Post on 18-Mar-2020
Functional evaluation of isoforms present in pDNA vaccines
Michael A. Connolly, Jeneice Hamilton and Timothy A. Coleman
Immunomic Therapeutics, 15010 Broschart Road, Rockville, MD 20850
Michael A. ConnollyImmunomic TherapeuticsEmail: mconnolly@immunomix.comWebsite: www.immunomix.comPhone: 301-968-3501 ext. 3505
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References
Nucleic acid vaccines (DNA or RNA) are experiencing a significant
resurgence in the treatment of infectious diseases, allergy and oncology.
Plasmid DNA (pDNA) provides the elements required for propagation in a
bacterial host (e.g. replication origin, selection marker) but more importantly,
they contain all the elements required for directing target-gene expression in
human patients. Our Unite™ technology provides an additional element, the
gene encoding Lysosomal Associated Membrane Protein (LAMP) to help
traffic the target gene into the MHC-II pathway thus eliciting a strong immune
response (1)(2)(3).
As with other parenteral drugs, pDNAs made for use in clinical studies must
meet minimal requirements for safety, identity, purity and potency(4). One
such attribute is Forms Analysis, which relates to the proportion of
supercoiled (SC), Open Circular (OC), Linear (L) or other forms like
concatamers that are routinely found in the pDNA product. While all these
forms are identical in nucleic acid composition, it is generally accepted that
preparations containing a higher proportion of SC pDNA will be taken up
more readily by cells and ultimately be more efficacious.
In the present study, we prepared different isoforms of pDNA encoding either
a GFP reporter or a LAMP fusion gene, confirmed the forms by Agarose Gel
Electrophoresis (AGE) and HPLC and analyzed their ability to direct gene
expression in vitro (transfection or electroporation) and in vivo (injection into
mice). Our results demonstrate that the OC form of pDNA is competent at
directing gene expression in vitro. Unexpectedly, a linear form with an intact
LAMP fusion gene was also able to direct gene expression. Confirmation of
these results in vivo, could be used to substantiate a reduced specification for
SC pDNA which would directly impact product yield and manufacturing
COGs.
Abstract
Preparation of pDNA Forms
pDNA clones were prepared from the same host strain. The cells were grown
in fermentation using a 3L Eppendorf BioFlo 120 Bioreactor. Bacterial cell
paste was harvested by centrifugation, and pDNA was prepared using an
alkaline lysis process with ion exchange chromatography. The concentration
of pDNA was determined spectrophotometrically, the identity was confirmed
by restriction digestion and AGE and the pDNA was tested for the presence
of endotoxin prior to further evaluation.
Opened Circular pDNA was prepared by digesting the pDNA with Nb.Btsl
(New England Biolabs). Nb.BtsI is a nicking endonuclease that cleaves only
one strand of DNA at specific sites (3’ CGTCAC^NN 5’) resulting in the
formation of OC-pDNA.
Linear pDNA was prepared by digestion with restriction endonucleases that
cut at a single site in each plasmid. Two different endonucleases were used,
one enzyme linearized upstream from the promoter (XbaI) thus keeping the
gene intact. The second enzyme was selected to disrupt the transcriptional
unit. pGFP was digested with BtsαI which has a cut site within the eGFP
gene. pLAMP-Fusion was digested with XhoI which has a cut site within the
fusion gene (see Figures 1 and 2).
Following digestion, the pDNA isoforms were phenol:chloroform extracted to
remove the restriction enzymes as well as other proteins introduced in the
reaction buffers. The pDNA isoforms were precipitated with 100% ethanol,
washed with 70% ethanol to remove salts, etc. and resuspended in PBS.
The concentration of each preparation was determined using a Little Lunatic
spectrophotometer (Unchained Labs). Purity of the resuspended isoforms
was determined by AGE and HPLC analysis.
BtsαI
pGFP3725 bp
XbaI
XhoI
pLAMP-Fusion6097 bp
Figure 1: (A) pGFP circular plasmid map. (B) Linear plasmid map. Digestion with XbaI linearizesthe plasmid upstream from the promoter. Digestion with BtsαI linearizes the plasmid within thegene. (C) AGE was used to confirm isoform preparation.
A M SC OC XbaI BtsαIC A
B
Figure 2: (A) 293T cells were transfected with pGFP isoforms using Lipofectamine 2000 (Invitrogen)or electroporated (B) using an ECM 830 Electroporation System (BTX). Cells were incubated for 48hours at 37°C with 5% CO2. GFP-producing cells were visualized using a fluorescent microscope.The SC, OC, and XbaI digested pDNAs were competent at directing GFP expression. Surprisingly,the BtsαI digested pDNA was also able to direct GFP production albeit at a lower level than theother isoforms. (C) The HPLC analysis confirms that the enzymatic treatments produced theexpected isoforms.
