Journal of Pathology

J Pathol 2006; 208: 290–298

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/path.1865

Review Article

Bacterial gene therapy strategies

Georges Vassaux,* Josianne Nitcheu, Sarah Jezzard and Nick R Lemoine
Cancer Research UK Molecular Oncology Unit, Barts and The London School of Medicine and Dentistry, John Vane Science Centre, Charterhouse
Square, London EC1M 6BQ, UK

*Correspondence to: Abstract

Georges Vassaux, Cancer
Research UK Molecular Oncology
Unit, Barts and The London
School of Medicine and Dentistry,
John Vane Science Centre,
Charterhouse Square, London
EC1M 6BQ, UK.
E-mail:
georges.vassaux@cancer.org.uk
The ability of bacteria to mediate gene transfer has only recently been established and
these observations have led to the utilization of various bacterial strains in gene therapy.
The types of bacteria used include attenuated strains of Salmonella, Shigella, Listeria,
and Yersinia, as well as non-pathogenic Escherichia coli. For some of these vectors, the
mechanism of DNA transfer from the bacteria to the mammalian cell is not yet fully
understood but their potential to deliver therapeutic molecules has been demonstrated
in vitro and in vivo in experimental models. Therapeutic benefits have been observed in
vaccination against infectious diseases, immunotherapy against cancer, and topical delivery
of immunomodulatory cytokines in inflammatory bowel disease. In the case of attenuated
Salmonella, used as a tumour-targeting vector, clinical trials in humans have demonstrated
the proof of principle but they have also highlighted the need for the generation of strains
with reduced toxicities and improved colonization properties. Altogether, the encouraging
results obtained in the studies presented in this review justify further development of bacteria
as a therapeutic vector against many types of pathology.
Copyright  2006 Pathological Society of Great Britain and Ireland. Published by John
Wiley & Sons, Ltd.
Keywords: gene therapy; bacteria; Salmonella; Shigella; Listeria; Yersinia; cancer; vacci-
nation; inflammatory bowel disease

Introduction With bacterial vectors, this is achieved through entry

Direct injection of naked DNA has been shown to
allow transgene expression in muscle [1]. However,
for most gene therapy applications, the need for an
efficient and relevant vector for gene transfer is now
widely acknowledged. Classically, these vectors are
classified into non-viral and viral vectors. The non-
viral vectors are usually chemically defined com-
pounds such as liposomes that complex the genetic
material and mediate gene transfer [2]. Viral vectors,
replicating or not, are recombinant viruses in which
all or part of the viral genome has been replaced by
the expression cassette encoding the therapeutic trans-
gene. Gene transfer can also occur from bacteria to a
very broad range of recipients that include yeast [3]
and plants [4] and several laboratories have reported
a relatively high frequency of functional gene trans-
fer from bacteria to mammalian cells [5–11]. From
these experimental studies, a third type of vector has
been proposed: bacterial gene transfer vectors. In this
review, we will describe the mechanism of gene trans-
fer and provide examples of in vitro and in vivo appli-
cations of this technology.
Mechanism of gene transfer by bacterial
vectors
The first limiting step of gene transfer lies in the
entry of the genetic material into mammalian cells.
of the entire bacterium into the target cells. When
professional phagocytic cells such as macrophages
or dendritic cells are targeted, this entry is likely
to happen through phagocytosis but, in the case of
non-phagocytic cells, there are two major strategies
for bacteria to gain entry into a eukaryotic cell [12].
For certain genera such as Salmonella or Shigella,
contact between the bacteria and the host results in the
secretion by the bacteria of a set of invasion proteins
that triggers intracellular signalling events. This leads
to cytoskeletal rearrangement, membrane ruffling, and
bacterial uptake by pinocytosis. For other genera such
as Yersinia or Listeria, binding of a single bacterial
protein to a particular ligand on the host cell surface
is necessary and sufficient to trigger entry by a zipper-
like mechanism.
