Biological Gene Delivery Vehicles: Beyond Viral Vectors

Abstract

Gene therapy covers a broad spectrum of applications, from gene replacement and knockdown for genetic or acquired diseases such as cancer, to vaccination, each with different requirements for gene delivery. Viral vectors and synthetic liposomes have emerged as the vehicles of choice for many applications today, but both have limitations and risks, including complexity of production, limited packaging capacity, and unfavorable immunological features, which restrict gene therapy applications and hold back the potential for preventive gene therapy. While continuing to improve these vectors, it is important to investigate other options, particularly nonviral biological agents which include bacteria, bacteriophage, virus-like particles (VLPs), erythrocyte ghosts, and exosomes. Exploiting the natural properties of these biological entities for specific gene delivery applications will expand the repertoire of gene therapy vectors available for clinical use. Here, we review the prospects for nonviral biological delivery vehicles as gene therapy agents with focus on their unique evolved biological properties and respective limitations and potential applications. The potential of these nonviral biological entities to act as clinical gene therapy delivery vehicles has already been shown in clinical trials using bacteria-mediated gene transfer and with sufficient development, these entities will complement the established delivery techniques for gene therapy applications.

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© The American Society of Gene Therapy
Molecular Therapy vol. 17 no. 5, 767–777 may 2009 767
INTRODUCTION
Nucleic acids and their analogs have many therapeutic applica-
tions, ranging from correction of genetic defects to gene augmen-
tation for chronic disease including cancer to acting as adjuvants
for vaccination. Nucleic acids have been exploited to deliver
genes as DNA plasmids, to mediate gene knockdown via RNA
interference (RNAi) mechanisms or to alter pre-mRNA splicing
to ameliorate disease-causing mutations. Although most nucleic
acid–based technology is currently used as therapeutics, there is
potential for gene therapy in disease prevention by replacing dis-
ease-predisposing alleles with innocuous versions before the onset
of disease. Moreover, as the knowledge of underlying genetic risk
factors accrues, pre-emptive gene therapy will increasingly be fea-
sible in order to reduce the burden of chronic disease.
Naked therapeutic genetic molecules are generally dicult to
deliver primarily due to rapid clearance,
1
nucleases which limit
serum half-life of unmodied small interfering RNA to 5–60
minutes
2
and DNA to 10 minutes,
3
the lack of organ-specic
distribution, and the low eciency of cellular uptake following
systemic delivery. Although nucleic acid modications, includ-
ing incorporation of targeting ligands and the use of physical
delivery systems, such as hydrodynamic injection, can over-
come some of these limitations, specialized gene delivery vehi-
cles (GDVs) that improve delivery eciency and cell-specicity
whilst protecting against immune recognition are preferred. In
addition, GDVs can enhance the therapeutic value of the trans-
gene by providing complementary eects such as codelivery of
inammatory suppressors to reduce cytokine production trig-
gered by plasmid DNA.
4
Viral vectors and cationic liposomes are at the forefront of
GDV technology with a large number already in clinical trial.
5
Despite their potential, limitations remain (Tab le 1 ), with immune
recognition
6–8
for most viral GDVs, mutagenic integration
9
for
some viruses, and inammatory toxicity and rapid clearance for
liposomes
10
being the most signicant. For example, immune
activation can require the concomitant use of immunosuppressive
strategies to overcome uptake and readministration problems with
current GDVs.
11–13
Antibodies generated against the GDVs can
also dramatically decrease transgene expression on readministra-
tion.
14
Furthermore, viral vectors have packaging size constraints
limiting their genetic cargo capacity. is is particularly important
given that additional plasmid maintenance and replication genes
are required for nonintegrating DNA vectors to maintain persis-
tent expression within their host cells (reviewed in ref. 15).
e inherent risks and limitations of current GDVs have gen-
erally limited their application to life-threatening diseases,
16
in
which the benets of therapy clearly outweigh the risks, to diseases
in special tissue environments, for example immune-privileged
sites such as the eye,
17
or for genetic vaccination.
