseases. 8th ed. New York: McGraw-Hill; 2001. p. 5121e88.
[4] Alton EW, Stern M, Farley R, Jaffe A, Chadwick SL, Phillips J, et al. Cationic
lipid-mediated CFTR gene transfer to the lungs and nose of patients with
cystic fibrosis: a double-blind placebo-controlled trial. Lancet 1999;353:
947e54.
[5] Ruiz FE, Clancy JP, Perricone MA, Bebok Z, Hong JS, Cheng SH, et al. A clinical
inflammatory syndrome attributable to aerosolized lipid-DNA administration
in cystic fibrosis. Hum Gene Ther 2001;12:751e61.
[6] Freimark BD, Blezinger HP, Florack VJ, Nordstrom JL, Long SD, Deshpande DS,
et al. Cationic lipids enhance cytokine and cell influx levels in the lung
following administration of plasmid: cationic lipid complexes. J Immunol
1998;160:4580e6.
[7] Li S, Wu SP, Whitmore M, Loeffert EJ, Wang L, Watkins SC, et al. Effect of
immune response on gene transfer to the lung via systemic administration of
cationic lipidic vectors. Am J Physiol 1999;276:L796e804.
[8] Norman J, Denham W, Denham D, Yang J, Carter G, Abouhamze A, et al.
Liposome-mediated, nonviral gene transfer induces a systemic inflammatory
response which can exacerbate pre-existing inflammation. Gene Ther 2000;7:
1425e30.
[9] Scheule RK, St George JA, Bagley RG, Marshall J, Kaplan JM, Akita GY, et al.
Basis of pulmonary toxicity associated with cationic lipid-mediated gene
transfer to the mammalian lung. Hum Gene Ther 1997;8:689e707.
[10] Hyde SC, Pringle IA, Abdullah S, Lawton AE, Davies LA, Varathalingam A, et al.
CpG-free plasmids confer reduced inflammation and sustained pulmonary
gene expression. Nat Biotechnol 2008;26:549e51.
[11] Yew NS, Zhao H, Wu IH, Song A, Tousignant JD, Przybylska M, et al. Reduced
inflammatory response to plasmid DNA vectors by elimination and inhibition
of immunostimulatory CpG motifs. Mol Ther 2000;1:255e62.
[12] Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, et al. A Toll-like
receptor recognizes bacterial DNA. Nature 2000;408:740e5.
[13] Abdullah S. Studies on the persistence of transgene expression in the murine
airways. Oxford: University of Oxford; 2005.
[14] Yew NS, Wang KX, Przybylska M, Bagley RG, Stedman M, Marshall J, et al.
Contribution of plasmid DNA to inflammation in the lung after administration
of cationic lipid:pDNA complexes. Hum Gene Ther 1999;10:223e34.
[15] UKCFGTC. The UK Cystic Fibrosis Gene Therapy Consortium - http://www.
cfgenetherapy.org.uk/clinical.html.
[16] Zabner J, Cheng SH, Meeker D, Launspach J, Balfour R, Perricone MA, et al.
Comparison of DNA-lipid complexes and DNA alone for gene transfer to cystic
fibrosis airway epithelia in vivo. J Clin Invest 1997;100:1529e37.
[17] Caplen NJ, Alton EW, Middleton PG, Dorin JR, Stevenson BJ, Gao X, et al.
Liposome-mediated CFTR gene transfer to the nasal epithelium of patients
with cystic fibrosis. Nat Med 1995;1:39e46.
[18] Gill DR, Southern KW, Mofford KA, Seddon T, Huang L, Sorgi F, et al. A placebocontrolled study of liposome-mediated gene transfer to the nasal epithelium
of patients with cystic fibrosis. Gene Ther 1997;4:199e209.
[19] Porteous DJ, Dorin JR, McLachlan G, Davidson-Smith H, Davidson H,
Stevenson BJ, et al. Evidence for safety and efficacy of DOTAP cationic lipo-
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
9865
some mediated CFTR gene transfer to the nasal epithelium of patients with
cystic fibrosis. Gene Ther 1997;4:210e8.
Hyde SC, Southern KW, Gileadi U, Fitzjohn EM, Mofford KA, Waddell BE, et al.
Repeat administration of DNA/liposomes to the nasal epithelium of patients
with cystic fibrosis. Gene Ther 2000;7:1156e65.