Once inside the cells, the bacteria are localized
in the phagosomal vacuoles and are targeted for
degradation. Therefore, in order for the plasmid to
be delivered to the nucleus, escape from the vacuolar
compartment must be achieved. In the case of bacterial
delivery vectors that remain in the phagosome such as
Salmonella typhimurium, the mechanism of plasmid
DNA escape in the cytosol remains unclear [11]. A
recent study suggests that part of the eukaryotic gene
expression reported with this type of vector may in
fact be due to a substantial activity of some eukaryotic
promoters such as the immediate-early promoter of
cytomegalovirus or the Rous sarcoma virus promoter

Copyright  2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Bacterial gene therapy strategies
in the bacteria, leading to protein instead of gene
transfer [13]. By contrast, the process of phagosomal
escape is particularly well understood for vectors
based on bacteria replicating in the cytosol (Listeria
monocytogenes) [8,14]. After internalization, these
Gram-positive bacteria are able to escape into the
cytosol thanks to the action of the pore-forming toxin
Listeriolysin O (LLO). Once in the cytosol, wild-type
L. monocytogenes will replicate [15]. An attenuated L.
monocytogenes strain has been engineered to undergo
self-destruction in the cell cytosol by production of
a phage lysine under the control of the promoter
of actA, which is preferentially activated when the
bacteria are in the cytosol [8]. This bacterial vector
was able to deliver a plasmid carrying the gene for
a chloramphenicol acetyltransferase or part of the
chicken ovalbumin gene (ova) under the control of the
CMV promoter into the P388D macrophage cell line.
Functional gene transfer was in the range of 0.2% and
both chloramphenicol acetyltransferase activity and
presentation of ova epitopes were detected. Plasmid
DNA recovered from macrophage clones cultured in
selective medium for more than 12 weeks was found
to be integrated into the macrophage genome at a
frequency of 10−7 . Another bacterial vector taking
advantage of the pore-forming activity of the LLO
protein is the invasive E. coli [9,10]. In addition to the
hly locus from L. monocytogenes encoding the LLO
protein, the inv locus encoding the invasin protein
from Yersinia pseudotuberculosis has been inserted
into this bacterium [9]. Invasin binds to β1-integrin
expressed at the surface of mammalian cells and
this binding is necessary and sufficient for entry of
the whole bacterium into the mammalian cell [16].
Therefore, invasin expression restricts the tropism of
these bacteria to non-phagocytic cells expressing β1-
integrin [17], while invasin-negative recombinant E.
coli can be used to target professional phagocytic cells
[18,19]. Figure 1 shows Caco-2 cells invaded by these
invasive E. coli. In these bacteria, LLO is engineered
to be intra-bacterial as opposed to secreted in wild-type
L. monocytogenes [9,10,17,20]. Therefore, once in the
cell, the bacterium is degraded in the lysosome. This
process releases LLO in the lysosomal compartment
and the bacterial content that includes the plasmid to
be delivered can escape in the cytosol through the
pores formed by LLO.
Finally, as there is no active mechanism of transport
of the plasmid from the cytosol to the nucleus, a likely
way for the plasmid to gain access to the nucleus is
disruption of the plasma membrane during mitosis.
Applications of bacterial vectors:
immunotherapy/vaccination
Delivery of plasmid-encoded antigens under the tran-
scriptional control of eukaryotic promoters by live,
attenuated bacteria is a strategy that has been used
successfully in vaccination. The methodology relies
291
Figure 1. Invasion of colonic epithelial cells by invasive E. coli.
Caco-2 cells were incubated for 2 h in the presence of invasive
bacteria, at a ratio of ten bacteria per mammalian cell. The
cells were then washed extensively and cultured as previously
described [17]. Twenty-four hours later, the cells were fixed,
permeabilized, and stained using a standard protocol [99], and
then observed using confocal microscopy. The blue colour
represents DAPI staining of the nucleus; the red phalloidin
staining of F-actin; and the green is the green fluorescent
protein expressed in the invasive bacteria
on the delivery of the plasmid in which a eukaryotic
promoter drives the expression of the antigen into
macrophages and/or dendritic cells. At the same
time, pathogen-associated molecular patterns (PAMPs)
present in the bacteria will stimulate these antigen-
presenting cells and trigger an innate immune response
in the form of the production of reactive oxygen,
pro-inflammatory cytokines, and nitrogen species, as
well as up-regulation of co-stimulatory molecules.
These responses promote the maturation and migra-
tion of dendritic cells to secondary lymph nodes
[21] and, in this way, PAMPs amplify the immune
response against the antigen and act as adjuvants.
PAMPs exert their action through binding to recep-
tors of the Toll-like receptor (TLR) family [21].
PAMPs include compounds such as lipopolysac-
charide (LPS) [22,23], bacterial DNA containing
unmethylated cytosine-phosphate-guanine (CpG) din-
ucleotides [24], flagellin [25] or bacterial lipoproteins
[26].
