Dna Important

Cole et al.
Cancer Nanotechnology (2015) 6:2

DOI 10.1186/s12645-015-0010-5
REVIEW Open Access
DNA vaccination for prostate cancer: key

concepts and considerations
Grace Cole, Joanne McCaffrey, Ahlam A. Ali and Helen O. McCarthy*
* Correspondence:
[email protected] Abstract
School of Pharmacy, Queen’s
University Belfast, 97 Lisburn Road, While locally confined prostate cancer is associated with a low five year mortality
Belfast BT9 7BLNorthern Ireland, UK rate, advanced or metastatic disease remains a major challenge for healthcare
professionals to treat and is usually terminal. As such, there is a need for the
development of new, efficacious therapies for prostate cancer. Immunotherapy
represents a promising approach where the host’s immune system is harnessed to
mount an anti-tumour effect, and the licensing of the first prostate cancer specific
immunotherapy in 2010 has opened the door for other immunotherapies to gain
regulatory approval. Among these strategies DNA vaccines are an attractive option in
terms of their ability to elicit a highly specific, potent and wide-sweeping immune
response. Several DNA vaccines have been tested for prostate cancer and while they
have demonstrated a good safety profile they have faced problems with low efficacy
and immunogenicity compared to other immunotherapeutic approaches. This review
focuses on the positive aspects of DNA vaccines for prostate cancer that have been
assessed in preclinical and clinical trials thus far and examines the key considerations
that must be employed to improve the efficacy and immunogenicity of these vaccines.
Keywords: Prostate cancer; DNA vaccine; Prophylactic; Therapeutic; Tumour associated
antigens
Introduction
Prostate cancer represents a major challenge to healthcare and accounts for 25 %
of all new diagnoses in males in the UK annually [1]. Localised prostate cancer
may be treated with prostatectomy or radiotherapy, which aims to remove or re-
duce the tumour load and is associated with favourable overall survival [2, 3].
However, typically anywhere from 20–30 % of patients experience a recurrence or
present with locally advanced or metastatic disease [4]. The first line treatment for
these patients is androgen deprivation therapy (ADT) which is associated with un-
pleasant side-effects such as urinary and erectile dysfunction [1, 3], and after an
initial response, the majority of cases eventually progress to castration resistant
prostate cancer (CRPC). Docetaxel is the gold standard treatment for CRPC but is
not curative and is associated with only a moderate (2.4 months) survival advan-
tage [5, 6]. As such, there is a clinical need for newer, highly effective treatment
options for patients with CRPC.
Immunotherapy is a strategy for cancer treatment that has received increasing at-
tention over the last few decades. The goal of immunotherapy is to harness the
© 2015 Cole et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly credited.
Cole et al. Cancer Nanotechnology (2015) 6:2 Page 2 of 23
immune system to mount a response against tumour associated antigens (TAAs),

normal proteins expressed by or upregulated in cancer cells [7]. In order to be suc-
cessful the vaccine must be capable of generating a tumour specific T cell response to
weakly immunogenic “self-antigens” [7, 8]. The vaccine must also overcome the
mechanisms of immune evasion employed by cancer cells, such as, the immunosup-
pressive microenvironment, downregulation of major histocompatibility complex
(MHC) antigen presentation, upregulation of regulatory T cells and co-inhibitory sig-
nalling pathways [8, 9].
Prostate cancer is an ideal candidate for immunotherapy for a number of reasons.
For example, the slow growing nature of cancer within the prostate [10] allows suffi-
cient time for the immune system to mount an anti-tumour response following a
prime/boost or multiple immunisation strategy. In addition, prostate cancer expresses
numerous TAAs which include the Prostate Specific Antigen (PSA) [11, 12], Prostatic
Acid Phosphatase (PAP) [13], Prostate Specific Membrane Antigen (PSMA) [12, 14],
Prostate Stem Cell Antigen (PSCA) [15] and Six Transmembrane Epithelial Antigen of
the Prostate (STEAP) [16]. All of these TAA’s provide multiple potential immunological
targets [17] and indeed the ideal combination of antigens has yet to be elucidated. Fur-
thermore, the presence of PSA in patient serum enables the malignancy to be detected
early and in some cases even before tumours are radiologically detectable [9]. This in
turn facilitates earlier treatment [18]. Circulating T cells that react with prostate TAAs
have previously been detected, which suggests that self-tolerance towards these anti-
gens can be overcome [18]. The prostate is considered to be a non-essential organ and
therefore immunological treatments utilising prostate TAAs will not cause acute off-
target toxicity [9, 18]. Finally and perhaps most importantly, the first prostate cancer
specific immunotherapy, Sipuleucel-T (Provenge®, Dendreon Corporation, Seattle,
WA), has recently been licensed by the US Food and Drug Administration (FDA) in
2010 for asymptomatic or minimally symptomatic CRPC [19]. Sipuleucel-T consists of
autologous peripheral blood mononuclear cells with antigen presenting dendritic cells
that have been activated ex vivo with a recombinant fusion protein (PA2024) consist-
ing of PAP linked to granulocyte-macrophage colony stimulating factor (GM-CSF)
[19]. In a phase III trial, CPRC patients receiving Sipuleucel-T had a 22 % reduction in
mortality [20]. The success of the therapeutic Sipuleucel–T has paved the way for
other immunotherapeutic prostate cancer vaccines to be granted regulatory approval
and enter the market.
Other immunotherapeutic cancer vaccine approaches which have been clinically
investigated for prostate cancer include the administration of whole tumour cells
[21], dendritic cells (DCs) loaded with peptides or tumour cell lysate [22], peptide
vaccines [23] and the administration of antibodies [24]. This review examines the
progress of DNA vaccines specifically for prostate cancer and focuses on the key
considerations required for successful development. Only the most recent studies
are included in this review to bring the reader up to date with the field. Clinical
trials that utilise DNA vaccines in prostate cancer therapeutically are summarised
in Table 1, while DNA vaccines administered prophylactically in preclinical models
prior to tumour challenge are summarised in Table 2. In addition, ongoing Phase
II or III clinical trials utilising DNA vaccines in prostate cancer are detailed in
Table 3.
Table 1 Summary of therapeutic clinical trials utilising DNA vaccines for prostate cancer
Vaccine/ targeted Phase trial/ number Delivery Immunological response/ clinical outcome/ Ref
antigen of patients route PSA DT
PAP: pTVG-HP (100 μg) Phase II i.d No. with tripling of T-Cell [99]
with rhGM-CSF (200 μg) (NCT00849121) specific antibodies- Group
N = 17 1: 3/8 Group 2: 6/8
No. with doubling of PSA
DT- Group 1: 3/8 Group 2: 4/9
PAP: pTVG-HP (100 μg, Phase I/IIa i.d. No. with PAP-specific IFNγ-secreting [31]
500 μg or 1500 μg) with (NCT00582140) CD8+ T-cells- 3/22
GM-CSF (200 μg) N = 22
No. with tripling of CD4+ and/or
CD8+ T-cell proliferation – 9/22
No. with doubling of PSA DT- 7/22
PSA: PROSTVAC with Phase II s.c. Overall survival- PROSTVAC group: [75]
GM-CSF (100 μg) (NCT00078585) 25/82 Control: 7/40
N = 125
Median survival- PROSTVAC group:
25.1 months Control: 16.6 months
PSA: Ad/PSA (106, 107, Phase I (IND #9706) s.c. No. with anti-PSA T cell responses- [74]
108pfu) N = 32 15/28
No. with increased PSA-DT- 13/28
PSA: pVAXrcPSAv531 Phase I i.d. with EP No. with prolongation of PSA-DT [47]
(rhPSA) (50–1600 μg) (NCT00859729) (DERMAVAX) by at least 50 % during study- 4/15
N = 15
PSMA: DOM-PSMA27 Phase I/II N = 30 i.m. with or No. with detectable anti-PSMA27 [48]
(800–3200 μg) without EP CD8+ T cells response- 16/30
No. with doubling of PSA-DT- 14/30
DNA vaccines
One of the key goals in a cancer vaccine is to induce a cell-mediated immune response,
primarily through the activation of TAA-specific cytotoxic T lymphocytes (CTLs).
Therapeutic responses arise from activation of these antigen specific CTLs which cause
destruction of TAA expressing cells. This could potentially eradicate disseminated de-
posits of prostate cancer for which current treatment options are limited following the
Table 2 Summary of preclinical prophylactic prostate cancer tumour challenge studies utilising
DNA vaccines
Vaccine/ targeted antigen Model Delivery route Clinical outcome Ref
PSCA/STEAP: pCI-neo-mPSCA C57 BL/6 i.m. prime Significant reduction in [17]
and/or pCI-neo-mSTEAP1 tumour volume
(100 μg) prime plus MVA-mPSCA
TRAMP C-1 i.p. boost Significant delay in time to
and/or MVA-mSTEAP1 (1X107
form tumours
pfu) boost
hPSA: phPSA (50 μg) with or C57 BL/6 i.m. with EP Significant delay in appearance [46]
without CpG of tumours
TRAMP C-1/hPSA Significantly prolonged survival
PSMA/PSCA/STEAP: rAd/PSMA, C57 BL/6 i.v. prime Tumour Growth Significantly [112]
rAD/PSCA, rAd/STEAP prime delayed
(1X108 PFU); TRAMP C-1 pulsed
DCs (2X106 cells) boost TRAMP C-1 s.c. boost
STEAP: mSTEAP DN A (2 μg) C57 BL/6 i.d. (gene gun) Significantly prolonged survival [56]
prime with: mSTEAP DN A (2 μg)
TRAMP C-2 s.c. Significantly delayed tumour
or mSTEAP-VRP (106 IU) boost;
growth
or mSTEAP-VRP (106 IU) prime
and boost
Table 3 Summary of ongoing or unpublished clinical trials utilising DNA vaccines for prostate
cancer
Vaccine/ targeted antigen Phase trial/ estimated Delivery Primary objectives Ref
enrolment route
PAP: Sipuleucel-T with or without Phase II (NCT01706458) i.d. PAP-specific Immunological [113]
pTVG-HP (100 μg)/ rhGM-CSF N = 30 response
(200 μg)
PAP: rhGM-CSF (200 μg) with or Phase II (NCT01341652) i.d. Metastasis-free survival [114]
without pTVG-HP (100 μg) N = 56
PSA: PROSTVAC with or without Phase III (NCT01322490) s.c. Overall Survival [77]
GM-CSF (100 μg) N = 1200
PSA: Flutamide with or without Phase II (NCT00450463) s.c. Time to Treatment Failure [115]
PROSTVAC N = 53
PSA: Adenovirus/PSA (1X108 pfu Phase II (NCT00583024) s.c. PSA-DT Response [76]
in gelfoam) N = 66
PSA: Adenovirus/PSA (1X108 pfu Phase II (NCT00583752) s.c. PSA-DT Response [116]
in gelfoam) with or without ADT N = 70
onset of castrate resistance. It has been well documented that DNA vaccination is a
highly potent strategy for inducing both prophylactic and therapeutic responses [25].
However, in order for the desired antigen to be expressed the plasmid DNA needs to
be delivered to the nucleus of the cell. There is also a need to ensure that the DNA is
delivered to antigen-presenting cells (APCs), so that the antigen expressed in the cyto-
plasm can be presented via the MHC class I complex [26]. Presentation via the MHC
class I complex will result in a much more potent cell-mediated therapeutic immune
response. Additionally, APCs are capable of internalising and processing exogenous an-
tigens from neighbouring apoptotic cells that have been transfected. Such antigens are
again presented via the MHC class I pathway and this process is known as cross-
presentation [26]. DNA that is delivered to non-APCs will give rise to exogenous anti-
gens that activate the more common MHC class II pathway which is characterised by a
humoral response and a subsequent prophylactic action (Fig. 1).
DNA vaccines confer many advantages over conventional treatments: (1) DNA vac-
cines are capable of eliciting host humoral and cellular immunity, leading to a potent,
wide-sweeping immune response to TAAs [25]; (2) Genes encoding the full length of
the TAA can be introduced, ensuring that the correct post-translational modifications
occur in the cell, thus presenting multiple potential antigenic epitopes to the immune
system [27, 28]; (3) The TAAs confer high specificity which renders DNA vaccines safe
compared to conventional treatments [29] and safety has been demonstrated in animals
and in several clinical trials [30, 31]; (4) DNA is relatively easy to produce and purify
and is highly reproducible, therefore DNA vaccines should be cost-effective for large
scale manufacture [27, 29]; (5) Lastly, in contrast to conventional live attenuated vac-
cines there is no reversion risk to pathogenicity in vivo.
The worldwide DNA vaccine market is projected to increase to $2.7 billion by 2019,
yet there are only four commercially available DNA vaccines licensed for use and these
are in animals. Licensed vaccines include the West Nile-Innovator® DNA (Pfizer),
Apex®-IHN (Novartis Aqua Health), ONCEPT™ (Vical) and LifeTide® SW 5 (VGX
Animal Health). Of these DNA vaccines only ONCEPT™ has been licensed (2007) for
use in cancer, specifically for the treatment of malignant melanoma in dogs [32]. Des-
pite the promise shown by DNA vaccines in preclinical models, success has proven
Fig. 1 Schematic representation of immune responses elicited following DNA vaccination. DNA may be
taken up by bystander cells (e.g., muscle cells, keratinocytes) or APCs at the site of immunisation resulting in
production of host-synthesised antigens capable of eliciting immune responses via both MHC‐I and MHC‐II
pathways. APCs have a central role in the induction of immunity following vaccination, either by direct
transfection of the APCs or cross-presentation through bystander cell associated exogenous antigens resulting
in presentation of antigen on MHC class-I molecules, eliciting CD8+ T cell expansion. Additionally, exogenous
antigens, secreted from bystander cells, captured and processed by APCs are presented via MHC class‐II
molecules resulting in CD4+ T cell expansion resulting in a cascade of cellular responses and B cell activation
and antibody production
difficult to reproduce in larger animals and clinical trials [33]. This lack of efficacy is
thought to be due to low immunogenicity and cellular uptake of DNA. Nevertheless, if
the barriers preventing the translation of this therapy to humans can be overcome, the
impact of DNA vaccination on the treatment of cancer could be revolutionary.
In order to be efficacious, DNA must reach the cell nucleus in quantities sufficient to
produce enough antigen to overcome self-tolerance. After introduction to the host
there are a number of biological barriers to nuclear delivery that contribute to a low
clinical success rate. At the cell surface DNA must undergo internalisation across the
cell membrane, which frequently results in endosomal entrapment. In the endosome,
DNA is vulnerable to degradation by intracellular nucleases and must escape into the
cytoplasm. From the cytoplasm, the DNA must be actively transported into the cell nu-
cleus where the cell can begin to transcribe and translate the DNA to produce the anti-
gen of interest [34–36]. Vectors can be utilised to improve DNA delivery. Such vectors
can be employed to condense and protect DNA from clearance and degradation in
addition to overcoming the extra and intracellular barriers (Fig. 2).
Strategies to improve DNA vaccine efficacy

