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Plasmid Biopharmaceuticals
DUARTE MIGUEL F. PRAZERES
1
and GABRIEL A. MONTEIRO
1
1
IBB, Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering,
Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal
ABSTRACT Plasmids are currently an indispensable molecular
tool in life science research and a central asset for the modern
biotechnology industry, supporting its mission to produce
pharmaceutical proteins, antibodies, vaccines, industrial
enzymes, and molecular diagnostics, to name a few key
products. Furthermore, plasmids have gradually stepped up in
the past 20 years as useful biopharmaceuticals in the context of
gene therapy and DNA vaccination interventions. This review
provides a concise coverage of the scientific progress that has
been made since the emergence of what are called today
plasmid biopharmaceuticals. The most relevant topics are
discussed to provide researchers with an updated overview of
the field. A brief outline of the initial breakthroughs and
innovations is followed by a discussion of the motivation behind
the medical uses of plasmids in the context of therapeutic and
prophylactic interventions. The molecular characteristics and
rationale underlying the design of plasmid vectors as gene
transfer agents are described and a description of the most
important methods used to deliver plasmid biopharmaceuticals
in vivo (gene gun, electroporation, cationic lipids and polymers,
and micro- and nanoparticles) is provided. The major safety
issues (integration and autoimmunity) surrounding the use of
plasmid biopharmaceuticals is discussed next. Aspects related to
the large-scale manufacturing are also covered, and reference is
made to the plasmid products that have received marketing
authorization as of today.
INTRODUCTION
The contributions of plasmids to biology and their im-
pact in biotechnology and discovery have been immense.
Together with restriction enzymes, plasmids were one
of the key molecular tools at the heart of the invention
and development of DNA cloning and recombinant
DNA by Hebert Boyer and Stanley Cohen (1,2). These
fundamental technologies shaped molecular biology and
paved the way to the development of the modern, mul-
tibillion dollar biotechnology industry (2,3). The ability
to produce unlimited amounts of proteins via the cloning
of the corresponding gene into a plasmid and subsequent
transformation of a microbial host made it possible to
develop a range of medically and industrially relevant
products and applications. The development of molec-
ular diagnostics and protein biopharmaceuticals, for
example, would have been impossible without plasmids.
However, few would have suspected in the earlier years
of recombinant DNA that plasmids could one day as-
sume the role of biopharmaceuticals themselves (4).
The breakthrough that sparked the development of
plasmid biopharmaceuticals came in 1990, when Wolff
and colleagues injected saline solutions of plasmids
containing genes for chloramphenicol acetyltransferase,
luciferase, and β-galactosidase into the skeletal muscle
of live mice (Fig. 1)(5). The authors found that the
reporter transgenes encoded in such a “naked”plas-
mid DNA molecule were expressed within the muscle
cells and concomitantly envisaged the use of plasmid-
mediated gene transfer into human muscle as a means
of improving the effects of genetic diseases of muscle.
Transfection by direct injection of naked DNA was
subsequently found in tissues other than skeletal muscle,
like liver (6), heart (7), and brain (8), and in species
as varied as fish (9), chicken (10), and cattle (11). The
proximate discovery that mice could elicit antibodies
Received: 8 November 2013, Accepted: 16 December 2013,
Published: 7 November 2014
Editors: Marcelo E. Tolmasky, California State University, Fullerton,
CA, and Juan Carlos Alonso, Centro Nacional de Biotecnología,
Cantoblanco, Madrid, Spain
Citation: Prazeres DMF, Monteiro GA. 2014. Plasmid
biopharmaceuticals. Microbiol Spectrum 2(6):PLAS-0022-2014.
doi:10.1128/microbiolspec.PLAS-0022-2014.
Correspondence: Duarte Miguel F. Prazeres,
miguelprazeres@ist.utl.pt
© 2014 American Society for Microbiology. All rights reserved.
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(12) and generate cytotoxic T lymphocytes (13) in re-
sponse to the direct administration of naked plasmid
DNA molecules encoding an antigen showed that, in
principle, plasmids could also be used to immunize ani-
mals against pathogens. This innovative approach, later
termed DNA vaccination, constituted a radical depar-
ture from conventional immunization methodologies,
which relied on the industrial production of the vacci-
nating antigens before their administration.
The seminal discoveries of the early 1990s that opened
up the possibility of using plasmids as biopharmaceu-
ticals for therapy (5) or prophylaxis (12,13) were fol-
lowed by 10 years of major innovations (Fig. 1)(14). The
following are noteworthy examples of those milestones:
the delivery of plasmid DNA by particle bombardment
(15), the application of electroporation for in vivo de-
livery (16), the coexpression of cytokines alongside with
the target genes (17), the addition of immunostimula-
tory CpG motifs to plasmid backbones (18), prime (with
DNA vaccine)-boost (with non-DNA vaccine) vaccina-
tion (19), the use of targeting sequences to enhance
the immunogenicity of DNA vaccines (20), the encap-
sulation of plasmids in microparticles (21), the design
of minimal plasmids (so-called minicircles) containing
only the functional elements required for expression of
the transgene (22), the compaction of single molecules
of DNA into minimally sized nanoparticles (23), and
the systemic in vivo administration of plasmid DNA by
rapid injection of large volumes of solution (24). These
formative years (Fig. 1) were followed by intensive re-
search efforts directed toward the adaptation of the
major concepts developed earlier to new applications
and to the expansion and accumulation of the scientific
know-how related to the mechanisms of action of plas-
mid biopharmaceuticals in vivo, via laboratorial, pre-
clinical, and clinical experimentation. Major innovations
also took place on the “process”side as the industry
sensed the increase in the maturity of the product
prototypes and concepts and prepared for clinical de-
velopment and manufacturing (14).
PLASMIDS IN DISEASE MANAGEMENT
Plasmids versus Viral Vectors
The use of plasmids as carriers of medically relevant
genes is usually considered on par with viral vectors.