18
However, for
genetic diseases that are chronic and debilitating but not neces-
sarily life-threatening, a much lower risk prole and the ability to
sustain corrective gene therapy for decades is required for cura-
tive intervention. An example of an unacceptable risk lies in the
aforementioned immunosuppressive strategy, and is highlighted
Correspondence: Matthew J Wood, Department of Physiology, Anatomy and Genetics, Le Gros Clark Building, University of Oxford, South Parks Road,
Oxford OX1 3QX, UK. E-mail: matthew.wood@dpag.ox.ac.uk
Biological Gene Delivery Vehicles:
Beyond Viral Vectors
Yiqi Seow
1
and Matthew J Wood
1
1
Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK
Gene therapy covers a broad spectrum of applications, from gene replacement and knockdown for genetic or
acquired diseases such as cancer, to vaccination, each with different requirements for gene delivery. Viral vec-
tors and synthetic liposomes have emerged as the vehicles of choice for many applications today, but both have
limitations and risks, including complexity of production, limited packaging capacity, and unfavorable immu-
nological features, which restrict gene therapy applications and hold back the potential for preventive gene
therapy. While continuing to improve these vectors, it is important to investigate other options, particularly
nonviral biological agents which include bacteria, bacteriophage, virus-like particles (VLPs), erythrocyte ghosts,
and exosomes. Exploiting the natural properties of these biological entities for specific gene delivery applica-
tions will expand the repertoire of gene therapy vectors available for clinical use. Here, we review the prospects
for nonviral biological delivery vehicles as gene therapy agents with focus on their unique evolved biological
properties and respective limitations and potential applications. The potential of these nonviral biological enti-
ties to act as clinical gene therapy delivery vehicles has already been shown in clinical trials using bacteria-me-
diated gene transfer and with sufficient development, these entities will complement the established delivery
techniques for gene therapy applications.
Received 22 December 2008; accepted 5 February 2009; published online 10 March 2009. doi:10.1038/mt.2009.41

768 www.moleculartherapy.org vol. 17 no. 5 may 2009
© The American Society of Gene Therapy
Biological Gene Delivery Vehicles
by the death of a healthy patient due to opportunistic infection
in a recent adeno-associated virus gene therapy trial for rheu-
matoid arthritis.
19
With an increasing number of common dis-
eases shown to possess a genetic component, there is potential
for safe and sustained pre-emptive genetic solutions. Hence, it is
imperative to develop ecient gene delivery technologies that
are able to avoid immune recognition and inammation. Despite
sustained eorts to improve the safety and ecacy of liposome
formulations and viral vectors, research into alternative GDVs
may provide the least complex solution for novel therapeutic
applications.
BIOLOGICAL GDVs
Biological entities are well suited for gene delivery. Many forms
of microorganism, exemplied by viruses, have evolved to infect
cells eectively and stably while evading host immune responses.
Furthermore, many such microorganisms are tolerated by the
immune system, including commensal bacteria in the gut and
transfusion-transmitted virus in the liver.
20
Numerous cell types
can also endocytose membrane-bound bodies, for example mac-
rophage phagocytosis of apoptotic cells
21
and mast cell uptake
of exosomes,
22
which could potentially serve as novel routes
for therapeutic cargo delivery (Figure 1). e ‘ideal’ biological
GDV should have the appropriate packaging size for its cargo,
the ability to evade immune recognition, target cell-specicity,
and achieve ecient cargo delivery. e requirements at each of
these steps dier between applications and thus dierent GDVs
are likely to be required. Although the most-developed GDVs
in this class, viruses, show great promise for a wide range of dis-
ease applications, they have the aforementioned limitations and
unconventional biological GDVs with unique properties might
ll therapeutic niches currently poorly served by mainstream
viral and liposomal GDVs, for example the use of gut bacteria to
deliver transgenes to gut epithelium
23
or exosomes for delivery
to mast cells.