A number of factors contribute to the overall transfection rate and therefore efficacy of
each DNA vaccine. With a plethora of delivery platforms and strategies designed to im-
prove the potency of DNA vaccines, it is difficult to elucidate the optimum delivery
strategy for the “best” TAA. Few studies include a direct comparison between the effi-
cacy of a delivery system against the current gold standard, with most studies examin-
ing a new delivery vehicle against control groups receiving “naked” DNA or no
Fig. 2 Schematic representation of extracellular and intracellular barriers to DNA delivery. DNA and DNA
complexes delivered in vivo must overcome a number of barriers to achieve successful gene expression in
the cell nucleus: (i) Endo and exonuclease degradation of DNA; (ii) Migration of DNA from the target tissue
into systemic circulation; (iii) Binding and aggregation of DNA via serum protein complexation; (iv) Immune
activation to delivered DNA; (v) Interaction and binding with erythrocytes; (vi) Clearance of DNA via spleen,
renal and hepatic systems; (vii) Migration of DNA through extracellular matrix in target organ; (viii) Cellular
uptake, mediated via endocytosis or passive entry; (ix) enzymatic degradation of DNA in lysosome;
(x) Nuclear localization of DNA for protein expression
treatment. This makes it particularly difficult to evaluate the true potential of any new
delivery strategies. This is further complicated by discrepancies in experimental design
and evaluation, which render it almost impossible to directly compare the variety of
approaches employed.
Injection of “naked” DNA is the simplest delivery strategy and has been shown to in-
duce humoral and cellular immune responses when administered to mouse models
[37]. However, this strategy offers little protection to DNA and transfection rates are
significantly reduced when upscaled to human studies [30]. Several delivery approaches
are undergoing investigation to improve DNA vaccine efficacy. Delivery platforms can
be broadly classified as physical or non-physical (vector-based) methods, which can be
further subcategorised to either viral or non-viral. In addition to the DNA delivery plat-
form, consideration must be given to the immunisation protocol, the co-administration
of adjuvants, which may be used to modify the cellular environment, and to the origin
and combination of DNA delivered which may play a central role in the induction of a
potent immune response. This review shall introduce and provide a brief discussion of
the most recent developments in each field, but shall focus on the strategies that are
most applicable to prostate cancer.
Physical delivery methods

Physical delivery methods act to overcome the extracellular barriers to gene delivery
and/or to temporarily disrupt the membrane of target cells, allowing DNA entry. Tat-
tooing [38], micro-injection [39], gene gun [40], ultrasound [41] and electroporation
(EP) [42] have been described as physical methods for gene delivery. Of these, EP, gene
gun and ultrasound have been used for gene delivery in prostate cancer models.
Electroporation
EP is a technique whereby DNA is delivered intradermally (i.d.) or intramuscularly (i.m.)
to the target site and a short electrical pulse or series of electrical pulses are applied locally
to the area. This results in a transient destabilisation of cell membranes in the target tissue
[35]. EP has been well documented as a potent means of enhancing transgene delivery,
with antigen expression reported to increase up to 1000 fold [28, 43, 42]. Antigen specific
responses have also been detected 25 weeks post immunisation [44]. A favourable safety
profile also makes EP an attractive option for in vivo immunisation.
EP has been used to enhance DNA vaccine immunogenicity in several prostate cancer
preclinical models. For example, Roos et al. demonstrated a significant increase in PSA
specific CD8+ T cells circulating in peripheral blood following i.d. injection of only
10 μg pVax-PSA accompanied by EP compared to those receiving no EP in C57 BL/6
mice following 2 immunisations [45]. EP has also demonstrated efficacy and safety in a
number of clinical trials [12, 46, 47]. For example, in a phase I/II dose escalation trial,
patients with biochemically recurrent prostate cancer were immunised i.m. with
pDOM-PSMA, a DNA fusion vaccine encoding a PSMA epitope, PSMA27, and DOM,
a domain of fragment C a tetanus toxin, without (Arm I) or with (Arm II) EP [48].
Patients received a total of five immunisations at weeks 0, 4, 8, 24 and 48, with follow-
up recorded up to week 72. At week 24, 11 of the 15 patients from Arm I switched to
Arm II due to a significantly higher anti-DOM antibody response in patients receiving
EP [48]. Vaccination with or without EP was associated with significant increases in de-
tectable DOM-specific CD4+ and PSMA27-specific CD8+ T cells compared to baseline,
with a significant trend towards higher responses in those treated with EP up to week
24 [48]. Treatment was associated with a significant increase in PSA doubling time
(PSA-DT), an indication of disease progression, from 11.98 months pre-treatment to
16.82 months at 72 week follow-up, independent of whether the patient had received
EP or not. While the authors found in this case that vaccination and EP were well toler-
ated by patients, other reports have found that EP is associated with pain, inflammation
and bleeding, especially when given i.m. which could decrease clinical acceptability [49,
50]. It is likely that the delivery site (whether i.d. or i.m.) is key to the generation of the
immune response. For example, the high population of resident APCs in the skin have
increased the number of EP plus i.d. studies [42]. Eriksson et al. delivered pVAX plas-
mid encoding rhesus macaque PSA (rhPSA), pVAX/rhPSA, to patients with biochem-
ical evidence of prostate cancer relapse i.d. followed by EP with the DermaVax device
(BTX/Harvard Appartus) and monitored patients for evidence of decrease in PSA-DT
or generation of PSA-specific T cells [47]. However, no significant changes in PSA kin-
etics were observed in any patients and increased PSA-specific T cell reactivity was only
observed in patients in the highest dose cohorts (4 of 15) [47]. The authors speculated
that i.m. delivery elicits a more potent immunological response. However, as this is
more invasive it may be desirable to increase the potency of i.d. vaccination with higher
vaccine doses or with the use of adjuvants [47].
To conclude, EP is a well established means of enhancing transgene expression and
acts as an immune adjuvant [42] due to the inflammation and recruitment of DCs fol-
lowing application, largely due to local tissue damage. Damage is directly related to the
intensity of EP and higher intensities are associated with higher transfection efficacies
[42]. As such, there is a trade-off between increased efficacy and discomfort to the
patient, with the latter being the rate-limiting factor [42]. Despite these concerns EP
has been used safely in a number of key prostate cancer clinical trials [47, 48], provid-
ing an encouraging platform for DNA vaccine delivery. The availability of commercially
produced EP devices also provides a convenient, reproducible means for researchers to
administer their vaccines in preclinical and clinical trials.
The gene gun