Viral vectors are very effective at transferring genes
because of their natural ability to deliver and express
genes, while avoiding the different defense barriers of
the host organism and cells. For this reason, recombinant
viral vectors are the gene carriers of choice in more than
65% of the clinical trials of gene therapy registered as
of January 2014. (Data were extracted from The Journal
of Gene Medicine Gene Therapy Clinical Trials World-
wide website, http://www.wiley.co.uk/genmed/clinical
[last accessed on 4 February 2014].) However, even
though recombinant viruses used in gene transfer are
designed to minimize the toxicity and immunogenicity
of their natural counterparts, safety concerns associated
with the use of viral vectors remain high as a result of
a number of incidents and serious adverse events re-
corded during a number of gene therapy clinical trials
(25). Plasmids, on the other hand, are characterized by
an excellent safety profile (see below). For this reason,
close to 20% of the gene therapy clinical trials recorded
FIGURE 1 Plasmid biopharmaceuticals: the formative years. doi:10.1128/microbiolspec
.PLAS-0022-2014.f1
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up to 2014 had used plasmid DNA as a carrier of the
therapeutic/prophylactic transgenes (The Journal of
Gene Medicine Gene Therapy Clinical Trials Worldwide
website mentioned above).
Role of Transgene Products
Generically speaking, plasmid biopharmaceuticals are
used to transfer genes with the goal of managing disease
in humans and animals. The rationale behind this ap-
proach is that, once expressed in the target cells/tissues,
the products coded in the plasmid-borne transgenes will
act in such a way as to tackle and resolve the specific
disease or clinical condition under study. The different
functions exerted by plasmid-borne transgene products
can be broadly divided into five categories, as briefly
described next (26).
Boosting
Plasmids can be used to increase the expression of a
specific endogenous protein, whose level is otherwise
normal, by adding more copies of the coding genes.
This could contribute to accelerating the endogenous
response generated by our bodies in the context of a
specific disease (26). For example, an increase in the
expression of vascular endothelial growth factor (VEGF)
can accelerate the vascularization of ischemic tissue in
arterial diseases (27), and an increase in the expres-
sion of the hepatocyte growth factor (HGF) gene may
enhance the function of dopaminergic neurons in
Parkinson’s disease (28).
Replacement
When a hereditary defect in a single gene prevents the
body from functioning normally, e.g., as in cystic fibrosis
(29) or Duchenne muscular dystrophy (30), regular levels
of the normal protein can be supplemented by transfer-
ring the correct gene via plasmids (26). Replacement can
also be explored to compensate for the deterioration
of normal levels of a protein as a consequence of disease
(e.g., insulin in type 1 diabetes mellitus [31], erythro-
poietin in anemia [32]).
Immune stimulation
DNA vaccines can be designed on the basis of plasmids
that transfer genes whose products are able to recruit
the immune system (26,33). These vaccines can be ad-
ministered to prevent future episodes of the target dis-
ease (prophylactic vaccines) or to motivate the immune
system to fight cancer (therapeutic vaccines). In the first
case, the DNA vaccine carries the gene that codes for a
specific antigen of the causative infectious agent (e.g.,
AIDS [34], malaria [35], tuberculosis [36], influenza
[37]), whereas, in the second case, genes that code for
products that increase tumor immunogenicity and mo-
bilize immune cells to fight cancer are used (38). Unlike
in the case of traditional vaccines, antigens delivered by
DNA vaccines are synthesized endogenously, and, thus,
the process of antigen presentation that ensues may
mimic natural infection more closely.
Cytotoxicity
The plasmid-mediated transfer of genes can be used to
kill malignant cells. The therapeutic strategy is usually
designed so that the gene product plays an intermediate
role (e.g., by replacing a missing key protein, stimulating
the immune system into recognizing harmful cells, or
introducing a new functionality that contributes to kill
cells) in a more complex network of events that ulti-
mately result in the death of the target cells (26).
Blocking
The genetic information in a plasmid can also code for
short hairpin RNAs (shRNAs), which once expressed
will knock down the expression of the disease-related
target genes via RNA interference pathway (39,40,41).
Plasmids for Therapy
Gene transfer via plasmid molecules has been studied as
a possibility to treat both hereditary disorders that are
characterized by deficiencies at the single-gene level and
diseases that are caused by a combination of environ-
mental factors and genetic predisposition.
Hereditary disorders
In this case, the expectation is that the plasmid-mediated
delivery of the correct genes results in the restoration of
normal levels of the faulty protein and hence in the
halting of the course of the disease. The management of
a genetic disorder by using plasmids will inevitably rely
on chronic administration, since plasmids are typically
cleared by the cell machinery after a certain amount of
time has elapsed. The possibility of the development of
autoimmune responses or immune tolerance is thus a
cause for concern. Examples of genetic disorders that
have been addressed by plasmid-based gene transfer in-
clude (i) hemophilia, a coagulation disorder associated
with defects in factor VIII (hemophilia A) and factor IX
(hemophilia B) (42,43); (ii) cystic fibrosis, a multiorgan
disease caused by an abnormal cystic fibrosis trans-
membrane regulator gene (29,44); and (iii) Duchenne
muscular dystrophy, a neuromuscular disorder associ-
ated with defects in the dystrophin gene (30,45). A key
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challenge in the management of these diseases is to en-
sure that the corrective genes are delivered to the proper
cell. One of the strategies studied to achieve this target-
ing relies on the use of plasmid delivery vehicles modified
with ligands for specific receptors of the target cells. In
other situations, the relevant tissue can be targeted by
direct administration of the plasmid to the relevant tissue
(e.g., aerosol delivery of plasmids to the lungs in the case
of cystic fibrosis [29], intramuscular injection in the case
of muscular dystrophy [30]).
Multifactorial diseases
Clinical research on plasmid biopharmaceuticals has fo-
cused strongly on multifactorial diseases that result from
a combination of environmental factors and genetic pre-
disposition. For example, in the context of coronary and
peripheral arterial diseases, plasmids have been used to
deliver specific genes such as fibroblast growth factor
(FGF), VEGF, HGF, and AGGF1 that promote the for-
mation of new blood vessels and thus increase blood flow
to the affected ischemic tissues (myocardium, limbs) (46,
47,48,49).