22
Such a specialized delivery philosophy may allow
the immune and inammatory problems that have plagued other
delivery systems to be eliminated. Here, we review the current
state-of-the-art in unconventional biological GDVs, including
bacteria, bacteriophage, virus-like particles (VLPs), erythrocyte
ghosts, and exosomes, and how their unique properties might be
exploited for specic gene therapy applications.
BACTOFECTION
Bactofection refers to the use of bacteria for transgene delivery,
which are naturally or articially engineered to be invasive but
attenuated to prevent pathogenesis. Strains of bacteria currently
used include Listeria monocytogenes,
24–28
certain Salmonella
strains,
29–34
Bidobacterium longum,
35
and modied Escherichia
coli.
23,36,37
Cellular entry typically occurs through endocytosis
followed by endosomal escape into the cytoplasm. Once in the
cytoplasm, the transgene product can be expressed in two distinct
ways. e rst is via host cell–mediated expression of the genetic
cargo released by the bacteria; the alternative is the production
and secretion of the transgene product
33,38
or RNA
39
directly by the
bacteria, eciently acting as an expression cassette within the host
cell (Figure 3). Although the former can be achieved by other gene
delivery systems, the latter can only be accomplished by bacteria
because as functional organisms, they possess the full comple-
ment of RNA polymerase and transfer RNAs for the production
of mRNA, RNAi molecules, and proteins. When DNA is released
into a cell by GDVs, the presence of immunostimulatory unm-
ethylated CpG motifs in the exogenous plasmid DNA
40
and epi-
genetic silencing of integrated viral DNA
41
both result in reduced
transgene expression over time. Bacteria-mediated transgene or
RNAi expression may evade the host defence against exogenous
DNA because the DNA is enclosed within the bacteria, poten-
tially allowing for long-term expression of transgene. In support
of this idea, a Listeria mutant with low expression of membrane
pore–forming protein listeriolysin O is able to reach a ‘stalemate’
with the host cell innate defence mechanism without causing cell
death, allowing for active transcription and slow replication in
macrophages,
42
suggesting that stable long-term bacterial expres-
sion systems are attainable, even though most cells have innate
defenses against bacteria.
Gastrointestinal
Liver
Neurons
Circulatory
Exosomes
Herpesviruses
Erythrocyte ghosts
Exosomes
Transfusion transmitted virus
Hepatitis C virus
Hepatitis B virus
Herpesviruses
Escherichia coli
Bifidobacteria
Lactobacilli
Streptococci
Bacteriophages
Herpesviruses
Human papilloma viruses
Erythrocyte ghosts
Exosomes
Herpesviruses
AAV
Figure 1 Examples of potential biological vehicles tolerated natu-
rally in selected organs. Organisms and cell-derived particles are found
naturally in certain organs as indicated, and are well-tolerated. These
particles are promising delivery vehicles that can be exploited for gene
therapy. AAV, adeno-associated virus.
Table 1 Limitations of viral vectors and cationic liposomes
Vehicle Limitations References
Adeno-associated
viral vectors
Small packaging capacity
(~4.7 kb)
Reviewed in ref. 126
Allows only DNA-based cargo
Low probability of integration
Reduced ecacy of repeat
administration
127
Retroviral/lentiviral
vectors
Insertional mutagenesis 9,128
Transcriptional silencing
leads to reduced expression
over time
Reviewed in ref. 129
Adenoviral vectors Triggers strong immune
response against vehicle and
transgene
Reviewed in ref. 130
Cationic liposome Immune recognition 131, reviewed in ref. 132
Generally low transduction
eciency compared to viruses
Risk of inammatory toxicity 133

Molecular erapy vol. 17 no. 5 may 2009 769
© The American Society of Gene Therapy
Biological Gene Delivery Vehicles
DNA cargoes for gene therapy are normally bacteria-derived
plasmids. e bacterial delivery system hence merges the produc-
tion and packaging of cargo into a single step, both increasing
the speed of production and decreasing the cost. Additionally, as
naturally occurring plasmids in bacteria range from 2 to 200 kbps
in size, they can accommodate the inclusion of large constructs
and multiple genes for gene therapy. e potential of this system
has already been demonstrated with plasmids encoding prodrug
convertases
25,31,35
and short hairpin RNAs.