A second physical delivery strategy is the use of the “gene gun”, whereby naked DNA is
adhered to the surface of gold particles, which are accelerated under high pressure by a
ballistic device into the target tissue. Such high pressure is necessary to ensure penetra-
tion of cell membranes which is vital for intracellular DNA delivery [40]. The gene gun
has demonstrated superior gene expression compared to injection of naked DNA [51],
and is capable of enhancing specific humoral and cellular immunity [51]. In preclinical
trials the gene gun has also demonstrated comparable efficacy to EP in inducing a po-
tent cellular immune response following i.m. injection [52]. As such, the gene gun has
been used to induce antigen specific responses in several clinical trials [53, 54], most
notably to protect humans from influenza challenge [54]. However, its use as a delivery
agent in clinical trials for cancer treatment has been less successful, perhaps due to lim-
ited gene carrying capacity (~2 μg per dose), which often necessitates multiple immuni-
sations at different sites in the body, reducing patient compliance [52–54]. Despite this
dose limitation the gene gun is a simple and flexible device for in vivo gene delivery
and has been used for the delivery of prostate TAAs in several preclinical studies.
Gregor et al. [55] and Garcia-Hernandez et al. [56] have used the gene gun to deliver
prostate TAAs in murine preclinical studies and these are discussed subsequently.
To conclude, the gene gun is a flexible, fast and highly reproducible option for
in vivo gene delivery due to the availability of a commercial device (Helios Gene Gun,
Bio-Rad). The gene gun is however limited by the DNA carrying capacity, as well the
degree of tissue penetration, with penetration depths limited to 0.1 mm [40]. Therefore,
moving to larger animal models and humans may prove impractical.
Ultrasound
Although EP and gene gun have good efficacy in vivo there are newer, less invasive
physical delivery systems being developed. One such delivery enhancement strategy is
ultrasound. DNA is injected into the target tissue and ultrasonic waves are applied ex-
ternally, continually or in pulses, causing a transient, reversible increase in cell mem-
brane permeability, thus facilitating cellular entry of the DNA [41, 57]. Transfection
efficacy varies according to a number of factors including frequency, intensity and dur-
ation of application. However, optimal conditions have not yet been established and the
risk of cellular damage to the host tissue has not been fully elucidated [41]. Although
ultrasound has been shown to increase gene expression 10–15 fold in vivo compared to
“naked” DNA [39], levels of gene expression are still considerably lower than that
which can be achieved using either EP or gene gun approaches [39]. Nevertheless,
Yoshida et al. [58] utilised ultrasound to enhance delivery of mannose-modified bubble
lipoplexes containing ubiquitylated melanoma-related antigen (pUb-M) to APCs. In
combination with doxorubicin, this produced a robust CTL response following one im-
munisation and was able to significantly prolong the survival of C57 BL/6 mice with
established solid B16 tumours [58]. This study highlights the potential of ultrasound to
improve the therapeutic response to TAAs in cancer models in vivo. To date, ultra-
sound has not been used to deliver TAAs in a preclinical prostate cancer model, how-
ever, several authors have employed ultrasound to enhance gene delivery to prostate
cancer tumours in vivo.
Duvshani-Eshet et al. utilised ultrasound to enhance delivery of anti-angiogenic
hemopexin-like domain fragment (PEX) genes to prostate tumours in vivo [59]. The
group inoculated C57 BL/6 mice with PC-3 tumour cells and when the tumours
reached 100 mm3 treatment was initiated with intratumoural (i.t.) injection of naked
PEX expressing plasmid (pPEX) with or without therapeutic ultrasound (TUS). Tumour
burden was monitored every 2 days for 28 days. Following a single application TUS
significantly decreased tumour weight and volume compared to control (no treatment),
0.65 ± 0.15 g compared to 1.05 ± 0.25 g and 1300 ± 250 mm3 compared to 2000 ±
300 mm3, respectively. This effect was significantly improved by the addition of an ultra-
sound contrast agent, Optison. Optison is a microbubble composed of an albumin shell
with a gas core used to enhance the ultrasound backscatter in the target tissue. Subse-
quent studies involved tumour implantation followed by weekly treatment for four weeks
with pPEX and Optison with or without TUS. Repeated treatments of both pPEX or
pPEX + Optison alongside TUS significantly reduced prostate tumour burden and growth
by 80 % compared to a single treatment and control (no TUS). This study demonstrated
the promise of ultrasound as a gene delivery strategy in cancer. However, while ultrasound
can be easily directed towards specific tissues it is not always possible to inject therapy i.t.
This limitation needs to be overcome before clinical translation of this delivery system
can be achieved.
To conclude, ultrasound is a promising delivery option for the future, particularly for
use in combination with other immunotherapeutic approaches. The non-invasive nature
of ultrasound makes it an ideal candidate as a physical delivery system. At present the
higher efficacy of other systems, such as EP, make them more appealing to researchers. EP
and the gene gun have been used for DNA vaccination in numerous clinical trials and
have well established safety profiles, however, ultrasound has not been evaluated to the
same extent and the long term toxicity and efficacy still require elucidation.
Conclusions and future considerations

Physical delivery systems use the application of force to overcome the extra- and intra-
cellular barriers to gene delivery. In the majority of systems this enables the bypassing
of endocytosis and allows cellular entry of DNA through physically formed pores in the
cell membrane. This leads to a rapid and sustained gene expression, thus, physical de-
livery systems represent a convenient and efficacious method for gene delivery in vivo.
The advantages and disadvantages of these systems are summarised in Table 4. Delivery
methods such as EP and the gene gun are well established in terms of efficacy, but are
invasive and require the need for specialist equipment and training. This makes them
less desirable for widespread vaccination use in a clinical setting. Ultrasound, while less
invasive, suffers from a lack of efficacy compared to these established techniques and
also requires specialist equipment. Further optimisation and investigation into the effi-
cacy and cytotoxicity of this technique is required before it can be considered for rou-
tine use in gene delivery trials. However, ultrasound may have a role in enhancing the
Table 4 Summary of advantages and disadvantages of physical delivery strategies used in DNA
vaccination
Physical delivery strategy Advantages Disadvantages
Electroporation • High levels of transgene • Invasive
expression
• Long-lasting gene expression • Need for specialist equipment and training
• Safety demonstrated in numerous • Potential for tissue damage
clinical trials
• Commercially available delivery • Two-step delivery process
devices
Gene Gun • High levels of transgene • Invasive
expression
• Long-lasting gene expression • Limited DNA carrying capacity
• Safety demonstrated in clinical • Need for specialist equipment and
trials training
• Commercially available delivery • Often need for multiple administrations
devices
• One-step delivery process • Low tissue penetration
Ultrasound • Non-invasive • Low levels of transgene expression
• Can be targeted to specific • Need for specialist equipment and training
organs easily
• Two-step delivery process
• Safety not yet widely demonstrated in gene
therapy clinical trials
efficacy of gene delivery protocols when used in combination with other techniques.
For example, Yamashita et al. [60] used a combination of EP and ultrasound, termed
electro-sonoporation, to deliver plasmid DNA coding the luciferase reporter gene and
mouse Interleukin-12 (mIL-12) to the quadriceps of mice. The group found that two
days post delivery, mice that had undergone electro-sonoporation demonstrated lucifer-
ase expression levels two-fold higher than those that had received electroporation alone
[60]. Likewise, levels of serum mIL-12 were found to be two-fold higher in mice treated
with electro-sonoporation, with gene expression still detectable 28 days post adminis-
tration [60].
This study highlights the potential in using a combination of delivery strategies to im-
prove gene expression. While it is commonplace to enhance the immune response
through the administration of biological or chemical adjuvants, new strategies are
emerging using a combination of physical delivery systems to synergistically increase
gene delivery [61–63]. These two-tier or combinational approaches are likely to yield a
more efficacious gene delivery and thus, may prove necessary in larger animal models
to produce sufficient amounts of antigen to overcome self-tolerance to TAAs.
DNA delivery vector

DNA delivery vectors can be broadly classified as viral or non-viral. DNA delivery vec-
tors enhance the uptake of DNA and protect it from the intracellular barriers to gene
delivery. This process involves condensing the DNA to facilitate endocytosis, masking
the negative charge of the DNA and protecting it from degradation by nucleases. In
addition, several delivery vectors are capable of directly trafficking DNA to the nucleus
thus enhancing gene expression.
Viral vectors
Several types of viruses have been utilised as delivery vectors for DNA vaccines includ-
ing adenoviruses (Ad) [64], adeno-associated viruses (AAVs) [65, 66], herpes simplex vi-
ruses (HSV) [67, 68], retroviruses [69], lentiviruses [70] and poxviruses [71]. Viruses
have specifically evolved to overcome the barriers presented to gene delivery and as
such, they are associated with high transfection efficacy and are the current gold stand-
ard for gene delivery [72]. A number of DNA vaccines using viral vectors have been
used in prostate cancer preclinical and clinical trials and have proven safe and effica-
cious [67, 73–75].
Lubaroff et al. [74] recently reported encouraging results from a Phase I trial utilising
an adenoviral vector to deliver DNA coding human PSA (Ad/PSA). Patients with evi-
dence of metastatic castrate resistant disease received 1 × 106, 1 × 107 or 1 × 108 CFU of
Ad/PSA subcutaneously (s.c.) either as an aqueous suspension or as a Gelfoam collagen
matrix [74]. Patients were then observed for adverse effects, and at days 14, 21 and 2,
4, 8 and 12 months returned for assessment and to allow evaluation of antibody or T
cell specific responses to PSA. The group reported that 34 % of patients experienced an
increase in detectable anti-PSA antibodies, while 68 % of patients developed anti-PSA
T cells [74]. In addition, 46 % of patients experienced an increase in PSA-DT. The re-
sults of this small but encouraging study resulted in the commencement of a Phase II
trial to assess the benefit of Ad/PSA in patients with recurrent prostate cancer [76], al-
though results have yet to be published.
To date, perhaps the most successful prostate cancer DNA vaccination platform
is that of the PSA-targeting vaccine, PROSTVAC, consisting of a prime-boost strat-
egy with recombinant vaccinia virus and fowlpox virus vectors expressing PSA and
a triad of co-stimulatory molecules, B7.1, ICAM-1 and LFA-3, known as TRICOM
[71]. The success of a phase II clinical trial in men with metastatic CRPC demon-
strated a survival benefit of 8.5 months in patients who received PROSTVAC-VF
plus GM-CSF [75]. This has now led to a randomised double-blind phase III
clinical trial [77].
Despite this success, a number of limitations have been highlighted with these
vectors including time-consuming production, uncertain reproducibility, limited car-
rying capacity of transgenes, safety concerns such as toxicity, dose-dependent im-
munogenicity and potential integration into the host genome causing oncogene
activation [36, 72]. Many groups continue to develop recombinant viral vectors due
to their efficacy in vivo, and many DNA vaccines with these vectors continue to be
brought to trial. However, there has been a shift towards creating new, non-viral vec-
tors for DNA vaccination.
Non-viral vectors
Non-viral vectors, while attractive in terms of reproducibility and safety, are limited by
low transfection efficacy in vivo. Existing vectors include cationic lipids, polymers and
peptides [72]. Due to their cationic charge these vectors often spontaneously condense
DNA to form smaller cationic nanoparticles in addition to enhancing endocytosis and
protecting DNA from degradation. Despite ease of production, these vectors continue
to suffer from a lack of efficacy compared to viral vectors in vivo and so the challenge
is to improve non-viral characteristics to overcome the barriers to gene delivery.
Lipid/liposome delivery systems