Additionally, many clinical trials and a large body of
scientific research have been directed at treating cancer
with plasmids. Several therapeutic strategies can be de-
vised to kill cancer cells on the basis of genes including
tumor suppression, suicide gene therapy, antiangiogene-
sis, and immune stimulation. For example, cancer cells
can be driven to commit suicide by delivering a missing or
defective tumor suppressor gene like p53 (50,51,52,53).
The suicide gene therapy approach calls for plasmid-
encoded proteins to convert a coadministered prodrug
into an active, cytotoxic agent that kills the tumor cells.
The coupling of the cytosine deaminase gene with the
prodrug 5-fluorocytosine (54) and of the herpes simplex
virus thymidine kinase gene with the antiviral drug
ganciclovir (55) are two of the most researched enzyme-
prodrug systems. In this case, the cytotoxicity is not lim-
ited to the transfected cells, since the active drug can
diffuse and act on neighboring cells (bystander effect).
Cancer can hypothetically be treated by inhibiting the
formation of the network of blood vessels (i.e., angio-
genesis) that supply nutrients and oxygen to tumoral cells.
Plasmids have been used in this context to deliver genes
that code for antiangiogenic proteins, such as the VEGF
receptor sFLT147 (56), endostatin (41), or interleukin-12
to the affected tissues (57). Immune cells can be stimulated
to fight cancer cells by using DNA vaccines that promote
the expression of cell surface markers or cytokines. In the
first case, plasmids are used to deliver the genes that code
for tumor-associated antigens, like the prostate-specific
antigen (58) in prostate cancer and gp75/tyrosinase-
related proteins in melanoma (59). Once expressed, these
antigenic markers are adequately processed and dis-
played, recruiting cytotoxic T lymphocytes (CTLs) that
eventually kill the neoplastic cells (38,60). In the second
case, the regulatory role of interleukins, interferons, tu-
mor necrosis factors, and colony-stimulating factors in
the pathogenesis of cancer is explored as a means of
generating potent antitumor responses (42,61,62).
Further examples of multifactorial diseases in which
plasmid vectors have been used to deliver therapeutic
genes include Alzheimer’s(
63), anemia (32), arthritis (64),
burn wounds (65), dental caries (66), diabetes mellitus
(31), glaucoma (67), lupus (68), sepsis (69), spinal cord
injury (70), and wound healing (71).
Plasmids for Prophylaxis
One of the most appealing applications of plasmid bio-
pharmaceuticals is in the prevention of infectious dis-
eases. In principle, DNA vaccines can be designed to
immunize humans and animals against an enormous
range of diseases caused by viruses, bacteria, protozoans,
and fungi (33). Studies performed with animal models
have demonstrated that the protection conferred by DNA
vaccines against infectious agents occurs via the activa-
tion of the innate immune system and the induction of
CTLs, T-helper (Th) cells, and neutralizing antibodies
that are antigen specific(
72). DNA vaccines from the
earlier generation, in general, were poorly immunogenic
(33). Over the past years, several strategies have been
pursued to improve the immunogenicity of DNA vaccines
that include the optimization of codons in the antigen
gene (73), the coadministration ofgenes coding for immu-
nostimulatory functions (e.g., cytokines [74]), the use
of CpG motifs in plasmid backbones (75), the fusion of
antigens with sequences that target specific major his-
tocompatibility complex (MHC) pathways and Th-cell
responses (76), the design of heterologous prime/boost
immunization modalities (77), and the use of delivery
methodologies such as the gene gun (78). DNA vaccine
prototypes have been constructed by cloning genes coding
for antigens associated with a range of diseases including
AIDS (77), dengue (79), human papillomavirus (80), in-
fluenza (81), sleeping sickness (82), and tuberculosis (83).
MOLECULAR ASPECTS
Basic Components
Plasmid-based gene therapy and DNA vaccination rely
on an effective delivery and expression (in terms of level
and duration) of the transgene to the target cells. The
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typical plasmid vector (Fig. 2) is a covalently closed,
double-stranded DNA molecule derived from natural
plasmids, which is mostly found as a tightly twisted, super-
coiled topoisomer (84). It contains a set of prokaryotic
sequences necessary for plasmid amplification in a bacte-
rial host (replication origin, antibiotic resistance gene),
and an eukaryotic expression cassette, which includes
the therapeutic gene and the regulatory elements required
for expression in eukaryotic cells (e.g., promoter, poly-
adenylation sequence [Fig. 3]). The design of such a plas-
mid vector should take into consideration aspects such as
the stability of plasmids in vivo, the profile of transgene
expression, the impact of prokaryotic sequences, and the
response of the host immune system to the vector.
In Vivo Plasmid Stability
The plasmid journey to the cell nucleus is hindered by
several physical barriers, including the cytoplasmic mem-
brane, the network of cytoskeleton proteins and organelles
that overcrowd the cytoplasm, and the nucleus envel-
ope (85). Moreover, the degradation of plasmid DNA by
intra- and extracellular exo/endonucleases constitutes a
major barrier to gene expression (86,87).Onlyverysmall
amounts (0.1%) of the plasmid molecules that enter cells
reach the nucleus (86). The removal of secondary forming
sequences (e.g., homopurine-rich and cruciforms) from
the plasmid backbone (87,88) and the coadministration
of nuclease inhibitors like aurintricarboxylic acid (89)and
DMI-2 (90) have both been advocated as a means to im-
prove the resistance of plasmids to nucleases. Moreover,
plasmids containing direct and inverted repeats, insertion
sequences, and regions similar to genomic DNA can suffer
genetic rearrangements, such as deletions, duplications, in-
versions, translocations, and insertions, albeit at very low
frequencies (91,92,93,94). These rearrangements can
affect both plasmid production in Escherichia coli and
the efficiency of transgene expression. These and other
unstable regions should be removed from the vector or at
least changed whenever possible. Removal of nonessential
sequences from plasmids also reduces the size which per
se decreases the number of intrinsic nuclease-susceptible
or inhibitory regions and might increase the molecular
stability.
Transgene Expression
Upon successful arrival of the transgene to the nucleus
of the target cells, a reasonable amount of gene expression
during a more or less extended time period is required to
elicit the wanted therapeutic effect. The transgene ex-
pression profile can be modulated by judiciously selecting
the best promoter and transcriptional regulators. A few
promoters are currently used to drive transgene expres-
sion in the context of gene therapy and DNA vaccination.