Vaccination against bacterial antigens
Following the demonstration that strains of Shigella
flexneri mutated in a gene essential for cell wall syn-
thesis were able to deliver plasmids in vitro resulting
in β-galactosidase expression in mammalian cells [5],
nasal inoculation of these bacteria in mice led to the
induction of β-galactosidase-specific humoral and cel-
lular responses [27].
J Pathol 2006; 208: 290–298
292
Oral administration in mice of an attenuated Yersinia
enterocolitica carrying a eukaryotic expression plas-
mid encoding Brucella genes elicited humoral and
cellular responses. This immune response, in conjunc-
tion with potential cross-reacting antibodies against
LPS of both Yersinia and Brucella, induced protection
against a Brucella challenge [28].
Another set of studies exploited S. typhimurium as
a gene delivery vector [6]. When β-galactosidase was
used as a transgene, specific cytotoxic T-lymphocytes
(CTLs) and T-helper (Th) cells, mainly of the Th1
type, as well as specific antibodies could be detected
after a single oral vaccination [6]. A very similar
immune response was elicited when two virulence
factors of L. monocytogenes (LLO and ActA) were
encoded in S. typhimurium vectors [29]. Protection
of mice against a lethal challenge with L. monocyto-
genes was observed [6,30]. The route of administration
appears to affect these responses. β-Galactosidase as
well as a fusion antigen of the Pseudomonas aerogi-
nosa outer membrane protein with the fimbriae was
administered orally or nasally. Multiple nasal admin-
istrations were necessary to obtain a T-cell response
in the spleen compared with a single oral one [11,30].
Specific IgGs were observed in the gut, saliva, and
serum after oral administration, whereas nasal admin-
istration gave rise to antibodies in the lungs, saliva, and
serum, with hardly any antigen-specific IgA detected
[11,30]. Independently, partial protective responses
against Chlamydia were obtained in the lungs of
mice after oral administration of Salmonella encod-
ing the major outer membrane protein of Chlamydia
trachomatis [11].
Salmonella-mediated vaccination has essentially
been achieved with a strain of S. typhimurium
auxotrophic for aromatic amino acids, strain SL7207
[31], but intranasal vaccination with S. typhi was
also reported [32]. In this study, the guaBA-attenuated
S. typhi strain (CVD915) defective in guanine
biosynthesis carried a eukaryotic expression plasmid
encoding tetanus toxin fragment C. This vaccination
induced antigen-specific antibody responses that were
higher than those induced by a similar bacteria
carrying a prokaryotic expression plasmid in which the
expression of the same antigen was inducible in vivo
[32].
Vaccination against viral antigens
Viral infections have also been targeted through DNA
vaccination mediated by bacteria. Oral vaccination
with S. typhimurium encoding a hepatitis B virus sur-
face antigen (HbsAg) proved successful at inducing
CTLs in Balb/c mice [33] and a single administration
of these bacteria to transgenic mice expressing HbsAg
in the liver led to loss of expression of the antigen in
hepatocytes [34]. This loss of HbsAg was accompa-
nied by hepatic flare that subsided after 3 weeks, while
the suppression of HbsAg expression continued in the
absence of overt liver pathology for the remaining
J Pathol 2006; 208: 290–298
G Vassaux et al
duration of the experiment (12 weeks). This single
administration of the recombinant bacteria induced
CTLs, Th1 T cells, and HbsAg-specific IgG2 sub-
class antibodies as further proof that immune tolerance
against the viral antigen had been broken. A similar S.
typhimurium was also used in bacteria-mediated DNA
vaccination against the non-structural region 3 (NS3)
of hepatitis C virus in HLA-A2.1 transgenic mice [35].
A single oral administration induced A2.1-restricted
CTLs, INF-γ -producing T cells, and resistance against
a challenge with NS3-expressing vaccinia virus [35].
Recombinant S. typhimurium administered orally was
also reported to induce an immune response against
herpes simplex virus 2 [36] and human papillomavirus
16 [37,38].