Cationic lipids are capable of condensing DNA through electrostatic interactions into
small lipoplexes. These lipoplexes carry a positive surface charge which aids internalisa-
tion through cell membrane binding. However, highly charged particles have been dem-
onstrated to cause significant toxicity and aggregation with serum proteins which can
hinder efficacy in vivo. Some of these limitations have been addressed through the func-
tionalisation of liposomes by the addition of Poly-ethylene-glycol (PEG). PEG shields the
liposome, increases the circulation time and facilitates the addition of ligands to improve
targeting [78]. The addition of Mannose to liposomes has led to significant increases in
transfection of DCs and macrophages through targeting of the mannose receptor [79–81].
Targeting of APCs makes these vectors ideal for DNA vaccination, and mannosylated li-
posomes have been demonstrated to enhance gene expression and the antigen specific
immune response compared to non-mannosylated vectors [78, 79]. Liposome vectors
have not been used for DNA vaccination purposes with prostate TAAs to date.
Allen et al. [82] delivered lipoplexes containing the gene coding for the p75 neurotro-
phin receptor (p75NTR) i.t. to PC-3 xenografts implanted on SCID mice. p75NTR is a
known tumour suppressor gene in prostate cancer and therefore it was hypothesised
that transfection of established PC-3 tumours would result in an increase in cell apop-
tosis and a decrease in cell proliferation. The authors implanted SCID mice with 1x106
PC-3 cells s.c. and 5 days post implantation injected i.t. with Lipofectamine/ p75NTR
cDNA (1 μg, 5 μg or 10 μg) or Lipofectamine 3 times weekly for 5 weeks [82]. Tu-
mours treated with lipoplexes containing p75NTR cDNA were significantly smaller than
those treated with Lipofectamine or control (no treatment). In addition, tumour size
was reduced in a dose dependent manner with 5 μg or 10 μg of DNA decreasing
tumour volume significantly more than 1 μg of DNA [82]. The authors successfully
demonstrated the feasibility of this approach for gene therapy for prostate cancer. How-
ever, while it was demonstrated that the lipoplexes were capable of transfecting prostate
cancer cells in this model it is not always possible to administer i.t. in vivo, especially in
diseases such as prostate cancer where there may be disseminated disease. Therefore,
DNA vaccination protocols targeting prostate TAAs may be more relevant clinically,
and do not require systemic delivery.
In conclusion, liposomes enable enhanced transfection through complexation with
DNA, circumvent the tissue damage associated with physical delivery systems and do
not require additional specialist equipment. Liposomes can be modified to enhance sta-
bility, improve circulation times and target APCs, making them good candidates for
in vivo gene delivery. However, liposome vectors also continue to suffer from a lack of
efficacy compared to viral vectors and cellular toxicity remains an on-going problem. At-
tempts to reduce the limitations of unspecific cellular transfection and poor circulation
time through incorporation of PEG have also been undermined by the formation of PEG-
specific antibodies upon repeat administration [72]. Liposome vectors still require further
refinement before they become mainstream vectors for use in DNA vaccination.
Polymer delivery systems

Cationic polymers are also capable of condensing anionic DNA through electrostatic
interaction to form particles known as polyplexes, and have been extensively studied as
non-viral gene delivery agents. These synthetic polymers provide a simple method of
gene delivery and are easily modified to accommodate other stabilising polymers, tar-
geting ligands or drug conjugates [83]. Polyethylenimine (PEI) and poly (L-lysine) (PLL)
have been the most widely studied cationic polymers. Although both PLL and PEI en-
hance DNA transfection, PEI is most effective. The large buffering capacity of PEI enables
efficient endosomal escape via the ‘proton sponge effect’ [83]. However, the main limita-
tion of these cationic polymers is that increased transfection efficacy is correlated to a
higher molecular weight that results in a substantial increase in toxicity [83–85]. Attempts
to reduce the toxicity of these polymers while maintaining the transfection efficacy are on-
going with varying success [86–88]. Polyplexes have not yet been used as non-viral vectors
for DNA vaccine delivery in prostate cancer models, however several polyplexes have
been used to deliver gene therapy to tumours in clinical and preclinical trials [89, 90].
Hence prostate cancer may benefit from gene therapy delivered in this manner.
Similar to liposomes, polymer carriers may be modified to contain mannose moieties
that mediate delivery to APCs [61, 91]. Kim et al. [61] described a novel, two-tiered de-
livery system designed to enhance gene delivery to DCs in the dermal layer for DNA
vaccination. The system utilised solid microneedles coated with a pH-responsive layer
designed to release polyplexes when inserted into skin. Following insertion and release
into the skin uptake to APCs may be accommodated by mannosylated polyplexes en-
coding an antigenic amyloid beta monomer, Aβ 1–42, which enhances APC uptake
through interaction with mannose receptors. A single immunisation of BALB/c mice
with 10 μg of DNA was sufficient to induce detectable Aβ-specific antibodies one-week
post immunisation. Five weeks following immunisation mice challenged with Aβ 1–42
peptide produced a rapid and robust Aβ-specific humoral response, demonstrating the
ability of this approach to induce a long-lasting antigen specific humoural response.
This type of approach may prove beneficial in DNA vaccines for prostate cancer, where
targeted transfection of APCs is key for the development of a robust cellular response
to eradicate antigen expressing tumour cells.
In conclusion, while polymers provide a stable, efficacious vector for targeted gene
delivery, further development is still required. Although unspecific cell targeting, poor
circulation time and non-specific interaction of polyplexes with serum proteins have
been largely improved through the incorporation of PEG and targeting ligands, cytotox-
icity remains the rate limiting factor in vivo. Further development of newer polymers,
not limited by a trade-off between efficacy and cytotoxicity is required before these vec-
tors can be used widely in clinical trials for DNA vaccination.
Peptide delivery systems