The cytomegalovirus (CMV) immediate early promoter
is the most widely used in many vectors because of its
high strength in many different tissues. The activity of
the CMV promoter can be modulated by the presence
of specific cytokines (95) or by other proteins like p53
and Mekk1 (96). The downregulation of the promoter
activity might prevent transgene expression. Therefore,
the use of alternative promoters is often considered, e.g.,
viral promoters (e.g., Rous sarcoma virus, simian virus
40 [SV40]) and some cellular promoters (e.g., human
ubiquitin B [UbB], ubiquitin C, human elongation factor
1α), or chimeric promoters (e.g., CMV-chicken-β-actin,
CMV-UbB) (97,98,99).
The transcriptional inactivation of the promoters that
regulate transgenes is expected during the normal ho-
meostasis of the cells. A sustained and long-term ex-
pression depends on the promoter type that includes
regulatory elements such as enhancers, boundary ele-
ments, and silencers (98,99) and also on the cell type
FIGURE 2 Basic physical characteristics of plasmid vectors. Data presented is for 2,000- to
10,000-bp plasmids with a typicaldegree of supercoiling (Prazeres, 2011). Image is reprinted
with permission from reference 84 with permission from Wiley. Copyright 2011, John Wiley
and Sons, Inc. doi:10.1128/microbiolspec.PLAS-0022-2014.f2
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and physiological state at the time of transcription (96).
For example, the Kozak sequence (gccRccAUGG), which
includes the start codon, helps the transcript to bind to
ribosome to start translation. Another important regu-
latory sequence is the polyadenylation site (AAUAAA)
located at the 3′end of the mRNA that allows termina-
tion of transcription and is important for the nuclear ex-
port, stability of mRNA, and, consequently, translation.
Many eukaryotic expression vectors use the bovine
growth hormone, SV40, or rabbit β-globin terminator
sequences (88,98,99), or endogenous terminators that
are downstream from the open reading frame of the gene
of interest to ensure proper transcriptional termination.
Immune Response to Plasmid Vectors
The administration of plasmid vectors that contain bac-
terial sequences is likely to generate immune responses.
The innate immune system is able to discriminate mi-
crobial components and self-components by identifying
the pathogen-associated molecular patterns (PAMPs).
Depending on their composition (lipopolysaccharides,
nucleic acids, proteins, etc.), PAMPs are recognized by
different pattern-recognition receptors (PRRs), triggering
single, multiple, cooperative, or redundant specific sig-
naling pathways that set up an immune response (100,
101). Toll-like receptors (TLRs), like others PRRs, are
expressed in the cell surface or intracellularly (e.g., TLR9)
by various immune cell types (e.g., macrophages, den-
dritic cells, B cells) and also by nonprofessional immune
cells (e.g., fibroblasts, epithelial cells) (100,101). Usually,
sensing of PAMPs by PRRs upregulates the transcription
of type I interferons and proinflammatory cytokines.
DNAs of bacterial origin show a high frequency of
unmethlylated cytosine-phosphate-guanine (CpG) dinucle-
otides. In contrast, CpG motifs are infrequent in mam-
malian DNAs, and when present, are highly methylated
(5mCpG). Overall, DNA of bacterial origin is a PAMP that
is recognized by intracellular TLR9 and by stimulator of
interferon genes (STING) proteins, activating innate im-
munity response (101,102,103). The TLR9 signaling
cascade induces expression of type I interferons and in-
flammatory genes, mainly through activation of the tran-
scription factors interferon regulatory factor 7 (IRF7) and
NF-kB (101,102). STINGs are endoplasmic reticulum
translocon-associated transmembrane dimer proteins that
are critical for regulating the production of interferon in
response to cytoplasmic DNA (102,104). STINGs bind to
cytosolic double-stranded DNA without a requirement
for accessory molecules (103), activating the TANK-
binding kinase 1 (TBK1). STING appears essential for
escorting TBK1 to endosomal compartments for activa-
tion of the transcription factors IRF3/IRF7 and NF-kB,
leading to the expression of type I interferon and in-
flammatory cytokines (103).
Although this immunostimulatory property of CpG
motifs is undesirable when plasmids are used for gene
therapy purposes, it can be used favorably as an adju-
vant in DNA vaccination (105). The idea is that the
FIGURE 3 (A) Schematic representation of the recombination of a parental plasmid (PP)
into a minicircle (MC) and a miniplasmid (MP) via the excision of the eukaryotic expression
cassette that is flanked by two multimer resolution sites (MRS). (B) Agarose gel electro-
phoresis showing a parental plasmid before the induction of recombination (BR) and
minicircle and miniplasmid species after recombination (AR). Abbreviations: ORI, origin of
replication; GOI, gene of interest. doi:10.1128/microbiolspec.PLAS-0022-2014.f3
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type and the number of CpG motifs in a plasmid back-
bone can be designed to modulate the humoral immune
response triggered by DNA vaccines (106,107,108,
109). Several human clinical trials have used such CpG
adjuvants in the context of preventive and therapeutic
vaccination (105,110).
Optimized Vectors
Some of the functional elements (origin of replication,
prokaryotic resistance marker) found in a plasmid are
required only during the replication process that takes
place during the growth of the prokaryotic production
host. Once the cell culture is halted, those prokaryotic
sequences are no longer needed and may actually decrease
stability, uptake, and efficacy. Furthermore, even though
the presence of some prokaryotic sequences in plasmids is
approved by the FDA and European Medicines Agency,
their use can be detrimental both from a clinical and en-
vironmental point of view. For example, concerns have
been raised that a widely used selection marker like the
kanamycin resistance gene may be horizontally trans-
mitted to the recipient’s enteric bacteria (111,112). Thus,
and in line with the recommendations of regulatory agen-
cies to eliminate antibiotic resistance markers from plas-
mid vectors, a number of antibiotic-free selection systems
have been developed to produce safer and smaller (and
eventually more efficient) plasmids (99,112,113).