37
Well-characterized nutritional tropism and the ease of bacte-
rial genetic screens for nutritional and tissue-specic tropism can
provide favorable biodistribution proles restricting bactofection
to target tissues. For example, Pawelek et al.
31
demonstrated that
polyauxotrophic mutants of a highly invasive Salmonella typhimu-
rium, defective in purine and multiple amino-acid biosynthesis,
are able to invade nutrient-rich tumors in mice up to 9,000 times
more eectively than nutrient-poor liver tissue, allowing for spe-
cic delivery of a prodrug convertase gene to tumors. Natural pref-
erences for anaerobic conditions within tumors can also be found
with attenuated B. longum
35
and Vibrio cholerae;
43
while E. coli
(DH5α) and attenuated V. cholerae preferentially target the liver
aer systemic injection despite rapid clearance subsequently.
43
In
the absence of natural tropism, modication of bacteria surface
proteins by conjugation to a cell-type specic ligand can also alter
tissue specicity to suit the application. For example, Akin et al.
26
recently demonstrated a system for conjugating nonbiological
nanoparticles with N-acetylmuramidase-specic antibodies to
L. monocytogenes to target delivery of a uorescent transgene to
N-acetylmuramidase-expressing cells. Other benecial mutations
can also be exploited, such as those in a strain of E. coli K12 that
undergoes lysis to release the therapeutic cargo upon entry into
mammalian cells because of impaired cell wall synthesis due to
diaminopimelate auxotrophy.
23
To date, bacterial GDVs have been used mainly in cancer gene
therapy
25,31–33,35
and for DNA vaccination
27–29,34
but they have also
been used for the treatment of genetic diseases, e.g., cystic brosis.
36
Although transgene expression levels have been generally low
compared to other delivery methods, work by Zelmer et al.
44
sug-
gests that, at least for Listeria-mediated gene transfer, the plasmid
DNA is oen eectively released into the target cell but nuclear
transport and subsequent expression are inhibited by the macro-
molecular structure, perhaps related to the CpG content of the
plasmid. is implies that improved design of the plasmid, not the
bacterial GDV, may be what is required to improve the expression
of the transgene product, such as with CpG-free plasmids.
45
e intrinsic toxicity of bacteria, the most signicant risk in
using bactofection, varies between dierent bacterial strains. In
clinical gene transfer trials of direct intratumoral
33
and intrave-
nous
32
injection of attenuated Salmonella bacteria, no signicant
side eects were observed at low doses <7.6 × 10
7
colony forming
units over the course of 4 days for intratumoral injections and 10
8
colony forming units/m
2
of body surface area for single intrave-
nous injections. Although bacteremia-related symptoms devel-
oped at higher doses, these were readily resolved with antibiotics
and side eects were rapidly reversed without signs of toxic shock,
indicating that administration of attenuated bacteria could be safe
in humans if titres are kept below a certain threshold. However,
IgM, IgA, and IgG antibodies developed against Salmonella
in a substantial number of subjects following intravenous
administration,
32
resulting in strong immune priming that may
render this GDV unsafe and ineective due to the presence of
neutralizing antibodies. For intranasal applications with invasive
E. coli
36
in mice, septic shock resulted from high titres and reects
the low therapeutic indices for attenuated bacteria in nonnative
environments. However, deployment of bacteria into the gut, their
natural host environment, appears to be benign, based on the oral
administration of E. coli for gene delivery to the intestinal lining
in mice.
23
e immune system has also developed tolerance for
many of the gut-derived bacteria, probably due to host–commen-
sal interactions using pattern recognition receptors,
46
making
such gut-derived bacteria suitable for avoiding immune stimula-
tion. It has been recently shown that Lactococcus lactis adminis-
tered orally to mice can transduce intestinal epithelia to produce
β-lactoglobulin eectively to stimulate a transitory 1 immune
response in order to modulate the 2 response against milk pro-
tein allergens.