Vaccination against HIV was attempted using atten-
uated strains of Shigella and Salmonella carrying
eukaryotic expression plasmids encoding HIV env and
gp120, respectively [39,40]. Oral administration of
recombinant Salmonella as well as intranasal vacci-
nation with recombinant Shigella led to the activation
of antigen-specific CD8 T-cell responses. In addi-
tion, Shigella HIV gp120 DNA vaccination conferred
some protection against challenge with a vaccinia virus
expressing env [39]. A study comparing the ability
of the Shigella strain to different Salmonella strains
suggested that the induction of antigen-specific CD8
T-cell responses was superior with Shigella [41]. In
the same study, intranasal application of Shigella was
shown to induce a similar cellular response to intra-
muscular DNA vaccination but the mucosal, antigen-
specific IgA response induced by the bacterial vec-
tor was superior [41]. Finally, an attenuated Shigella
defective in LPS-O-antigen synthesis and carrying an
HIV gag DNA vaccine was used as a boost, after
priming by intramuscular DNA injection [42]. Gag-
specific T-cell responses were detected in the spleen
and lungs and the prime/boost strategy resulted in a
dramatically increased T-cell response in the lungs
[42].
Other DNA vaccinations mediated by bacteria
include vaccination against measles virus [43] and
influenza [44] with a recombinant strain of S. flexneri
and vaccination against pseudo-rabies virus using
either non-pathogenic E. coli administered intramus-
cularly [45] or a swine-adapted strain of Salmonella
choleraesuis [46].
Cancer immunotherapy
Salmonella strains have essentially been used to
deliver DNA for therapeutic applications in oncology.
In the first instance, ‘model’ tumour antigens such
as β-galactosidase or human gp100 (hgp100) were
encoded in eukaryotic expression vectors carried by
strains of Salmonella [7,47,48]. Oral administration of
these bacterial strains protected the mice against chal-
lenges with fibrosarcoma [7], renal carcinoma [47],
and melanoma [48] cells expressing the relevant model
antigen.
Bacterial gene therapy strategies
The next series of experiments assessed the effi-
cacy of oral administration of Salmonella DNA carrier
against autologous tumour antigens also expressed in
normal tissues. Successful protection was observed
with transgenes including the murine gp100 (mgp100)
fused to the invariant chain [49], epitopes of mgp100
and TRP2 fused to ubiquitin [50], a minigene encod-
ing epitopes of the tyrosine hydroxylase (TH) fused
to ubiquitin [51], the complete TH coding sequence
linked to a virally derived post-transcriptional reg-
ulatory element [52], and human carcinoembryonic
antigen (hCEA) in an hCEA mouse transgenic model
[53]. As all of these antigens were self-antigens,
these observations strongly suggest that Salmonella-
mediated DNA vaccination can break immunological
tolerance. In many cases, the protection observed was
strongly improved by co-administration of interleukin
(IL)-2 [49,52,53].
The vaccination against a tumour antigen can be
amplified by co-delivery of another plasmid encod-
ing a cytokine. This strategy was illustrated by the
vaccination of mice with a Salmonella strain carrying
eukaryotic expression plasmids encoding the transcrip-
tion factor Fos-related antigen 1 (Fra-1) overexpressed
in an aggressive murine breast carcinoma model and
IL-18, a cytokine known to suppress angiogenesis and
to stimulate INF-γ production by T and NK cells [54].
This multifunctional DNA vaccine proved effective in
protecting against growth and metastases of breast can-
cer by combining the action of immune effector cells
with suppression of tumour angiogenesis [54].
Genetic vaccination mediated by Salmonella has
also been used to target vascular endothelium growth
factor receptor 2 [55]. This receptor is up-regulated
on proliferating endothelial cells of the tumour vas-
culature and strong cellular immune responses were
elicited against these cells by oral administration of
the bacteria. This vaccination resulted in the sup-
pression of tumour vascularization and the vacci-
nated mice showed protection against tumour chal-
lenge and reduced growth of metastases. This effect
was enhanced when a strain of S. typhimurium car-
rying plasmids encoding the vascular endothelium
growth factor receptor 2 and the murine IL-12 gene
was used [56].
Applications of bacterial vectors:
bacteriolysis of tumours
Historical context
The preferential replication of bacteria in certain
experimental animal tumours was initially reported
in the 1960s when certain strains of Clostridia were
shown to proliferate exclusively in the tumour. It was
assumed that the anaerobic bacteria were replicating in
the necrotic centres of these tumours, leaving the well-
oxygenated normal tissues unaffected. This bacterial
growth was associated with lysis of large tumours
with a necrotic/hypoxic centre but had little effect on
293
small metastatic lesions. Some animals became ill and
died during the peak of oncolysis, presumably from
a combination of the systemic effects of the bacterial
inflammation and the release of necrotic tumour debris
[57–59]. These pre-clinical studies led to a clinical
trial in humans, using spores of Clostridia incapable
of producing toxins [60]. Most patients showed no
evidence of objective regression of the tumours but
abscesses in the tumour masses were detected in
some patients. Cultures of biopsies demonstrated the
presence of the injected micro-organism but further
studies were abandoned due to the lack of clinical
efficacy.
