functional evaluation of isoforms present in pdna …...functional evaluation of isoforms present in...

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