In 1988 it was discovered that HIV TAT trans-activating factor was able to traverse the
cell membrane and be taken up by a wide variety of cells [92]. This revelation led to
the development of a whole class of natural and synthetic peptides capable of delivering
cargo to a variety of cell types, known as cell penetrating peptides (CPPs) [93]. Further-
more, peptides are being developed that mimic viral sequences. These include peptides
that facilitate internalisation [94], endosomal escape [95], and nuclear localisation [96].
Such peptides are often rich in basic amino acids such as lysine and arginine which are
essential to condense DNA into nanoparticles via electrostatic interaction [95, 97]. The
advantages of these viral mimetic peptides include biocompatibility, low cytotoxicity
and versatility with respect to rational design resulting in tailored systems.
Zhang et al. recently demonstrated the feasibility of this approach in a mouse pros-
tate cancer model where an in-house cationic peptide [K] 18P9, composed of 18 lysine
residues and a human CTL PSCA epitope, was used to condense a plasmid encoding
the full-length human PSCA (hPSCA) gene for immunisation. HLA-A2.1/Kb Tg mice
were immunized with 25 nmol of DNA 3 times at 2 weekly intervals. Effector cells
from the immunised mice were subsequently intravenously (i.v.) injected into tumour
bearing nude mice once per week and tumour growth monitored. These results showed
significant retardation in tumour growth in those receiving cells from peptide/DNA
vaccinated animals compared to those immunised with DNA alone, thus confirming
administration of the DNA vaccine complexed with this peptide elicited superior im-
mune responses in vivo [98].
Peptide delivery systems confer a level of targeting and safety profile that is far super-
ior to any other non-viral vehicle. Perhaps the drawback of peptide delivery systems lies
in systemic administration where accumulation in the liver is a frequent event. How-
ever, given that most DNA vaccination strategies require either i.m. or i.d. injection,
peptides are ideally placed to deliver their cargo to APCs and have the potential to fill
that delivery void.
Conclusions
Non-physical delivery methods carry the advantage of enhancing gene delivery in a
non-invasive means to patients without requiring specialist equipment. Amongst the
non-physical delivery systems viral vectors remain the gold standard in terms of effi-
cacy. For DNA vaccination in larger animals and humans high transfection rates are es-
sential, and as such, the extensive use and success of viral vectors for gene delivery in
clinical trials makes them the most attractive vector for in vivo protocols. This success
has led to the PSA-targeting DNA vaccine PROSTVAC entering Phase III clinical trials,
the first prostate cancer specific DNA vaccine to do so. Despite these advantages there
continues to be limitations over the safety, immunogenicity and carrying capacity of
these vectors. Thus, there is a need to develop new, non-viral vectors capable of produ-
cing similar transfection efficacies. Unfortunately these vectors suffer from high toxicity
and poor transfection rates in vivo, though modification with ligands to improve APC
uptake is promising for DNA vaccination. Peptide delivery vectors hold promise in
terms of being able to mimic viral characteristics for DNA delivery. However, there is
still a significant gap in acquiring the necessary pre-clinical data to validate the peptide
delivery of DNA TAAs.
Co-stimulatory adjuvants
Co-administering immune enhancing molecules at the site of DNA vaccination either
directly or encoded in plasmids is primarily designed to improve vaccine immunogen-
icity. Few studies directly compare the efficacy of DNA vaccines with and without adju-
vant making it difficult to deduce the benefits of co-administration. In addition, there is
little consensus as to whether these adjuvants should be delivered as soluble protein or
as plasmids, making direct comparison between studies examining the effect of adju-
vant problematic. The most commonly co-administered molecules include chemokines,
cytokines and bacterial toxins [48].
GM-CSF is a cytokine commonly used as an adjuvant for DNA vaccination and has
been used in numerous clinical trials [30, 31, 73, 99]. In a Phase I/II trial, Mincheff
et al. demonstrated that 50 % of patients vaccinated i.d. at one weekly intervals with
100 μg of PSMA and CD86 encoding plasmid(s) showed signs of immunisation in the
form of delayed-type hypersensitivity (DTH). In contrast, 100 % of patients vaccinated
with PSMA and CD86 plasmids also receiving 40,000 IU of soluble GM-CSF i.d. showed
signs of DTH upon challenge [100]. While this study demonstrated that GM-CSF was
capable of enhancing the general immune response to DNA vaccination, it is unclear as to
whether GM-CSF enhanced the antigen specific response or improved patient outcomes.
As such, more studies directly comparing the effect of GM-CSF on the antigen-specific
cellular and humoral immune responses induced by prostate cancer DNA vaccines are ne-
cessary. Indeed, current studies with the PROSTVAC vaccine include a phase III random-
ized, double-blind trial to examine the effect with and without GM-CSF [77].
Several preclinical studies have demonstrated the benefits of the co-expression of
GM-CSF with DNA vaccines for tuberculosis [101], encephalitis [102], and melanoma
[103]. However, these studies highlighted that co-inoculation with plasmid GM-CSF
did not confer the same benefits, possibly due to unpredictable GM-CSF expression
and competition with plasmid DNA encoding antigens for cellular uptake. Therefore,
when considering the benefit of GM-CSF as an adjuvant it is crucial to consider the
mode of GM-CSF delivery and demonstrate a clear benefit of inclusion.
Xenogeneic DNA
A number of TAAs possess functional homologues in other animal species, where the
expression patterns and functions are similar to those of their human counterparts
[104, 105]. As well as providing suitable preclinical models for DNA vaccines in a “self”
model of prostate cancer, these xenoantigens have been used by groups in an effort to
increase the immunogenicity of DNA vaccines. As xenoantigens are highly homologous
to native peptides, they can be capable of eliciting a specific cross-reactive response to-
wards the host self-antigen that can overcome tolerance issues.
Johnson et al. immunised Lewis rats with naked pTVG-HP, a DNA plasmid encoding
full length human PAP (hPAP), at 2 weekly intervals, for up to six i.d. immunisations
with doses of 100 μg, 500 μg or 1500 μg of pTVG-HP [37]. Immunisation with pTVG-
HP elicited hPAP-specific CD4+ and CD8+ T cells at the lowest dose following two
immunisations. PAP-specific IgG antibodies were also detectable in all pTVG-HP
treated animals and levels increased with increasing DNA dose and frequency of immuni-
sations [37]. Further work from this group immunising rats with pTVG-RP, encoding rat
PAP (rPAP) elicited hPAP-specific T cell responses following 6 immunisations indicating a
larger number of immunisations are necessary to elicit a cross-reactive immune response
in this model [106].
To confirm the animals immunised with pTVG-RP elicited a rPAP specific response
in addition to hPAP specific responses splenocytes were isolated from the animals and
restimulated in vitro with hPAP or rPAP expressing DCs and the resultant levels of
interferon gamma (IFN-ϒ) were analysed. Immunisation with pTGV-RP did elicit a
rPAP-specific T cell response and a cross-reactive hPAP immune response. This indi-
cates that autologous PAP antigen is capable of overcoming tolerance to autologous
PAP [106]. Based on these preclinical data, and using the same immunisation schedule,
the group carried out a Phase I/IIa clinical trial in 22 patients with D0 prostate cancer
[31]. Patients were immunised with pTVG-HP, a self-antigen in humans. Responses
were found in 9 of the 22 patients with a tripling in PAP-specific CD4+ or CD8+ T cell
responses, and 7 of the 22 patients experienced at least doubling of the PSA-DT [31].
Upon completion of the initial study, two patients who had developed CD8+ T-cell
responses to PAP received monthly booster vaccinations of 100 μg pTVG-HP to deter-
mine whether this could augment the initial immune response [107]. Prior to continu-
ation of treatment neither patient had residual, detectable PAP-specific T cells, and
upon only two booster immunisations one patient developed detectable levels of CD4+
and CD8+ PAP-specific T cells suggesting that further immunisations could indeed
prove beneficial [107].
The group hypothesised that immunisation with a xenoantigen may require lower
numbers of immunisations to elicit immune responses against the host antigen, and
carried out immunisation of Lewis rats with pTVG-HP to determine whether rPAP
could be targeted through a cross-reactivity to immunisation with the human antigen
[108]. The group found that despite the high homology between rat and human PAP
peptides the xenoantigen was not capable of eliciting a cross-reactive immune response
to native rPAP [108], suggesting that vaccination of humans with xenoantigens may
not produce an advantageous immune response against the native antigen. The authors
suggested that this may not be the case with all xenoantigens, as the major determinant
of whether a foreign peptide is capable of inducing a cross-reactive immune response
may not be the overall homology of the foreign and self peptides, but the homology of
the epitopes presented to the immune system [108].
Following the promising results of their Phase I/IIa clinical trial and to evaluate the
benefit of further “booster” immunisations, 17 patients were recruited into a Phase II
clinical trial to assess the safety, immunological impact and clinical outcome of pTVG-
HP, administered with rhesus macaque GM-CSF (rhGM-CSF) as an adjuvant, in
patients with non-metastatic castrate resistant disease [99]. Patients were randomised
to receive 100 μg pTVG-HP with 200 μg rhGM-CSF i.d. biweekly for 12 weeks and
then subsequent boosters every 12 weeks until radiographic progression (Group 1) or
boosters every 2, 4 or 12 weeks depending on cellular immune response (Group 2).
From Group 1, 3 of 8 patients experienced at least a tripling in PAP-specific T cells
compared to 6 of 8 participants from Group 2 [99]. Additionally, 3 of 8 patients from
Group 1 and 4 of 9 patients from Group 2 experienced at least a doubling in their
PSA-DT [99]. Taken together these results suggest that further periodic booster immu-
nisations are of benefit to develop an immunological response and do not result in tol-
erance to the targeted antigen.
Several other groups have reported encouraging results using xenoantigens, Castelo-
Blano et al. treated mice bearing TRAMP-C2 prostate tumours over 10 days with 4
intraneoplastic injections of 1x107 PFU of oncolytic herpes simplex virus (oHSV) con-
structs expressing human PAP or mouse PAP (mPAP). Mice immunised with oHSV
expressing hPAP had significantly reduced tumour growth and prolonged survival com-
pared to those treated with oHSV expressing mPAP (p = 0.01) or control (p = 0.0008),
with a 10 day prolongation of survival to 39 days compared to the latter [67]. Further-
more Gregor et al. demonstrated that 5 intraperitoneal (i.p.) immunisations of mice
with human PSMA (hPSMA) encoding DNA vaccine or protein was capable of indu-
cing autoantibodies against native mouse PSMA (mPSMA) as demonstrated by ELISA.
However, immunisation with native PSMA encoding DNA vaccine elicited no detect-
able increase in immune response to human or murine PSMA [55]. Moving from this
preclinical study Slovin et al. carried out a Phase I trial immunising patients with meta-
static prostate cancer at 3 week intervals i.m. with 100 μg, 1500 μg or 4000 μg of xeno-
geneic or homologous DNA vaccine coding PSMA [109]. Patients received three
immunisations and subsequently patients who had received homologous vaccination
were immunised a further three times with xenogeneic DNA The vaccine has proven
safe, however, no high titer antibodies specific to PSMA were produced by any patients,
though further analysis of T cell reactivity is ongoing but has yet to be published. As
such, further studies are required to elucidate whether xenogeneic or autologous DNA
is optimal in the clinical setting.
Prime / boost strategies