Pre-clinical studies
More recently, investigators have attempted to use
the tumour-targeting properties of Clostridium for
the selective delivery of pro-drug-activating enzymes
[61–64]. In these studies, the E. coli enzyme cytosine
deaminase [62,64,65] and nitroreductase [61] were
expressed in Clostridium and were shown to convert
the non-toxic pro-drugs 5-fluorocytosine and CB1954,
respectively, into toxic compounds capable of dif-
fusing in the tumours and killing the cancer cells
through a bystander effect. Using a similar principle, a
strain of Clostridia was engineered in which a radio-
responsive promoter drove the expression of TNFα
[66]. In an attempt to optimize the obligate anaerobic
strain used, a screen of 26 different types of bacteria
was performed and Clostridium novyi appeared par-
ticularly promising [67]. A strain of C. novyi devoid
of its lethal toxin was then engineered (C. novyi-NT)
and when its spores were administered with conven-
tional chemotherapeutic drugs, necrosis of tumours
often developed within 24 h, resulting in a significant
and prolonged anti-tumour effect [67]. Encouraging
anti-tumour activities in pre-clinical tumour models
were also observed when C. novyi-NT was admin-
istered in combination with anti-microtubule agents
[68] or associated with radiation therapy [69]. Finally,
this bacteriolysis was shown to trigger a long-lasting
immune response amplifying the bacteriolytic action
of C. novyi-NT [70].
Based on the same principles, the Gram-positive
anaerobic bacteria Bifidobacterium have been shown
to colonize large tumours. In contrast to Clostridia,
Bifidobacteria are non-pathogenic, non-spore-forming,
and are found naturally in the digestive tract of
humans and other mammals. The first study was per-
formed in the 1980s, when Ehrlich ascites tumours
were implanted in the thigh muscles of mice [71].
Systemic injection of a suspension of Bifidobacte-
ria led to the colonization of tumours. This effect
was amplified by daily administration of lactulose, a
sugar substrate metabolized by bacteria but not by
mammalian cells. However, no anti-tumour effects
or increases in survival were found in these stud-
ies. More recently, B. adolescentis carrying a plas-
mid encoding the endostatin gene was shown to tar-
get subcutaneously implanted liver tumours in Balb/c
J Pathol 2006; 208: 290–298
294
mice, leading to inhibition of angiogenesis and growth
of the tumour [72]. The same group has also reported
that the oral administration of B. longum carrying
the endostatin gene was efficient in the same tumour
model. This effect was amplified by co-administration
of selenium, thought to act through an improved activ-
ity of NK and T cells [73]. Bifidobacteria may there-
fore represent a safer alternative to Clostridia.
Gram-negative Salmonella have also been proposed
as oncolytic agents. In contrast to obligate anaero-
bic bacteria such as Clostridia and Bifidobacteria,
Salmonella are facultative anaerobic bacteria and have
the potential to colonize oxygenated small metastatic
lesions as well as large tumours with a hypoxic centre.
The first demonstration of the potential of Salmonella
was provided in 1997, when Salmonella auxotrophs
injected into tumour-bearing animals were shown to
replicate preferentially in tumours. The ratio of bac-
teria in the tumour to bacteria in normal tissues
exceeded 1000/1 and this accumulation was accompa-
nied by a therapeutic effect [74]. In a separate study,
Salmonella were shown to inhibit melanoma metas-
tases, leading to a significant reduction in the size
and number of micrometastases [75]. To reduce the
possibility of lipopolysaccharide-induced septic shock
in patients, lipid-A-modified (msbB ) Salmonella aux-
otrophs (purI– ) were developed [76]. These mutants
showed attenuated toxicity in mice and swine, asso-
ciated with reduced host TNFα induction. This was
achieved without losing the targeting of the tumour
and the therapeutic effect in mice [76]. In terms of
mechanism, the Salmonella pathogenicity island 1
(SPI1) does not appear to be necessary for the anti-
tumour activity of the bacteria [77]. Disabling SPI1
dramatically reduced tumour cell invasion in vitro, but
did not alter the anti-tumour activity in vivo. This
observation strongly suggests that invasion of the can-
cer cells is not involved in this anti-tumour effect.