Noteworthy examples include plasmids with conditional
origin of replication and plasmids free of antibiotic resis-
tance, vectors expressing small RNA-OUT antisense RNA
(114), RNAI/RNAII-based plasmids containing ColE1-
type origin (115), and operator-repressor titration systems
(116). Additionally, the absence of antibiotic resistance
genes from bacterial origin in these minimized plasmids
may lead to reductions in innate immune responses and
minimize the risk of silencing of transgene expression.
The backbone of a plasmid-based DNA vaccine can be
designed with the amount of correct unmethylated CpG
sequences that maximizes immune response. On the con-
trary, an effective plasmid vector for gene therapy can be
designed by avoiding unmethylated CpGs or by adding
CpG antagonists of TLR9 (e.g., containing a (5-methyl-
dC)p(7-deaza-dG) or (5-methyl-dC)p(arabino-G) motif)
(117). Another strategy involves the methylation of the
plasmid before its administration. While several methyl-
ases (SssI, HpaII, or HaeIII) have been used in vitro, the
costs associated with this strategy have prompted re-
searchers to explore the possibility of performing meth-
ylation in vivo, for example, using SssI methylase. This
approach has shown prolonged transgene expression by
circumventing immune recognition (118).
Avoiding the presence of CpG sequences within reg-
ulatory sequences can also increase the level and the
duration of expression (97,119). Long-term gene ex-
pression (at least 19 months) has been reported by Wolff
et al. (120), suggesting that plasmid DNA could stably
persist and be expressed in nondividing muscle cells.
However, strategies like chromosomal integration or
episomal replication are usually required to obtain long
expression periods. Interestingly, the Kay’s group (121)
has shown that the administration of plasmid DNA
containing CpG motifs (methylated or not) to the mouse
liver will only lead to transgene silencing and innate
immune responses if those sequences are covalently
linked to the transgene. These authors hypothesized
that the absence of DNA sequences devoid of tran-
scriptional enhancers that maintain an active transcrip-
tion state are prone to form repressive heterochromatin
on the plasmid DNA backbone, which then spreads and
inactivates the transgene in cis, but not in trans (121).
Kay’s group also suggests that it is the length (>1 kb) and
not the sequence of the extragenic DNA flanking the
transgene expression cassette that leads to transgene
silencing (122). This is a very complex subject from which
much more insights are needed to understand the com-
plex nature of regulation of gene expression.
The presence of bacterial sequences triggers an asso-
ciation with inactive forms of chromatin. Episomal DNA
constructs with persistent expression have a slight greater
abundance of histone H3 lysine 4 dimethylated, while
unexpressed constructs showed enrichment in histone
H3 lysine 9 trimethylated (123,124,125). Interestingly,
AT-rich scaffold or matrix attachment regions, which
facilitate opening and maintenance of euchromatin, can
be incorporated near promoters to allow enhanced and
persistent transgene expression (126).
Other plasmid derivatives that are devoid of bacterial
sequences have been designed and tested successfully.
Minicircles, for example, are double-stranded and super-
coiled expression eukaryotic vectors devoid of bacterial
sequences such as the origin of replication and the anti-
biotic resistant marker (22). Minicircles are produced in
E. coli by excising the desired expression cassette from a
parental plasmid. This excision takes place by promoting
the in vivo recombination between two recombinase tar-
get sites strategically located in the parental plasmid
backbone (Fig. 3). Several recombinases acting under
the regulation of inducible promoters have been used to
catalyze this excision, including λ-integrase, Cre recombi-
-integrase, Cre recombinase, φC31 integrase, and Par
resolvase (112,113). The recombination event generates
two products, a replication-deficient minicircle, which
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contains the mammalian expression cassette, and a mini-
plasmid, which contains the undesired antibiotic resis-
tance gene and the bacterial origin of replication (Fig. 3).
The selection of the recombination system should be
aimed at providing the best balance between recombi-
nation efficiency and the yield of supercoiled minicircle
species. Since both products will coexist inside E. coli
cells once the process is terminated, adequate method-
ologies must be developed to isolate the therapeutically
useful minicircle from its counterpart (as well as from
the usual bacterial impurities). This separation consti-
tutes a challenge on its own owing to the similarity of
the physical-chemical characteristics of the two DNA
rings. One of the strategies devised to purify minicircles
relies on the placement of lactose operator sites in the
minicircle moiety and on the use of an affinity chroma-
tography matrix with bound lac repressor (LacI). When
a mixture of minicircles and miniplasmids is contacted
with this affinity column, minicircles will bind to LacI,
whereas miniplasmids are washed away in the flow-
through. Minicircles can subsequently be recovered by
eluting the column with isopropyl-β-D-1-thiogalacto-
pyranoside (127). Experimental evidence has shown that
adequately purified minicircles are able to generate a per-
sistent and high-level transgene expression in vivo (22).
The concept of transforming plasmids into minimal-
size gene transfer units also fostered the development of
short eukaryotic expression cassettes called Minimalistic
Immunogenically Defined Gene Expression (MIDGE)
vectors. Unlike the case of minicircles, however, the
final vector is a small, linear molecule that is covalently
closed at the extremities by two short hairpin oligonu-
cleotide sequences. The linear DNA fragments are gen-
erated either by restriction digestion of conventional
therapeutic vectors or by PCR-mediated amplification
(128,129).
ADMINISTRATION AND DELIVERY
Barriers
The transportation of plasmids from the outside of the
body of a patient into the cell nuclei and the subsequent
expression of the gene cargo are critical for the success
of a plasmid-mediated gene transfer intervention (130).
However, this process is difficult to achieve because of
the existence of a series of barriers that DNA molecules
encounter during their journey across the entry route,
capillaries, interstitial spaces, tissues, body fluids, mem-
branes, and cells cytoplasm and that contribute to re-
duce the number of molecules arriving at the cell nucleus
(131). Examples of such barriers include mononuclear
phagocytes, blood components, low pH, plasma and
cellular endonucleases, cellular membranes, endosomes,
lysosomes, and narrow nuclear pore complexes (131,
132). The efficiency of plasmid vectors is critically de-
pendent of the use of delivery systems adequate to over-
come these barriers and guarantee that a significant
fraction of the administered pool of plasmid molecules
arrives safely and ready for transcription into the cell
nucleus (Fig. 4).