47
erefore, the use of native bacterial ora to target
tissues appears to improve the therapeutic index and fulll the cri-
teria of an ‘ideal’ delivery vehicle within certain organs, including
the gut, upper respiratory tract, and vagina.
48
Although this selec-
tive approach has been successful in animal models, signicantly
more research is required before clinical adoption to ascertain
safety in human subjects. Unlike revertant replication-competent
viruses potentially generated from vector production, if the atten-
uated bacteria unexpectedly spread beyond the target organ, via
increase in pathogenic potential or causing severe bacteremia, the
resultant infection can easily be brought under control with an
established arsenal of antibiotics.
ere is growing interest in the use of bacteria as GDVs with
currently at least three bactofection Phase I clinical trials for dier-
ent cancers delivering either tumor antigens for vaccination with
S. typhimurium or cytotoxic genes with L. monocytogenes, and one
Phase II trial for ulcerative colitis involving delivery of interleu-
kin-10 by L. lactis.
5
e inexpensive production, the diverse natu-
ral and modied tropism proles, the large and diverse packaging
capacity of bacteria, coupled with their immunological tolerance
in certain target organs, and relative ease of control in the case
of adverse events make bactofection an attractive alternative to
consider for gene delivery to the gastrointestinal, respiratory, and
urogenital tracts. For application of bactofection beyond these
native tissue environments, the problem of immune activation
and the development of methods to achieve immune tolerance
will require further study.
BACTERIOPHAGE
A second GDV inspired by prokaryotic systems is the bacterio-
phage. Bacteriophages are natural viruses that exclusively infect
bacteria and are ingested without serious side eect within fer-
mented food, e.g., yoghurt.
49
Bacteriophages are very resistant
to nonenzymatic degradation when compared to other vehicles,
allowing for longer and less costly storage. Bacteriophage lambda
in particular has been shown to retain infectivity and, by exten-
sion, structural integrity for up to 6 months at 4 °C in water,
which is longer than any other biological vehicle including virus-
es.
50
Although bacteriophages are still susceptible to enzymatic

770 www.moleculartherapy.org vol. 17 no. 5 may 2009
© The American Society of Gene Therapy
Biological Gene Delivery Vehicles
degradation, their ability to withstand a wide range of pH, from
3 to 11, for up to 24 hours could make them suitable for oral
delivery.
50
Over the past century, the use of bacteriophage in research
has placed many tools at our disposal. Development of phagemid
vectors, plasmids with a bacteriophage origin of replication that
are packaged into phage particles upon addition of helper phages
(Figure 2), has made the genetic manipulation of bacteriophage
of similar ease to plasmids used in production of viral vectors.
51
e phagemid system allows for precise control over the rela-
tive composition of the wild type and modied coat proteins,
47
while eliminating the genes of the potentially immunogenic bac-
teriophage proteins. e baceteriophage has generous packaging
capacity beyond what most viral vectors like adenovirus (8 kb),
adeno-associated virus (4.7 kb), or lentiviruses (8–10 kb) (ref. 52)
oer. Lambda phage is capable of holding 53 kb (ref. 53) while
M13 phage does not seem to possess a dened packaging limit.
54
is allows for the insertion of mammalian plasmid maintenance
sequences, such as the EBNA1/oriP system
55
and endogenous
control regions for the transgene.
e coat proteins of the most commonly used bacteriophage
species for gene delivery, namely M13 lamentous phage
51,56–59
and lambda phage,
60
can be engineered to incorporate target-
ing ligands without aecting structure signicantly. Such ligands
incorporated to date include a single-chain variable fragment anti-
body directed against HER2 receptor,
57
growth factors, e.g., epider-
mal growth factor and broblast growth factor 2 (refs. 51,56,58),
metabolites, e.g., galactose and succinic acid,
61
adenovirus penton
base,
60
and targeting peptides, e.g., against gliomas.