By contrast, disabling SPI2 led to loss of the anti-
tumour activity after either intravenous or intratu-
moural injections [77]. SPI2 has a crucial role in sys-
temic growth of Salmonella in its host and is required
for survival within macrophages and epithelial cells
[78]. However, an SPI2-negative Salmonella strain did
not suppress tumour growth in CD18-deficient mice
with defective macrophages and neutrophils, suggest-
ing that the loss of effect of this bacterial strain was not
solely a function of increased susceptibility to immune
clearance. The precise molecular mechanisms there-
fore remain to be determined. The anti-tumour effect
mediated by Salmonella was shown to be enhanced
by radiation [79] and low doses of cisplatin [80]. To
amplify the anti-tumour effect, new strains of genet-
ically engineered Salmonella armed with therapeuti-
cally relevant genes have been produced. They include
strains capable of delivering the herpes simplex thymi-
dine kinase protein [74,76] as well as the genes cod-
ing for endostatin [81] and thrombospondin-1 [82].
In addition, diagnostic imaging is an unexpected and
potentially powerful application of tumour-targeting
J Pathol 2006; 208: 290–298
G Vassaux et al
Salmonella [83,84]. The strategy involves the ability
of bacteria expressing the herpes simplex thymidine
kinase protein to phosphorylate radiolabelled nucleo-
side analogues. In this way, the radiotracer becomes
trapped in the bacteria and as Salmonella accumulates
in tumours, the radioactive signal that can be moni-
tored by positron emission tomography [83] provides
information on the localization of tumour sites.
Studies with direct clinical relevance
The anti-tumour activity of the Salmonella strain
VNP20009 was tested in dogs with spontaneous neo-
plasia, in the context of a phase I dose escalation
trial [85]. VNP20009 is a genetically modified strain
of S. typhimurium that includes genetically stable
attenuated virulence (a deletion in the purI gene),
reduction of septic shock potential (a deletion in the
msbB gene), and antibiotic susceptibility [86]. Intra-
venous administration of VNP20009 at doses with
acceptable toxicity resulted in detectable bacterial col-
onization of tumour tissue and significant anti-tumour
activity, with four complete responses out of 41 ani-
mals treated [85]. The same strain was administered
to 24 patients with metastatic melanoma and one
patient with metastatic renal carcinoma [87]. The
results established a maximum tolerated dose of 3 ×
108 cfu/m2 and dose-limiting toxicity was observed
in patients receiving 109 cfu/m2 , with symptoms that
included thrombocytopenia, anaemia, persistent bac-
teraemia, hyperbilirubinaemia, diarrhoea, and vomit-
ing. VNP20009 induced a dose-related increase in
the circulation of pro-inflammatory cytokines. Tumour
colonization occurred in three patients but no objec-
tive tumour regression was observed [87]. Another
clinical trial involved the intratumoural injection of
attenuated Salmonella expressing the E. coli cytosine
deaminase gene (3 × 106 − 3 × 107 cfu/m2 ) in three
refractory cancer patients, followed by administration
of 5-fluorocytosine [88]. No significant adverse events
related to the treatment were observed. Two patients
had evidence of bacterial colonization of the tumour
that persisted for at least 15 days after the initial injec-
tion. Conversion of 5-fluorocytosine to 5-fluorouracil
as a result of cytosine deaminase expression was
demonstrated in these two patients [88]. These two
clinical trials highlight the need for the generation of
strains with reduced toxicities and improved tumour
colonization properties.
Applications to gastro-intestinal diseases
Inflammatory bowel disease (IBD) is a significant
health-care problem characterized by diarrhoea, pain,
other intestinal symptoms, and life-long relapses. The
pathogenesis is complex and relies on the inter-
action between three essential factors: genetic sus-
ceptibility, intestinal bacteria, and the gut mucosal
immune response. Despite significant progress in drug
Bacterial gene therapy strategies
therapy, most strategies that use immunomodulation
share the same limitations, which include lack of
organ specificity, that result in unpleasant side effects.