A: Transfection
B: Electroporation
Figure 4: 293T cells were transfected with pLAMP-Fusion isoforms using Lipofectamine 2000(Invitrogen) or electroporated using an ECM 830 Electroporation System (BTX). Cells wereincubated for 48 hours at 37°C with 5% CO2. Total cellular protein was prepared using RIPAbuffer and quantified by BCA. 1μg of protein was electrophoresed and blotted to nitrocellulose.Expression of the LAMP-Fusion protein was determined via Western Blot using an anti-LAMPantibody (Sino). Western Blot results from Transfection (A) and Electroporation (B) are shown.The SC, OC, and XbaI digested isoforms were able to direct comparable levels of the fusion geneexpression. Similar to the GFP results, the XhoI digested plasmid was able to direct geneexpression at a lower level. (C) The HPLC analysis confirms that the enzymatic treatmentsproduced the expected isoforms.
6000
3000
1000
6000
3000
1000
A: Transfection
B: Electroporation
Conclusions• pDNA isoforms were prepared and confirmed via AGE and HPLC analysis
• SC and OC isoforms were shown to be competent at directing target gene
expression in vitro using both lipid mediated transfection and electroporation
• Linear pDNA in which the gene remains intact is also capable of directing
gene expression
• Linear pDNA in which the gene is ‘disrupted’ can direct gene expression
albeit at a somewhat reduced level
• Results from the in vivo study are pending
• Taken together these results could be used to substantiate a reduced SC
specification for a pDNA vaccine
Figure 5: In vivo study layout. Four groups of Balb/c mice (n=8) were injected with thedifferent isoforms of pDNA. A control group was also included in the study. A total ofthree ID/EP injections were given. On day 28, blood and spleens were harvested toassess the overall immune response. Results from ELISpot analysis indicative of T-Cellactivation and ELISA for specific antibody production are pending.
M SC OC XbaI XhoI ( + ) ( - )
SC (93%) OC (99.3%) XbaI (100%) XhoI (100%)
220
120
80
220
120
80
C: HPLC Analysis
SC (93%) OC (99.5%) XbaI (100%) Btsαl (100%)
C: HPLC Analysis
LAMP Fusion Gene
M SC OC XbaI XhoI
B
C
GFP
GFP
Figure 3: (A) pLAMP-Fusion circular plasmid map. (B) Linear plasmid map. Digestion with XbaIlinearizes the plasmid upstream from the promoter. Digestion with XhoI linearizes the plasmidwithin the fusion gene. (C) AGE was used to confirms isoform preparation.
D=0 D=7 D=14 D=28
1st injection 2nd injection 3rd injection Analysis
SCOC
XbaIXhoI
14 16 18 20 22
mAU
0
100
200
300
400
500
600
DAD1 A, Sig=260,4 Ref=360,100 (20180523_DNA_STAT 2018-05-23 16-50-23\033-0901.D)
17.4
52
19.2
46
20.2
66
14 16 18 20 22
mAU
0
50
100
150
200
250
300
350
400
DAD1 A, Sig=260,4 Ref=360,100 (20180524_DNA_STAT 2018-05-24 17-24-48\033-0901.D)
17.0
35
18.1
40
14 16 18 20 22
mAU
0
50
100
150
200
250
300
350
400
DAD1 A, Sig=260,4 Ref=360,100 (20180524_DNA_STAT 2018-05-24 17-24-48\032-0601.D)
17.2
52
14 16 18 20 22
mAU
0
50
100
150
200
250
DAD1 A, Sig=260,4 Ref=360,100 (20180523_DNA_STAT 2018-05-23 16-50-23\034-1201.D)
17.5
58
mAU
DAD1 A, Sig=260,4 Ref=360,100 (20180212_DNA_STAT 2018-02-12 17-42-59\032-0901.D)
18 19 20 21
0
100
200
300
400
500
17.9
27
19.6
14
20.7
93
14 16 18
mAU
0
50
100
150
200
250
300
DAD1 A, Sig=260,4 Ref=360,100 (20180731_DNA_STAT 2018-07-31 16-33-48\034-1201.D)
15.8
12
16.7
81
14 16 18
mAU
0
100
200
300
400
500
600
700
DAD1 A, Sig=260,4 Ref=360,100 (20180731_DNA_STAT 2018-07-31 16-33-48\033-0901.D)
16.2
31
14 16 18 20
mAU
0
100
200
300
400
500
DAD1 A, Sig=260,4 Ref=360,100 (20181003_DNA_STAT 2018-10-03 16-42-44\033-0901.D)
18.7
97
XbaI