Plasmid biopharmaceuticals can be administered
through different routes depending on the disease and
therapeutic intervention planned. For example, intra-
tumoral administration is used in the treatment of solid
tumors (133), the airways are preferred when tackling
lung diseases (134), and muscular or skin tissues are
favored when administering prophylactic DNA vaccines
(72). Virtually all organs and tissues in the human body
have been used as entry points for plasmids (130). The
administration route, to a large extent, will constrain
the choice of the delivery system used to carry the plas-
mid from the vial on the shelf into the cell nucleus.
Likewise, certain delivery systems and devices are spe-
cifically designed to serve defined entry routes.
FIGURE 4 The intracellular barriers to plasmid-
based gene transfer. In their journey to the
nucleus, plasmids have to cross the phospho-
lipidic cell membrane through endocytosis (1),
escape entrapment and degradation in endo-
somes and lysosomes (2), survive degradation
by cytosolic nucleases, traffic the overcrowded
cytoplasm (3), and translocate across the nu-
clear envelope (4). doi:10.1128/microbiolspec
.PLAS-0022-2014.f4
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Plasmid DNA
Plasmids can be delivered to cells of a living recipient via
the needle injection of a saline solution of the plasmid
into muscle (Fig. 5), as originally experimented by Wolff
and coworkers (5). While this naked DNA approach is
simple, easy to execute, and safe, in most cases, the effi-
ciency of expression of transgenes in the skeletal muscle
of nonhuman primates is inferior in comparison with
alternative delivery methodologies (135). The key prob-
lem relies on the fact that plasmids are rapidly cleared
from the injection site because of the action of endoge-
nous nucleases. This means that only a fraction of the
injected molecules will transfect cells and that this is es-
sentially restricted to the injection site. The exact mech-
anism by which naked plasmids cross cell membranes
is unclear, but suggestions have been made regarding a
receptor-mediated formation of plasmid vesicles at the
cell surface and subsequent formation of endosomes
and then fusion to lysosomes (136,137). Plasmids must
then escape endosomes/lysosomes and travel through
the cytoplasm toward the cell nucleus, most likely via a
mechanism of active transport involving microtubule
networks (138). The final crossing of the nuclear enve-
lope is facilitated if cells are engaged in division, but
in nondividing cells the nuclear pore complex (NPC)
becomes the only gate to access the nucleus (138,139). In
the latter case, the presence of nuclear localization signals
has been shown to increase the translocation of plasmids
through NPCs to a certain extent (140).
Naked DNA can be delivered more effectively via
the rapid intravenous injection of a large volume (8%
to 10% of the body weight) of a plasmid-containing sa-
line solution, a procedure that favors the transfection
of hepatocytes (24,141). However, this so-called hy-
drodynamic injection is an inherently invasive and com-
plex procedure difficult to transfer to the clinic (142).
Naked plasmid DNA can also be delivered via liquid jet
injectors, devices that generate fine (∼76 to 360 μm)
high-pressure jets that puncture through the skin at high
velocities (100 m s
−1
) and deposit solutions in the tissue
beneath (141,143).
Gene Gun
Particle bombardment (aka biolistics) is one of the most
effective ways to deliver plasmids to living cells and
tissues (Fig. 5)(72,144,145). The method relies on a
hand-held device (the gene gun) that uses pressurized
gases such as helium to propel plasmid-coated nonpo-
rous metallic microparticles (0.1 to 5 μm). Cartridges
must first be prepared with a dry powder of the plasmid-
coated particles and then inserted into the gene gun.
Gold has been the metal of choice for medical appli-
cations. When the device is triggered, a gas jet crosses
the cartridge, releasing and accelerating the particles
with a speed that allows penetration of target tissues
or organs. Gene guns have been often used to obtain
strong immune responses on delivery of DNA vaccines
to the antigen-presenting cells residing in the top layers
of skin. The microparticles ejected by the gene gun
cross past the outermost layer of the epidermis and
puncture through the membranes and across the cyto-
plasm and nuclei of cells (72,144,145). Following ex-
pression, the antigens encoded in the plasmid are then
processed, eliciting primary cellular responses and fos-
tering the production of antibodies (72,145). A number
of preclinical and human clinical trials have been con-
ducted to study the outcome of gene-gun-delivered DNA
vaccines in the context of immunization against infec-
tious diseases. These studies have indicated that sub-
stantially smaller doses of DNA vaccine (∼1to10μg)
are required to obtain immune responses (antibody titers
and CD8+ T cells) in mice and primates in comparison
with intramuscular or intradermal injections (72,145,
146).
FIGURE 5 In vivo plasmid delivery. Plasmid DNA can be combined and formulated with
buffers, stabilizers, and inorganic or organic matrices and molecules to produce: (i) a saline
solution of plasmid, (ii) gold particles coated with plasmid, (iii) plasmids complexed with
cationic lipids or polymers, (iv) polymeric microparticles with encapsulated or surface-
adsorbed plasmid, or (v) nanoparticles of compacted plasmid. doi:10.1128/microbiolspec
.PLAS-0022-2014.f5
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Electroporation
The low efficiency of gene expression that is associated
with the injection of naked plasmid DNA can be im-
proved with electric fields generated by high-energy
pulses, i.e., via electroporation. Allegedly, such electric
fields transiently increase the transmembrane potential,
leading to the opening up of ephemeral (microseconds
to seconds) transbilayer electropores (<10 nm), or to the
creation of structural defects in the membranes (147,
148). Suggestions have also been made that electropho-
retic effects are created that actively drive the negatively
charged plasmids and foster their passage across pores
and into the cell cytoplasm (147,148). Other authors,
however, advocate that transit across the destabilized
membranes occurs by passive diffusion (149)orthat
charged plasmid vesicles or stable DNA/membrane com-
plexes are formed and subsequently endocytized (150).