To address these limitations, a recombinant Lacto-
coccus lactis strain was engineered to produce the
anti-inflammatory cytokine IL-10 [89]. Administration
of this strain led to local production of the anti-
inflammatory cytokine and prevented the development
of colitis in different mouse models [89]. This strain
was also modified to carry a mutation disabling the
thymidylate synthase, resulting in a strain dependent
on the availability of thymine or thymidine in the local
micro-environment, which was less likely to accumu-
late in the environment [90]. Following the demon-
stration of proof of principle, further studies are now
focusing on characterization of the best possible trans-
gene (reviewed in ref 91).
Another bacterial strain used in the treatment of
experimental IBD is the invasive E. coli [9], express-
ing invasin from Yersinia and LLO from Listeria and
carrying a eukaryotic expression plasmid in which
the constitutive CMV promoter drives expression of
the immunomodulatory cytokine TGF-β1 [92]. Oral
administration of these bacteria, which is known to
allow gene transfer in vitro [9,17], led to a signifi-
cant reduction of the severity of experimental colitis
in mice, with vector-specific transcripts detected in
colonic and extra-colonic tissues such as the lungs,
liver, and spleen. To avoid expression in these extra-
colonic tissues, the CMV promoter driving the expres-
sion of TGF-β1 was replaced by the inflammation-
inducible IL-8 promoter. This substitution led to the
restriction of expression of TGF-β1 in the inflamed
colon without affecting the therapeutic effects [92],
demonstrating that targeted gene expression and ther-
apeutic benefits can be obtained using bacterial gene
transfer in the gut.
Applications of bacterial vectors: emerging
technologies
Delivery of large DNA molecules/artificial
chromosomes
One of the problems encountered when trying to trans-
fect mammalian cells with large DNA molecules is
the possibility of mechanical breakage of these large
molecules during the purification process. In that con-
text, the utilization of bacteria to transfer large DNA
molecules would simplify the procedure. The delivery
of bacterial artificial chromosomes was first demon-
strated into HeLa cells using an invasive E. coli [93].
Direct DNA transfer of up to around 1 Mb was demon-
strated and as the bacterial vector is equipped with
an inducible recombination system, modifications of
the bacterial artificial chromosome sequences should
be possible. More recently, efficient transfer of an
alpha-satellite DNA cloned into a P1-based artificial
chromosome was stably delivered into the HT1080
cell line and efficiently generated human artificial
295
chromosomes de novo [94]. In the same report, a
160 kb construct containing the cystic fibrosis trans-
membrane conductance regulator (CFTR) gene was
transferred into the same cells, where it was tran-
scribed and correctly spliced [94]. In a study in mice,
large DNA molecules carrying the viral genome of
the murine cytomegalovirus (MCMV) were transferred
using E. coli and S. typhimurium as delivery vectors
[95]. This transfer led to a productive virus infection
that resulted in elevated titres of specific anti-MCMV
antibodies, protection against lethal MCMV challenge,
and strong expression of additional genes introduced
into the viral genome. Thus, the reconstitution of infec-
tious virus from live attenuated bacteria presents a
novel concept for multivalent virus vaccines launched
from bacterial vectors.
Delivery of small interfering RNA (siRNA)
The utilization of double-stranded RNA to silence
target genes (RNA interference: RNAi) has potential
therapeutic applications that are widely acknowledged
[96]. Bacteria-mediated induction of RNAi was estab-
lished by demonstrating target-specific gene silencing
after transfer of double-stranded RNA from E. coli in
the nematode Caenorhabditis elegans [97]. RNA sta-
bility in the bacterial vector and transfer into eukary-
otic cells were enhanced when the E. coli vector was
rendered deficient in RNAse III [98]. In the light of
these results obtained in the nematode, the delivery of
double-stranded RNA or eukaryotic expression plas-
mids encoding siRNAs into mammalian cells can be
envisaged in the near future.
Conclusion
The utilization of bacteria in gene therapy is a recent
strategy but pre-clinical studies have already demon-
strated the potential of this approach in infectious
diseases, where they can be used as vectors for vac-
cination; in oncology, where they have potential in
immunotherapy and tumour targeting; and in gastroen-
terology, where they could be used as vectors for top-
ical delivery of immunomodulatory cytokines. Some
of these bacteria have been tested in humans and their
safety profile is acceptable. However, improvement in
their tolerability will have to be made to increase the
dose injected and achieve real efficacy.
Acknowledgement
The research in the author’s laboratory is supported by Cancer
Research UK.
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