Many DNA vaccines have proven efficient at elicitation of immune responses, however,
in some cases these responses are suboptimal to provide protection against the antigen.
Thus, a number of immunisation regimens involving “priming” with DNA and subse-
quently “boosting” with a heterologous agent such as a different antigen delivery plat-
form or protein with the aim of improving immunogenicity have been investigated.
This approach has recently been explored using murine STEAP1 (mSTEAP1) and
murine PSCA (mPSCA) in mice for prophylaxis of prostate cancer [17]. The group
primed mice with recombinant DNA (mSTEAP1 and/or mPSCA) and boosted with
modified vaccinia virus ankara (MVA) vector expressing the same antigen(s). The group
found that in a tumour challenge study, mice immunised against either antigen using
this protocol demonstrated significantly inhibited tumour growth compared to control,
49.8 % following immunisation against mPSCA and 41.7 % against mSTEAP1 [17]. An-
other group immunised mice against mSTEAP using several vaccination protocols in-
cluding a gene gun mSTEAP prime/ s.c. mSTEAP-Virus Replicon Particle (VRP) boost;
gene gun mSTEAP prime/ s.c. mSTEAP DNA boost and mSTEAP-VRP prime and
boost [56]. Mice were then challenged 10 days post boost with TRAMP C-2 prostate
cancer cells. Tumour growth was monitored twice weekly and survival followed until
tumours reached volumes over 1000 mm3. While survival was significantly increased
with all mSTEAP vaccination protocols, the most significant effect was seen in mice
vaccinated with mSTEAP DNA and boosted with mSTEAP-VRP. In a phase I/II trial
Mincheff et al. found that all patients immunised with a replication deficient adenoviral
vector expressing PSMA and later boosted with plasmid PSMA showed signs of
immunisation (by DTH), while only 50 % of patients vaccinated with plasmid PSMA/
CD86 alone showed signs of immunisation [100]. Indeed PROSTVAC employs two dif-
ferent viral vectors, upon priming with the recombinant vaccinia virus expressing PSA,
neutralizing antibodies are formed to the vector, making subsequent boosting doses un-
feasible. However, boosting with a fowlpox vector overcomes this limitation [71] and
similar strategies should be implemented in the design of new DNA vaccines.
Priming with DNA and boosting with protein has also proven successful. Gregor
et al. immunised C57 BL/6 mice with a DNA vaccine encoding hPSMA once weekly
for 5 weeks. Sera from these animals was subsequently analysed for affinity to mPSMA.
The mouse with the best response was subsequently boosted with 10 μg hPSMA pro-
tein and found resultant antibody specificity for hPSMA and cross-reactivity for
mPSMA [55]. A preclinical study carried out by Yong et al. demonstrated that C57 BL/
6 mice immunised with a DNA vaccine encoding gastrin-releasing peptide (GRP) (three
doses of 50 μg DNA), followed by boosting with HSP65-GRP6 protein resulted in in-
creased immunogenicity as compared to those receiving DNA vaccination alone, as in-
dicated by an increased titre of anti-GRP antibodies and inhibition of prostate tumour
growth (tumour weight 0.962 ± 0.462 and 1.536 ± 0.497 g respectively) [110].
Multivalent strategies
Several recent preclinical studies in rodents have focused on the effects of DNA vac-
cines coding multiple TAAs, the authors hypothesise that delivering multiple TAAs will
result in a wider and more potent immune response, targeting tumour cells with a syn-
ergistic effect [12]. Ferraro et al. used a dual antigen approach to immunise mice, PSA
and PSMA were co-delivered i.m. followed by EP. The immunisation elicited a robust
vaccine-specific CD4+ and CD8+ T cell response, indicating that the approach may
hold clinical promise [12]. In the tumour challenge study carried out by Krupa et al.
described previously [17], the DNA prime/MVA boost strategy significantly delayed
tumour growth upon challenge with TRAMP C-1 tumours. At day 55, the most signifi-
cant effect was seen in mice immunised with both mSTEAP1 and mPSCA, with
tumour volume 76.5 % lower than control mice vaccinated with empty plasmid vectors.
The group chose to immunise TRAMP mice (a more relevant and aggressive prostate
cancer model) using this prime/boost strategy. The vaccinated mice had significantly
reduced primary tumour burden and at 24 weeks showed lower histological grade tu-
mours, indicating that this approach is capable of breaking tolerance to self-antigens
[17]. While these preclinical data demonstrate promise for this strategy an earlier pre-
clinical study by Kim et al. primed mice with recombinant adenoviruses expressing
mSTEAP, mPSCA and mPSMA and then boosted with DCs pulsed with tumour lysate
[111]. This prime/boost strategy effectively delayed tumour growth following TRAMP
C-1 tumour challenge, however, only high levels of mSTEAP-specific CD8+ T cells were
found in the blood and spleens indicating that the anti-tumour effect was predominantly
due to mSTEAP vaccination. Furthermore, it was found that vaccinating against mSTEAP
alone was as effective as the triple antigen approach [112]. These results suggest that the
use of multiple TAAs, while demonstrating promise in some studies is not generalisable,
and in vitro and preclinical models should be used to determine the most effective com-
bination of TAAs for each individual delivery platform.
Conclusions
As discussed, DNA vaccination represents a promising platform capable of offering
both prophylactic protection and therapeutic treatment of prostate cancer. Currently
there are a significant number of pre-clinical and clinical trials underway utilising a
range of DNA plasmids, encoding a variety of TAAs and being delivered by an array of
delivery methods. However, there are a number of questions still to be answered in
order to achieve optimal immune responses clinically following vaccination. Primarily,
is there an optimal dose of DNA and dosing regimen capable of provoking strong cellu-
lar and humoural responses to the antigens produced while avoiding exacerbation of
pro-inflammatory responses to the vaccine and delivery vehicles utilised themselves.
Additionally, improved formulation and superior delivery technologies are essential
for the enhancement of clinical data which has thus far proved disappointing when
compared to the promising results elicited in vitro and in rodent models in vivo. It is
becoming increasingly common for researchers to use a combination of delivery strat-
egies to achieve higher transfection rates in vivo. These two-tier delivery systems enable
synergy between the delivery systems and may prove necessary to achieve sufficient
gene expression to provoke robust cellular and humoral immune responses [58,
61–63]. Among these strategies the use of microneedles to localise DNA delivery into
the APC rich dermal layer has been commonly reported [61–63]. These devices provide
a non-invasive means of overcoming the Stratum Corneum and do not require special-
ist training for use. This provides a significant benefit over other delivery platforms,
especially where repeated administration or large scale vaccination is required. In con-
clusion, further investigation and advancement in the use of DNA vaccination for the
protection against, and treatment of prostate cancer is necessary before a fully validated
prostate cancer vaccine is clinically available.
Abbreviations
AAV: Adeno-associated viruses; Ad: Adenovirus; APC: Antigen-presenting cell; CRPC: Castration resistant prostate
cancer; CTLs: Cytotoxic T lymphocytes; DNA: Deoxyribonucleic acid; DOM: Domain of fragment C tetanus toxin;
DTH: Delayed-type hypersensitivity; EP: Electroporation; FDA: US food and drug administration; GM-CSF: Granulocyte
macrophage- colony stimulating factor; GRP: Gastrin-releasing peptide; hPSA: Human prostate specific antigen;
hPSMA: Human prostate specific membrane antigen; HSV: Herpes simplex virus; I.D.: Intradermal; IFN-γ: Interferon
gamma; I.M.: Intramuscular; I.P.: Intraperitoneal; I.T.: Intratumoural; hPAP: Human prostatic acid phosphatase;
MHC: Major histocompatibility complex; mPSCA: Murine prostate stem cell antigen; mPSMA: Murine prostate specific
membrane antigen; mSTEAP: Murine six transmembrane epithelial antigen of the prostate; MVA: Modified Vaccinia
virus ankara; oHSV: Oncolytic herpes simplex virus; p75NTR: p75 neurotrophin receptor; PAP: Prostatic acid phosphatase;
PEG: Poly-ethylene-glycol; PEX: Antiangiogenic hemopexin-like domain fragment; PSA: Prostate specific antigen; PSA-
DT: Prostate specific antigen doubling time; PSCA: Prostate stem cell antigen; PSMA: Prostate specific membrane
antigen; rhGM-CSF: Rhesus macaque granulocyte macrophage- colony stimulating factor; rPAP: Rat prostatic acid
phosphatase; rhPSA: Rhesus macaque prostate specific acid; S.C.: Subcutaneous; STEAP: Six transmembrane epithelial
antigen of the prostate; STEAP1: Six transmembrane epithelial antigen of the prostate 1; TAAs: Tumour associated
anitgens; TRAMP: Transgenic adenocarcinoma of the mouse prostate; TUS: Therapeutic ultrasound; VRP: Virus replicon
particle.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
GC drafted the manuscript, tables and aided with the figures. JMcC drafted the manuscript and amended figures. AA
drafted the figures and aided with the manuscript. HMcC conceived and helped to draft the manuscript and was
responsible for design and co-ordination. All authors read and approved the final manuscript.
Acknowledgements
Prostate Cancer UK funds GC on a PhD studentship.
Received: 29 December 2014 Accepted: 16 May 2015
References
1. C. R. UK (2014) Cancer incidence for common cancers. Cancer Research, UK
2. Horwich A, Parker C, Bangma C, Kataja V (2010) Prostate cancer: ESMO Clinical Practice Guidelines for diagnosis,
treatment and follow-up. Ann Oncol 21(Suppl 5):v129–33
3. Saad F, Miller K (2014) Treatment options in castration-resistant prostate cancer: current therapies and emerging
docetaxel-based regimens. Urol Oncol 32(2):70–9
4. Rosenbaum E, Partin A, Eisenberger MA (2004) Biochemical relapse after primary treatment for prostate cancer:
studies on natural history and therapeutic considerations. J Natl Compr Canc Netw 2(3):249–56
5. Schurko B, Oh WK (2008) Docetaxel chemotherapy remains the standard of care in castration-resistant prostate
cancer. Nat Clin Pract Oncol 5(9):506–7
6. Schietinger A, Philip M, Schreiber H (2008) Specificity in cancer immunotherapy. Semin Immunol 20(5):276–85
7. Thakur A, Vaishampayan U, Lum LG (2013) Immunotherapy and immune evasion in prostate cancer. Cancers
(Basel) 5(2):569–90
8. Agarwal N, Padmanabh S, Vogelzang NJ (2012) Development of novel immune interventions for prostate cancer.
Clin Genitourin Cancer 10(2):84–92
9. Coffey DS, Isaacs JT (1981) Prostate tumor biology and cell kinetics–theory. Urology 17(Suppl 3):40–53
10. Cunha AC, Weigle B, Kiessling A, Bachmann M, Rieber EP (2006) Tissue-specificity of prostate specific antigens:
comparative analysis of transcript levels in prostate and non-prostatic tissues. Cancer Lett 236(2):229–38
11. Balk SP, Ko Y-J, Bubley GJ (2003) Biology of prostate-specific antigen. J Clin Oncol 21(2):383–91