Pringle IA, Raman S, Sharp WW, Cheng SH, Hyde SC, Gill DR. Detection of
plasmid DNA vectors following gene transfer to the murine airways. Gene
Ther 2005;12:1206e14.
Gill DR, Smyth SE, Goddard CA, Pringle IA, Higgins CF, Colledge WH, et al.
Increased persistence of lung gene expression using plasmids containing the
ubiquitin C or elongation factor 1alpha promoter. Gene Ther 2001;8:
1539e46.
Poulsen TT, Pedersen N, Juel H, Poulsen HS. A chimeric fusion of the hASH1
and EZH2 promoters mediates high and specific reporter and suicide gene
expression and cytotoxicity in small cell lung cancer cells. Cancer Gene Ther
2008;15:563e75.
Pringle IA, McLachlan G, Collie DD, Sumner-Jones SG, Lawton AE, Tennant P,
et al. Electroporation enhances reporter gene expression following delivery of
naked plasmid DNA to the lung. J Gene Med 2007;9:369e80.
FDA. Guidance for industry: consideration of plasmid DNA vaccines for infectious
disease indications, http://www.fda.gov/downloads/BiologicsBloodVaccines/
GuidanceComplianceRegulatoryInformation/Guidances/Vaccines/
ucm091968pdf; 2007.
EMEA. Note for guidance on the quality, preclinical and clinical aspects of
gene trasnfer medicinal products; 2001.
Cai Y, Rodriguez S, Hebel H. DNA vaccine manufacture: scale and quality.
Expert Rev Vaccines 2009;8:1277e91.
Lee ER, Marshall J, Siegel CS, Jiang C, Yew NS, Nichols MR, et al. Detailed
analysis of structures and formulations of cationic lipids for efficient gene
transfer to the lung. Hum Gene Ther 1996;7:1701e17.
Hebel H, Attra H, Khan A, Draghia-Akli R. Successful parallel development and
integration of a plasmid-based biologic, container/closure system and electrokinetic delivery device. Vaccine 2006;24:4607e14.
Emerson M, Renwick L, Tate S, Rhind S, Milne E, Painter HA, et al. Transfection
efficiency and toxicity following delivery of naked plasmid DNA and cationic
lipid-DNA complexes to ovine lung segments. Mol Ther 2003;8:646e53.
Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M. The chemokine
system in diverse forms of macrophage activation and polarization. Trends
Immunol 2004;25:677e86.
Krieg AM. CpG motifs in bacterial DNA and their immune effects. Annu Rev
Immunol 2002;20:709e60.
Zhao H, Wu I-H, Hemmi H, Akira S, Cheng SH, Scheule RK, et al. Role of the
toll-like 9 receptor in the acute inflammatory response to nonviral vectors.
Mol Ther 2003;7:S209.
Wooddell CI, Subbotin VM, Sebestyen MG, Griffin JB, Zhang G, Schleef M, et al.
Muscle damage after delivery of naked plasmid DNA into skeletal muscles is
batch dependent. Hum Gene Ther 2011;22:225e35.
Cai Y, Rodriguez S, Rameswaran R, Draghia-Akli R, Juba Jr RJ, Hebel H.
Production of pharmaceutical-grade plasmids at high concentration and high
supercoiled percentage. Vaccine 2010;28:2046e52.
Wu F, Levchenko I, Filutowicz M. A DNA segment conferring stable maintenance on R6K gamma-origin core replicons. J Bacteriol 1995;177:6338e45.
Muruve DA, Petrilli V, Zaiss AK, White LR, Clark SA, Ross PJ, et al. The
inflammasome recognizes cytosolic microbial and host DNA and triggers an
innate immune response. Nature 2008;452:103e7.
Takaoka A, Wang Z, Choi MK, Yanai H, Negishi H, Ban T, et al. DAI (DLM-1/
ZBP1) is a cytosolic DNA sensor and an activator of innate immune response.
Nature 2007;448:501e5.
Hagele H, Allam R, Pawar RD, Reichel CA, Krombach F, Anders HJ. Doublestranded DNA activates glomerular endothelial cells and enhances albumin
permeability via a toll-like receptor-independent cytosolic DNA recognition
pathway. Am J Pathol 2009;175:1896e904.
Takaoka A, Taniguchi T. Cytosolic DNA recognition for triggering innate
immune responses. Adv Drug Deliv Rev 2008;60:847e57.