59
Beyond the
standard ligands, the use of phage display, a high-throughput pep-
tide library screening technique using bacteriophage particles,
has the potential to identify novel peptides, which when conju-
gated to contextually relevant bacteriophage proteins can target
unknown cell surface molecules
59
and perform novel functions
such as skin penetration.
62
Phage nanoparticles with multiple pep-
tides engineered for dierent functions can then be produced with
phagemid technology to enhance gene delivery eciency.
A signicant hurdle to clinical application is that unmodi-
ed bacteriophage particles delivered systemically are rapidly
eliminated by the reticuloendothelial system (RES),
61,63
with sig-
nicant degradation occurring in the liver, spleen, and lungs.
61
Although long-circulating lambda phage variants with mutations
in their D and E capsid proteins have been developed to escape
such fate in mice,
64
the mechanism has not been elucidated and
it is not yet known whether such mutants will retain function in
human subjects. On the other hand, with the incorporation of
targeting ligands, specicity and delivery eciency can be dra-
matically improved. For example, Molenaar et al. have shown that
M13 phage uptake in the parenchymal and Kuper cells of the
liver is dramatically enhanced when the phage surface lysines
are chemically conjugated to lactose and this eect was shown
to be mediated by specic lactose receptors.
61
Oral delivery of
an E. coli phage in rabbits also results in transient localization of
the phage in major organs especially the spleen,
65
a property that
can be exploited to deliver genetic adjuvants, genes that enhance
the immune response against an antigen, or antigenic genes for
vaccination. Unfortunately, the generation of antibodies against
phage particles, which are potent antigens, is likely to signicantly
reduce the ecacy of readministered phage. However, no adverse
eect has been observed in healthy individuals
66
or immunocom-
promised patients
67
challenged with ~10
11
bacteriophage ϕX 174
particles as a single intravenous administration. Further in vivo
phage display panning experiments for weakly immunogenic
phage mutants may overcome the phage’s intrinsic immunogenic
nature.
In another related development, MS2 bacteriophage particles
assembled from bacteriophage proteins produced in a cell-free
expression system have been shown to encapsulate exogenous
RNA.
68
Although MS2 bacteriophage particles have been gen-
erated previously in E. coli cells, the cell-free expression system
allows the packaging of large amounts of exogenous therapeutic
RNAs ex vivo. Wu et al. achieved similar results with antisense
oligonucleotides,
69
opening the door to RNAi-based and antisense
therapeutics delivered through bacteriophage GDVs (Figure 3).
e principal advantages of the bacteriophage system—the
ease of developing and incorporating novel functionalities into
the phage particle, the large packaging capacity, the ability to take
non-DNA cargoes, and relative safety in humans—suggest that
bacteriophage vectors can be considered as potential therapeutic
agents. However, the potent antibody response limits the ability to
readminister these vectors and hence, restricts their current use
to the delivery of genes for vaccination purposes or to single-use
applications especially those poorly served by existing strategies
using viral vectors.
Virus-like particles
Mammalian VLPs can also be used as GDVs. Although viral
vectors are normally made replication-incompetent by separat-
ing their essential components into dierent plasmids, the vec-
tors usually possess their full complement of viral proteins when
packaged into a virion particle and the cargo plasmid containing
the therapeutic gene is packaged within the host cell. In contrast,
VLPs are made by transfecting a production cell line with a single
plasmid encoding only viral structural proteins, followed by puri-
cation of the resultant particle and encapsulation of the cargo
ex vivo (Figure 2).
One of the advantages of this method over conventional viral
vectors is that the VLPs can be produced in dierent cellular sys-
tems, including bacterial,
68,70
plant,
71
insect
72–77
and yeast cells
78–80
in
addition to standard mammalian cell lines, potentially lowering the
cost of mass production. Like viral vectors, clinical use of this sys-
tem invariably involves the complication of removing residual cellu-
lar components, but because VLPs can be denatured and reformed,
they can typically tolerate harsher purication conditions than viral
vectors. However, the key advantage of VLPs over the viral vectors
is their ability to separate loading from VLP purication as this
allows for unusual cargoes, such as modied oligonucleotides, to be
loaded.