12. Ferraro B, Cisper NJ, Talbott KT, Philipson-Weiner L, Lucke CE, Khan AS, Sardesai NY, Weiner DB (2011) Co-delivery
of PSA and PSMA DNA vaccines with electroporation induces potent immune responses. Hum Vaccin 7:120–7
13. Graddis TJ, McMahan CJ, Tamman J, Page KJ, Trager JB (2011) Prostatic acid phosphatase expression in human
tissues. Int J Clin Exp Pathol 4(3):295–306
14. Ghosh A, Heston WDW (2004) Tumor target prostate specific membrane antigen (PSMA) and its regulation in
prostate cancer. J Cell Biochem 91(3):528–39
15. Raff AB, Gray A, Kast WM (2009) Prostate stem cell antigen: a prospective therapeutic and diagnostic target.
Cancer Lett 277(2):126–32
16. Hubert RS, Vivanco I, Chen E, Rastegar S, Leong K, Mitchell SC, Madraswala R, Zhou Y, Kuo J, Raitano AB,
Jakobovits A, Saffran DC, Afar DE (1999) STEAP: a prostate-specific cell-surface antigen highly expressed in human
prostate tumors. Proc Natl Acad Sci U S A 96(25):14523–8
17. Krupa M, Canamero M, Gomez CE, Najera JL, Gil J, Esteban M (2011) Immunization with recombinant DNA and
modified vaccinia virus Ankara (MVA) vectors delivering PSCA and STEAP1 antigens inhibits prostate cancer
progression. Vaccine 29(7):1504–13
18. McNeel DG, Nguyen LD, Ellis WJ, Higano CS, Lange PH, Disis ML (2001) Naturally occurring prostate cancer antigen-
specific T cell responses of a Th1 phenotype can be detected in patients with prostate cancer. Prostate 47(3):222–9
19. Sims RB (2012) Development of sipuleucel-T: autologous cellular immunotherapy for the treatment of metastatic
castrate resistant prostate cancer. Vaccine 30(29):4394–7
20. Kantoff PW, Higano CS, Shore ND, Berger ER, Small EJ, Penson DF, Redfern CH, Ferrari AC, Dreicer R, Sims RB, Xu Y,
Frohlich MW, Schellhammer PF (2010) Sipuleucel-T immunotherapy for castration-resistant prostate cancer. N Engl
J Med 363(5):411–22
21. Simons JW, Sacks N. Granulocyte-macrophage colony-stimulating factor-transduced allogeneic cancer cellular
immunotherapy: the GVAX vaccine for prostate cancer. Urol Oncol. 24(5):419–24.
22. Fong L, Brockstedt D, Benike C, Breen JK, Strang G, Ruegg CL, Engleman EG (2001) Dendritic Cell-Based Xenoantigen
Vaccination for Prostate Cancer Immunotherapy. J Immunol 167(12):7150–6
23. Noguchi M, Moriya F, Suekane S, Matsuoka K, Arai G, Matsueda S, Sasada T, Yamada A, Itoh K (2012) Phase II study
of personalized peptide vaccination for castration-resistant prostate cancer patients who failed in docetaxel-based
chemotherapy. Prostate 72(8):834–45
24. Milowsky MI, Nanus DM, Kostakoglu L, Sheehan CE, Vallabhajosula S, Goldsmith SJ, Ross JS, Bander NH (2007)
Vascular targeted therapy with anti-prostate-specific membrane antigen monoclonal antibody J591 in advanced
solid tumors. J Clin Oncol 25(5):540–7
25. Rice J, Ottensmeier CH, Stevenson FK (2008) DNA vaccines: precision tools for activating effective immunity
against cancer. Nat Rev Cancer 8(2):108–20
26. Fehres CM, Unger WWJ, Garcia-Vallejo JJ, van Kooyk Y (2014) Understanding the biology of antigen cross-presentation
for the design of vaccines against cancer. Front Immunol 5:149
27. Wolchok JD, Gregor PD, Nordquist LT, Slovin SF, Scher HI (2003) DNA vaccines: an active immunization strategy
for prostate cancer. Semin Oncol 30(5):659–66
28. Sardesai NY, Weiner DB (2011) Electroporation delivery of DNA vaccines: prospects for success. Curr Opin
Immunol 23(3):421–9
29. Moss RB (2009) Prospects for control of emerging infectious diseases with plasmid DNA vaccines. J Immune
Based Ther Vaccines 7(1):3
30. Pavlenko M, Roos A-K, Lundqvist A, Palmborg A, Miller AM, Ozenci V, Bergman B, Egevad L, Hellström M, Kiessling
R, Masucci G, Wersäll P, Nilsson S, Pisa P (2004) A phase I trial of DNA vaccination with a plasmid expressing
prostate-specific antigen in patients with hormone-refractory prostate cancer. Br J Cancer 91(4):688–94
31. McNeel DG, Dunphy EJ, Davies JG, Frye TP, Johnson LE, Staab MJ, Horvath DL, Straus J, Alberti D, Marnocha R,
Liu G, Eickhoff JC, Wilding G (2009) Safety and immunological efficacy of a DNA vaccine encoding prostatic acid
phosphatase in patients with stage D0 prostate cancer. J Clin Oncol 27(25):4047–54
32. Ottnod JM, Smedley RC, Walshaw R, Hauptman JG, Kiupel M, Obradovich JE (2013) A retrospective analysis of the
efficacy of Oncept vaccine for the adjunct treatment of canine oral malignant melanoma. Vet Comp Oncol
11(3):219–29
33. Kutzler MA, Weiner DB (2008) DNA vaccines: ready for prime time? Nat Rev Genet 9(10):776–88
34. Brown MD, Schätzlein AG, Uchegbu IF (2001) Gene delivery with synthetic (non viral) carriers. Int J Pharm
229(1–2):1–21
35. Pouton CW, Seymour LW (2001) Key issues in non-viral gene delivery. Adv Drug Deliv Rev 46(1–3):187–203
36. Douglas KL. Toward development of artificial viruses for gene therapy: a comparative evaluation of viral and
non-viral transfection. Biotechnol. Prog. 24(4):871–83.
37. Johnson LE, Frye TP, Arnot AR, Marquette C, Couture LA, Gendron-Fitzpatrick A, McNeel DG (2006) Safety and
immunological efficacy of a prostate cancer plasmid DNA vaccine encoding prostatic acid phosphatase (PAP).
Vaccine 24(3):293–303
38. Oosterhuis K, van den Berg JH, Schumacher TN, Haanen JBAG (2012) DNA vaccines and intradermal vaccination
by DNA tattooing. Curr Top Microbiol Immunol 351:221–50
39. K. Jinturkar, M. Rathi, A. N. Misra, “Gene Delivery using Physical Methods,” in Challenges in Delivery of Therapeutic
Genomics and Proteomics, A. Misra, Ed. Elsevier, London, 2010, pp. 83–126.
40. Lin MT, Pulkkinen L, Uitto J, Yoon K (2000) The gene gun: current applications in cutaneous gene therapy. Int J
Dermatol 39(3):161–70
41. Suzuki R, Oda Y, Utoguchi N, Maruyama K (2011) Progress in the development of ultrasound-mediated gene
delivery systems utilizing nano- and microbubbles. J Control Release 149(1):36–41
42. Bolhassani A, Khavari A, Orafa Z (2014) “Electroporation-Advantages and Drawbacks for Delivery of Drug, Gene
and Vaccine,” in Application of Nanotechnology in Drug Delivery, Sezer AD, Ed. InTech, London, p. 369–398.
43. Drunen Littel-van den Hurk S (2010) Electroporation for DNA immunization: clinical application. Expert Rev
Vaccines 9(5):503–17
44. van Drunen Littel-van S, den Hurk A, Luxembourg B, Ellefsen D, Wilson A, Ubach DH, van den Hurk JV (2008)
Electroporation-based DNA transfer enhances gene expression and immune responses to DNA vaccines in cattle.
Vaccine 26(43):5503–9
45. Roos A-K, Moreno S, Leder C, Pavlenko M, King A, Pisa P (2006) Enhancement of cellular immune response to a
prostate cancer DNA vaccine by intradermal electroporation. Mol Ther 13(2):320–7
46. Ahmad S, Casey G, Sweeney P, Tangney M, O’Sullivan GC (2010) Optimised electroporation mediated DNA
vaccination for treatment of prostate cancer. Genet Vaccines Ther 8(1):1
47. Eriksson F, Tötterman T, Maltais A-K, Pisa P, Yachnin J (2013) DNA vaccine coding for the rhesus prostate specific
antigen delivered by intradermal electroporation in patients with relapsed prostate cancer. Vaccine 31(37):3843–8
48. Chudley L, McCann K, Mander A, Tjelle T, Campos-Perez J, Godeseth R, Creak A, Dobbyn J, Johnson B, Bass P,
Heath C, Kerr P, Mathiesen I, Dearnaley D, Stevenson F, Ottensmeier C (2012) DNA fusion-gene vaccination in
patients with prostate cancer induces high-frequency CD8(+) T-cell responses and increases PSA doubling time.
Cancer Immunol Immunother 61(11):2161–70
49. Daud AI, DeConti RC, Andrews S, Urbas P, Riker AI, Sondak VK, Munster PN, Sullivan DM, Ugen KE, Messina JL,
Heller R (2008) Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma. J Clin
Oncol 26(36):5896–903
50. Durieux A-C, Bonnefoy R, Busso T, Freyssenet D (2004) In vivo gene electrotransfer into skeletal muscle: effects of
plasmid DNA on the occurrence and extent of muscle damage. J Gene Med 6(7):809–16
51. Yoshida A, Nagata T, Uchijima M, Higashi T, Koide Y (2000) Advantage of gene gun-mediated over intramuscular
inoculation of plasmid DNA vaccine in reproducible induction of specific immune responses. Vaccine 18(17):1725–9
52. Davtyan H, Ghochikyan A, Movsesyan N, Ellefsen B, Petrushina I, Cribbs DH, Hannaman D, Evans CF, Agadjanyan
MG (2012) Delivery of a DNA vaccine for Alzheimer’s disease by electroporation versus gene gun generates
potent and similar immune responses. Neurodegener Dis 10(1–4):261–4
53. Boudreau EF, Josleyn M, Ullman D, Fisher D, Dalrymple L, Sellers-Myers K, Loudon P, Rusnak J, Rivard R, Schmaljohn C,
Hooper JW (2012) A Phase 1 clinical trial of Hantaan virus and Puumala virus M-segment DNA vaccines for
hemorrhagic fever with renal syndrome. Vaccine 30(11):1951–8
54. Jones S, Evans K, McElwaine-Johnn H, Sharpe M, Oxford J, Lambkin-Williams R, Mant T, Nolan A, Zambon M, Ellis J,
Beadle J, Loudon PT (2009) DNA vaccination protects against an influenza challenge in a double-blind randomised
placebo-controlled phase 1b clinical trial. Vaccine 27(18):2506–12
55. Gregor PD, Wolchok JD, Turaga V, Latouche J-B, Sadelain M, Bacich D, Heston WDW, Houghton AN, Scher HI
(2005) Induction of autoantibodies to syngeneic prostate-specific membrane antigen by xenogeneic vaccination.
Int J Cancer 116(3):415–21
56. Garcia-Hernandez MDLL, Gray A, Hubby B, Kast WM (2007) In vivo effects of vaccination with six-transmembrane
epithelial antigen of the prostate: A candidate antigen for treating prostate cancer. Cancer Res 67(3):1344–51
57. Mehier-Humbert S, Bettinger T, Yan F, Guy RH (2005) Ultrasound-mediated gene delivery: kinetics of plasmid
internalization and gene expression. J Control Release 104(1):203–11
58. Yoshida M, Kawakami S, Kono Y, Un K, Higuchi Y, Maruyama K, Yamashita F, Hashida M (2014) Enhancement of
the anti-tumor effect of DNA vaccination using an ultrasound-responsive mannose-modified gene carrier in
combination with doxorubicin-encapsulated PEGylated liposomes. Int J Pharm 475(1–2):401–7
59. Duvshani-Eshet M, Benny O, Morgenstern A, Machluf M (2007) Therapeutic ultrasound facilitates antiangiogenic
gene delivery and inhibits prostate tumor growth. Mol Cancer Ther 6(8):2371–82
60. Yamashita Y, Shimada M, Tachibana K, Harimoto N, Tsujita E, Shirabe K, Miyazaki J, Sugimachi K (2002) In vivo
gene transfer into muscle via electro-sonoporation. Hum Gene Ther 13(17):2079–84
61. Kim NW, Lee MS, Kim KR, Lee JE, Lee K, Park JS, Matsumoto Y, Jo D-G, Lee H, Lee DS, Jeong JH (2014)
Polyplex-releasing microneedles for enhanced cutaneous delivery of DNA vaccine. J Control Release 179:11–7
62. Zhang D, Das DB, Rielly CD (2014) Potential of microneedle-assisted micro-particle delivery by gene guns: a
review. Drug Deliv 21(8):571–87
63. Choi S-O, Kim YC, Park J-H, Hutcheson J, Gill HS, Yoon Y-K, Prausnitz MR, Allen MG (2010) An electrically active
microneedle array for electroporation. Biomed Microdevices 12(2):263–73
64. Tatsis N, Ertl HCJ (2004) Adenoviruses as vaccine vectors. Mol Ther 10(4):616–29
65. Mahadevan M, Liu Y, You C, Luo R, You H, Mehta JL, Hermonat PL (2007) Generation of robust cytotoxic T
lymphocytes against prostate specific antigen by transduction of dendritic cells using protein and recombinant
adeno-associated virus. Cancer Immunol Immunother 56(10):1615–24