Critical electric parameters that can be manipulated to
control plasmid delivery by electroporation include the
number, length (few microseconds to milliseconds), volt-
age (50 to 1500 V), and waveform (exponential decay
or square wave) of the pulses (148). A few companies have
designed and developed electroporation devices to meet
the requirements for a safe and consistent clinical deliv-
ery of plasmid DNA. Such devices typically combine a
system that delivers the required electric pulses with an
injection needle for intramuscular administration (151,
152). In general, preclinical and clinical data have shown
that in vivo uptake of plasmids by tissues like the skin
and muscle and transgene expression can be increased
by electroporation (153,154). Negative aspects that have
been associated with electroporation include muscle stim-
ulation, patient discomfort, and tissue damage (148).
Cationic Lipids and Polymers
The transfection ability of plasmid biopharmaceuticals
can be improved by formulating the plasmids with specific
molecules such as cationic lipids and soluble polymers
(Fig. 5). The methodology relies on the electrostatic inter-
action between the polyanionic plasmids with a cationic
lipid (e.g., DOTAP-1,2-dioleoyl-3-trimethylammonium
propane [155]) or polymer (e.g., polyethyleneimine [PEI],
polylysine [156]). As a result of this interaction, plasmids
collapse and condense, acquiring dimensions which are
substantially smaller than the size of the individual plas-
mids. This coating of the negatively charged plasmids with
cationic “envelopes”facilitates the fusion of the com-
plexes with the negatively charged cell membranes, thus
favoring internalization by endocytosis, and also protects
plasmids against the attack of lysosomal and cytosolic
nucleases.
Micro- and Nanoparticles
Gene delivery can also be accomplished by plasmid-
loaded polymeric microparticles of a defined size (0.5 to
10 μm; Fig. 5). A key advantage of these microparticles
is that they allow a more prolonged release of plasmids
instead of the bolus type of delivery that is characteristic
of the submicron plasmid/polymer complexes described
above (21,157,158). Two of the most popular polymers
used in this context are poly(DL-lactide-co-glycolide) and
poly(DL-lactic acid) owing to their biocompatible and
biodegradable nature. Plasmid molecules can be either
encapsulated (21,159,160) or adsorbed to the surface
of the microparticles (158). Plasmid-loaded micropar-
ticles can be administered via subcutaneous or intra-
muscular needle injection. Once in vivo, the particles are
phagocytosed by professional antigen-presenting cells
(macrophages, dendritic cells) and then transported to
the lymph nodes where plasmids are gradually released
(161). The usefulness of microparticles as in vivo plasmid
delivery agents has been described in the context of sev-
eral diseases. including cancer (162), hepatitis B (163),
and tuberculosis (164). In general, plasmid/microparticle
formulations are safe and able to increase gene expres-
sion and the immunogenicity of DNA vaccines (162,
163).
The lipid/polymer complexes and microparticles de-
scribed above have sizes in the 200-nm to 5-μm range
and typically contain several plasmid molecules. This
means that some kind of disaggregation or dismantling
process must take place in the cytoplasm for plasmid
molecules to be able to pass through the 25-nm-wide
nuclear pore complexes of the nuclear envelope of cells.
One way to overcome this need for dismantling before
nuclear entry is to produce plasmid nanoparticles with
sizes smaller than 100 nm (Fig. 5). These nanoparticles
can be prepared, for example, by using chitosan (165),
peptide-polyethylene glycol conjugates (134), or prot-
amine sulfate-calcium carbonate (166).
SAFETY ISSUES
Like other biopharmaceuticals, plasmids hold in them
the potential to injure recipients. The specific safety
issues that have been raised in association with the clin-
ical use of plasmids include (i) the potential of plas-
mids and derived fragments to integrate into the host
genomic DNA (167) and (ii) the stimulation of anti-DNA
antibodies and autoimmune reactions (168). These ques-
tions have been addressed during preclinical develop-
ment by performing pharmacological and toxicological
studies with adequate animal models in line with the
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recommendations of regulatory bodies (169,170). The
goals of such studies include the definition of safe starting
doses and escalation regimens and the identification of
organs at risk and parameters to monitor toxicity.
The potential for integration in the genome is minimal
since sequences that might drive homologous recombi-
nation and direct integration (e.g., insertion sequences,
retroviral-like long terminal repeats, sequences homol-
ogous to the packaging sequences of retroviruses) are
removed during the design of the plasmid molecule
(171). So far, results show that the risk for integration
of plasmid sequences is much lower than natural, ran-
dom mutations (172,173,174,175). Furthermore,
biodistribution and persistence studies have indicated
that most plasmids that are administered intramuscu-
larly (e.g., by needle injection, needleless jet, or particle-
mediated delivery) remain close to the injection site and
are rapidly degraded by endogenous nuclease within the
first minutes (174,176,177), reducing even further the
likelihood of integration.
A number of animal experiments have been con-
ducted to investigate whether the administration of plas-
mids and the concomitant expression of the encoded
transgenes in vivo could generate and promote the de-
velopment of autoimmunity and other deleterious im-
munological responses (168,178,179,180). One of the
specific safety concerns is whether plasmids can induce
the production of anti-DNA antibodies. Such anti-DNA
antibodies could form immune complexes with circu-
lating DNA, damaging various tissues and blood vessels
in critical areas of the body, as is characteristic of sys-
temic lupus erythematosus (171). However, no link be-
tween plasmid administration and changes in clinical
markers of autoimmunity has been found yet (33).
So far, none of the concerns highlighted above have
materialized, with scientific and clinical studies indicat-
ing that plasmid biopharmaceuticals are in general well
tolerated and safe (174,176,181,182,183). Another
reason for the favorable appreciation surrounding plas-
mid biopharmaceuticals is related to the fact that they
are, in the vast majority of cases, designed to promote
transient expression of the encoded protein in the target
human tissues.
PLASMID MANUFACTURING
Overview
The development of plasmid-manufacturing processes
is an undertaking that must occur in parallel with pro-
duct development, not only because it is required to
generate material for preclinical and clinical trials, but
also because the methodology that will ultimately pro-
duce the plasmid biopharmaceuticals for sale must be
established before market approval is received (184).