66. Daya S, Berns KI (2008) Gene therapy using adeno-associated virus vectors. Clin Microbiol Rev 21(4):583–93
67. Castelo-Branco P, Passer BJ, Buhrman JS, Antoszczyk S, Marinelli M, Zaupa C, Rabkin SD, Martuza RL (2010)
Oncolytic herpes simplex virus armed with xenogeneic homologue of prostatic acid phosphatase enhances
antitumor efficacy in prostate cancer. Gene Ther 17(6):805–10
68. Glorioso JC (2014) Herpes simplex viral vectors: late bloomers with big potential. Hum Gene Ther 25(2):83–91
69. Yi Y, Noh MJ, Lee KH (2011) Current advances in retroviral gene therapy. Curr Gene Ther 11(3):218–28
70. Escors D, Breckpot K (2010) Lentiviral vectors in gene therapy: their current status and future potential. Arch
Immunol Ther Exp (Warsz) 58(2):107–19
71. Madan RA, Arlen PM, Mohebtash M, Hodge JW, Gulley JL (2009) Prostvac-VF: a vector-based vaccine targeting PSA
in prostate cancer. Expert Opin Investig Drugs 18(7):1001–11
72. McCarthy HO, Wang Y, Mangipudi SS, Hatefi A (2010) Advances with the use of bio-inspired vectors towards
creation of artificial viruses. Expert Opin Drug Deliv 7(4):497–512
73. Eder JP, Kantoff PW, Roper K, Xu GX, Bubley GJ, Boyden J, Gritz L, Mazzara G, Oh WK, Arlen P, Tsang KY, Panicali D,
Schlom J, Kufe DW (2000) A phase I trial of a recombinant vaccinia virus expressing prostate-specific antigen in
advanced prostate cancer. Clin Cancer Res 6(5):1632–8
74. Lubaroff DM, Konety BR, Link B, Gerstbrein J, Madsen T, Shannon M, Howard J, Paisley J, Boeglin D, Ratliff TL,
Williams RD (2009) Phase I clinical trial of an adenovirus/prostate-specific antigen vaccine for prostate cancer:
safety and immunologic results. Clin Cancer Res 15(23):7375–80
75. Kantoff PW, Schuetz TJ, Blumenstein BA, Glode LM, Bilhartz DL, Wyand M, Manson K, Panicali DL, Laus R, Schlom J,
Dahut WL, Arlen PM, Gulley JL, Godfrey WR (2010) Overall survival analysis of a phase II randomized controlled
trial of a Poxviral-based PSA-targeted immunotherapy in metastatic castration-resistant prostate cancer. J Clin
Oncol 28(7):1099–105
76. “Phase II Study of Adenovirus/PSA Vaccine in Men With Hormone-Refractory Prostate Cancer (APP22).” [Online].
Available: http://clinicaltrials.gov/show/NCT00583024. [Accessed: 15-Dec-2014].
77. “A Randomized, Double-blind, Phase 3 Efficacy Trial of PROSTVAC-V/F +/− GM-CSF in Men With Asymptomatic or
Minimally Symptomatic Metastatic Castrate-Resistant Prostate Cancer (Prospect).” [Online]. Available:
https://clinicaltrials.gov/ct2/show/NCT01322490.
78. Kong F, Zhou F, Ge L, Liu X, Wang Y (2012) Mannosylated liposomes for targeted gene delivery. Int J
Nanomedicine 7:1079–89
79. Vyas SP, Goyal AK, Khatri K (2010) Mannosylated liposomes for targeted vaccines delivery. Methods Mol Biol 605:177–88
80. Jeong H-S, Na KS, Hwang H, Oh P-S, Kim DH, Lim ST, Sohn M-H, Jeong H-J (2014) Effect of space length of
mannose ligand on uptake of mannosylated liposome in RAW 264.7 cells: In vitro and in vivo studies. J Biomed
Mater Res A 102(12):4545–53
81. Kawakami S, Sato A, Nishikawa M, Yamashita F, Hashida M (2000) Mannose receptor-mediated gene transfer into
macrophages using novel mannosylated cationic liposomes. Gene Ther 7(4):292–9
82. Allen J, Khwaja F, Djakiew D. Gene therapy of prostate xenograft tumors with a p75NTR lipoplex. Anticancer Res.
24(5A):2997–3003.
83. Hosseinkhani H, Abedini F, Ou K-L, Domb AJ (2015) Polymers in gene therapy technology. Polym Adv Technol
26(2):198–211
84. Lv H, Zhang S, Wang B, Cui S, Yan J (2006) Toxicity of cationic lipids and cationic polymers in gene delivery.
J Control Release 114(1):100–9
85. Godbey WT, Wu KK, Mikos AG (1999) Size matters: molecular weight affects the efficiency of poly(ethylenimine) as
a gene delivery vehicle. J Biomed Mater Res 45(3):268–75
86. Yuan T, Wang Y, Cao W, Sun Y, Liang J, Fan Y, Zhang X (2014) Reducible cationic PAA-g-PEI polymeric micelle/
DNA complexes for enhanced gene delivery. J Bioact Compat Polym 29(5):458–73
87. Breunig M, Lungwitz U, Liebl R, Goepferich A (2007) Breaking up the correlation between efficacy and toxicity for
nonviral gene delivery. Proc Natl Acad Sci U S A 104(36):14454–9
88. Min S-H, Park KC, Il Yeom Y (2015) Chitosan-mediated non-viral gene delivery with improved serum stability and
reduced cytotoxicity. Biotechnol Bioprocess Eng 19(6):1077–82
89. Coll JL, Chollet P, Brambilla E, Desplanques D, Behr JP, Favrot M (1999) In vivo delivery to tumors of DNA
complexed with linear polyethylenimine. Hum Gene Ther 10(10):1659–66
90. “Gene Therapy of Pancreatic Ductal Adenocarcinoma (TherGAP).” [Online]. Available: https://clinicaltrials.gov/ct2/
show/NCT01274455. [Accessed: 03-Mar-2015].
91. Jiang H-L, Kang ML, Quan J-S, Kang SG, Akaike T, Yoo HS, Cho C-S (2008) The potential of mannosylated chitosan
microspheres to target macrophage mannose receptors in an adjuvant-delivery system for intranasal
immunization. Biomaterials 29(12):1931–9
92. Frankel AD, Pabo CO (1988) Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55(6):1189–93
93. Copolovici DM, Langel K, Eriste E, Langel Ü (2014) Cell-penetrating peptides: design, synthesis, and applications.
ACS Nano 8(3):1972–94
94. Dom G (2003) Cellular uptake of Antennapedia Penetratin peptides is a two-step process in which phase transfer
precedes a tryptophan-dependent translocation. Nucleic Acids Res 31(2):556–61
95. McCarthy HO, McCaffrey J, McCrudden CM, Zholobenko A, Ali AA, McBride JW, Massey AS, Pentlavalli S, Chen K-H,
Cole G, Loughran SP, Dunne NJ, Donnelly RF, Kett VL, Robson T (2014) Development and characterization of self-
assembling nanoparticles using a bio-inspired amphipathic peptide for gene delivery. J Control Release 189:141–9
96. Truant R, Cullen BR (1999) The Arginine-Rich Domains Present in Human Immunodeficiency Virus Type 1 Tat and
Rev Function as Direct Importin beta -Dependent Nuclear Localization Signals. Mol Cell Biol 19(2):1210–7
97. Martin ME, Rice KG (2007) Peptide-guided gene delivery. AAPS J 9(1):E18–29
98. Zhang K, Yang F, Ye J, Jiang M, Liu Y, Jin F, Wu Y (2012) A novel DNA/peptide combined vaccine induces PSCA-
specific cytotoxic T-lymphocyte responses and suppresses tumor growth in experimental prostate cancer. Urology
79(6):1410.e7–13
99. “Two-Arm Study of a DNA Vaccine Encoding Prostatic Acid Phosphatase (PAP) in Patients With Non-Metastatic
Castrate-Resistant Prostate Cancer.” [Online]. Available: https://clinicaltrials.gov/ct2/show/NCT00849121. [Accessed:
15-Dec-2014].
100. Mincheff M, Tchakarov S, Zoubak S, Loukinov D, Botev C, Altankova I, Georgiev G, Petrov S, Meryman HT (2000) Naked
DNA and adenoviral immunizations for immunotherapy of prostate cancer: a phase I/II clinical trial. Eur Urol 38(2):208–17
101. Zhang X, Divangahi M, Ngai P, Santosuosso M, Millar J, Zganiacz A, Wang J, Bramson J, Xing Z (2007)
Intramuscular immunization with a monogenic plasmid DNA tuberculosis vaccine: Enhanced immunogenicity by
electroporation and co-expression of GM-CSF transgene. Vaccine 25(7):1342–52
102. Gao N, Chen W, Zheng Q, Fan D, Zhang J, Chen H, Gao GF, Zhou D, An J (2010) Co-expression of Japanese
encephalitis virus prM-E-NS1 antigen with granulocyte-macrophage colony-stimulating factor enhances humoral
and anti-virus immunity after DNA vaccination. Immunol Lett 129(1):23–31
103. Sun X, Hodge LM, Jones HP, Tabor L, Simecka JW (2002) Co-expression of granulocyte-macrophage colony-stimulating
factor with antigen enhances humoral and tumor immunity after DNA vaccination. Vaccine 20(9–10):1466–74
104. Aggarwal S, Ricklis RM, Williams SA, Denmeade SR (2006) Comparative study of PSMA expression in the prostate
of mouse, dog, monkey, and human. Prostate 66(9):903–10
105. Yang D, Holt GE, Velders MP, Kwon ED, Kast WM (2001) Murine six-transmembrane epithelial antigen of the
prostate, prostate stem cell antigen, and prostate-specific membrane antigen: prostate-specific cell-surface
antigens highly expressed in prostate cancer of transgenic adenocarcinoma mouse prostate mice. Cancer Res
61(15):5857–60
106. Johnson LE, Frye TP, Chinnasamy N, Chinnasamy D, McNeel DG (2007) Plasmid DNA vaccine encoding prostatic
acid phosphatase is effective in eliciting autologous antigen-specific CD8+ T cells. Cancer Immunol Immunother
56(6):885–95
107. Becker JT, Olson BM, Johnson LE, Davies JG, Dunphy EJ, McNeel DG. DNA vaccine encoding prostatic acid
phosphatase (PAP) elicits long-term T-cell responses in patients with recurrent prostate cancer. J Immunother.
33(6):639–47.
108. Johnson LE, Frye TP, McNeel DG (2012) Immunization with a prostate cancer xenoantigen elicits a xenoantigen
epitope-specific T-cell response. Oncoimmunology 1(9):1546–56
109. Slovin S, Gregor P, Wolchok J, Pedraza A, Orlandi F, Jefferson M, Fernandez C, Rudolph J, Houghton A, Scher H
(2007) A xenogeneic PSMA DNA vaccine for patients (pts) with non-castrate metastatic (NCMPC) and castrate
metastatic prostate cancer (CMPC)–A phase I trial of proof of principle. ASCO Meet Abstr 25(18_suppl):3073
110. Lu Y, Ouyang K, Fang J, Zhang H, Wu G, Ma Y, Zhang Y, Hu X, Jin L, Cao R, Fan H, Li T, Liu J (2009) Improved
efficacy of DNA vaccination against prostate carcinoma by boosting with recombinant protein vaccine and by
introduction of a novel adjuvant epitope. Vaccine 27(39):5411–8
111. Kim S, Lee JB, Geon KL, Chang J (2009) Vaccination with recombinant adenoviruses and dendritic cells expressing
prostate-specific antigens is effective in eliciting CTL and suppresses tumor growth in the experimental prostate
cancer. Prostate 69(9):938–48
112. Kim S, Lee J-B, Chang J (2007) Vaccination with recombinant adenoviruses and dendritic cells expressing prostate-
specific antigens is effective in eliciting CTL and suppresses tumor growth in the experimental prostate cancer.
J Immunol 178:LB32, Meeting Abstracts
113. “Provenge With or Without pTVG-HP DNA Booster Vaccine in Prostate Cancer.” [Online]. Available: https://clinical
trials.gov/ct2/show/NCT01706458. [Accessed: 15-Dec-2014].
114. “Phase II PAP Plus GM-CSF Versus GM-CSF Alone for Non-metastatic Prostate Cancer.” [Online]. Available: https://
clinicaltrials.gov/ct2/show/NCT01341652. [Accessed: 15-Dec-2014].
115. “Vaccine Therapy With PROSTVAC/TRICOM and Flutamide Versus Flutamide Alone to Treat Prostate Cancer.”
[Online]. Available: https://clinicaltrials.gov/ct2/show/NCT00450463. [Accessed: 15-Dec-2014].
116. “Phase II Study of Adenovirus/PSA Vaccine in Men With Recurrent Prostate Cancer After Local Therapy APP21.”
[Online]. Available: https://clinicaltrials.gov/ct2/show/NCT00583752. [Accessed: 15-Dec-2014].
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Cole et al.

Cancer Nanotechnology (2015) 6:2

REVIEW Open Access

DNA vaccination for prostate cancer: key

immune system to mount a response against tumour associated antigens (TAAs),

Strategies to improve DNA vaccine efficacy

Physical delivery methods

The gene gun

Conclusions and future considerations

DNA delivery vector

Lipid/liposome delivery systems

Polymer delivery systems

Peptide delivery systems

Prime / boost strategies

Received: 29 December 2014 Accepted: 16 May 2015

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