The manufacturing of a plasmid biopharmaceutical pro-
duct will consist of a string of activities (Fig. 6) that are
set up and carried out with the aim of consistently pro-
ducing a defined amount (e.g., measured as biological
activity or mass) of a product that is safe and efficacious
(184). The preparation of cell banks containing the
plasmid of interest and the selection and testing of raw
materials are at the forefront of the activities. Cell cul-
ture and downstream processing unit operations are
then selected, arranged, designed, and operated to man-
ufacture unformulated (i.e., bulk) plasmid DNA (Fig. 6).
This purified plasmid product must then be adequately
formulated by considering aspects such as the method of
delivery, the final product form, ingredients (excipients,
adjuvants, stabilizers), dosage details, packaging, etc.
After the “filling and finishing”stage, the product is
ready for clinical testing or marketing (Fig. 6).
Cell Culture
Plasmids are produced by promoting replication in E. coli.
Before routine culture, a strain has to be chosen (e.g.,
DH5a, JM109) or developed (e.g., GALG20 [185]). While
the genetic background of these producer strains may
vary, mutations in the recA and endA genes to minimize
recombination events and plasmid DNA degradation,
FIGURE 6 An overview of the different activities and steps involved in the manufacturing
of plasmid biopharmaceuticals. doi:10.1128/microbiolspec.PLAS-0022-2014.f6
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respectively, are close to universal. Other important
genetic traits are related to genetic modifications (e.g.,
pkyA,pkyF,pgi) that increase the metabolic flux toward
the formation of nucleotide precursors (185). Once a
strain has been selected and transformed with the target
plasmid, high-production clones must be carefully se-
lected and isolated and used to establish master and
working cell banks with stocks of vials of the plasmid-
bearing cells. Growth medium composition, bioreactor-
operating variables, and cultivation strategy must then
be selected that maximize plasmid production. By ade-
quately combining these parameters with a high-copy-
number plasmid, volumetric plasmid yields up to 2.0 g/l can
be obtained (186).
Downstream Processing
The train of unit operations in the downstream pro-
cessing section is designed to recover plasmid DNA and
remove host impurities (genomic DNA, RNA, proteins,
etc.) until a level of purity compatible with human use is
met (184). The unit operations can be grouped into three
stages: primary isolation, intermediate purification, and
final purification. The starting point is typically a broth
with high cell densities (>55 g of dry cell weight/l) and
plasmid concentrations (1.6 to 2.0 g/l [186,187]]). In the
first stage, cells are harvested (e.g., by microfiltration)
and lysed (e.g., alkaline lysis, thermal lysis) to release
plasmid DNA. In the subsequent intermediate purifica-
tion stage, clarified lysates are processed by using oper-
ations such as tangential flow filtration, precipitation
(188), adsorption (189), and aqueous two-phase systems
(190) to reduce the impurity load and concentrate the
plasmid. Final purification aims to remove the more
recalcitrant impurities such as gDNA and pDNA vari-
ants. Traditional chromatographic modalities such as
gel filtration, anion exchange, and hydrophobic inter-
action (191) have all been used, mostly in the fixed-bed
mode, to purify plasmid. Efforts have also been made
to develop chromatographic operations based on amino
acid (192), thiophilic (193), and phenyl boronate (189)
ligands. Although dominating, chromatography is faced
with limitations (poor selectivity, coelution, low capac-
ity, and slow internal diffusion) that are related to the
structural nature of the stationary phases and molecules
(molecular mass >10
6
gmol
−1
,D=10
8
cm
2
/s) involved
(194,195). Larger capacity and faster internal mass
transfer can be achieved if chromatographic membranes
and monoliths are used instead of beads (195). Once
impurities have been reduced to levels below the speci-
fications, corrections to the plasmid concentration and
buffer exchange can be accomplished by operations like
ultrafiltration and alcohol precipitation (196). The final
step in the downstream processing train is usually sterile
filtration with 0.22-μmfilters (197).
THE ROAD TO THE MARKET
A handful of plasmid biopharmaceuticals have already
found their way into the market (198). In 2005, a vet-
erinary DNA vaccine designed and developed jointly
by the CDC and Fort Dodge Animal Health (Fort
Dodge, Iowa) to protect horses against West Nile virus
was licensed by the Center for Veterinary Biologics of
the U.S. Department of Agriculture, thus becoming the
first DNA vaccine to be registered with a governmental
regulatory body (199). The vaccine was subsequently
launched in the market in December 2008, under the
trade name West Nile Innovator DNA (200). In the same
year, a DNA vaccine developed by Novartis Animal
Health (Victoria, Canada) to protect farm-raised salmon
against Infectious Hematopoietic Necrosis virus (Apex-
IHN) also obtained regulatory approval and license
(201). In early 2007, the U.S. Department of Agriculture
conditionally approved a therapeutic DNA vaccine that
delivers the MHC gene to dog tumors to treat melanoma
in dogs (202). The vaccine hit the market under the trade
name Oncept (198). Finally, in 2008, an injectable plas-
mid DNA encoding for porcine Growth Hormone Re-
leasing Hormone (GHRH) developed by VGX Animal
Health, Inc. (The Woodlands, Texas) to decrease peri-
natal mortality and morbidity in pigs obtained market
entrance approval from the Australian Pesticides and
Veterinary Medicines Authority (203).
CONCLUDING REMARKS
Plasmid-mediated gene transfer has slowly materialized
as a possible solution for the management of an entire
constellation of veterinary and human diseases. The
investment made during the past 20 years in the devel-
opment of this new class of biopharmaceuticals has
generated a substantial amount of scientific and tech-
nological knowledge. Furthermore, plasmid biopharma-
ceutical prototypes are currently at the clinical stage of
development to tackle multifactorial diseases like cancer
and cardiovascular disorders and to prevent the onset
of infections like AIDS or influenza. So far, the data
accumulated have shown that plasmids, in general, are
well tolerated and safe. However, progress must be
made to increase the potency and efficacy of plasmid
molecules in vivo. Advances are clearly needed in the
delivery methodologies used to increase the number of
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administered plasmids that reach the cell nucleus. The
manipulation of plasmids and plasmid-related molecules
(e.g., minicircles, MIDGEs) is also likely to originate
molecules better adapted to bypass cell barriers and to
mediate the expression of therapeutic genes.
ACKNOWLEDGMENTS
Conflict of interest: We declare that we have no conflicts.
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