81
e ecacy of an unmodied papillomavirus L1 VLP in
transducing cell lines with a reporter plasmid is comparable to lipo-
somal methods,
82
the predominant in vivo delivery system for small
interfering RNAs. With an increasing interest in the use of modied
small interfering RNAs as therapeutics, the ability to package small
oligonucleotides and the high ecacy of transduction in vitro give
VLPs the potential to play a signicant role in RNAi delivery.

Molecular erapy vol. 17 no. 5 may 2009 771
© The American Society of Gene Therapy
Biological Gene Delivery Vehicles
is promise is tempered by the low yield of functional VLPs
as disassembly and reassembly of VLPs aer purication is com-
plex and inecient. An interesting concept proposed to address
this limitation is chemical self-assembly of VLPs, based on the
production of monomeric capsid precursors followed by ex vivo
assembly of the virus capsid upon activation to encapsulate its
desired cargo.
83
is approach may increase the consistency of the
VLP produced and the amount of cargo encapsulated; however, it
is yet to be experimentally tested.
VLPs that have been successfully shown to mediate trans-
fer of nucleic acids can be generated from many families of
viruses, including papillomaviruses,
15,66,68,72,78,84–86
hepatitis B and
E viruses,
74,87,88
and polyoma viruses.
73,77,89–93
Other virus fami-
lies from which VLPs have been derived include lentivirus,
76,94
rotavirus,
69
parvovirus,
70
and norovirus.
71
e diverse tropisms of
these viruses and their VLPs provide a range of natural target-
ing capabilities without the need for further genetic engineering.
ese tropisms include liver for hepatitis B VLPs,
75,88
spleen for
certain papilloma and polyoma VLPs,
82,90
antigen presenting cells
for certain papilloma VLPs,
83
and glial cells for JC virus VLPs.
77
Polyomavirus VP1 VLPs containing a β-galactosidase plasmid
administered via the subcutaneous, intravenous, intraperitoneal,
and intranasal routes also showed dierent β-galactosidase expres-
sion proles in the heart, lung, kidney, spleen, liver, and brain,
demonstrating how tropism of VLPs can also vary with delivery
routes.
90
e large number of permutations of natural VLPs and
delivery methods is likely to produce diverse distribution proles
suitable for many disease applications. Similarly to viral vectors,
VLPs can also be engineered to incorporate targeting ligands such
as epidermal growth factor
74
and antibodies,
86,87
and the genetic
and chemical modications available to virus vectors can also suc-
cessfully be applied to VLPs. Like bacteriophages, papillomavirus
VLPs delivered orally are transported to Peyer’s patches, lamina
propria, and the spleen, which also makes them suitable as oral
gene vaccine vehicles.
72,82
Empty VLPs were originally developed for vaccination as a
replacement for attenuated viruses because they were easier to
produce, hence most current gene delivery applications of VLPs
A. Bacteria
C. Virus-like particles
B. Bacteriophage
E. Exosomes
D. Erythrocyte ghost
Capsid
monomers
Purification
Producer cells
Purification
Patient-derived primary cells/
differentiated stem cells
Helper phage
Lysis +
purification
Phagemid
Transformation
Amplification
Production
vector
Assembly
Cytoplasm
removed
Patient-derived
erythrocytes
Erythrocyte
ghosts
Loading
Ghosts
reintroduced
into patients
MVB
Loading
Purification of
exosomes
Exosomes
reintroduced
into patients
Figure 2 Production of biological gene delivery vehicles. (A) Strains of bacteria with desirable properties are transformed with the plasmid
cargo and amplified to generate GDVs. (B) The phagemid, a modified bacterial plasmid with phage sequences within, is used as the cargo and
transformed into bacteria. The bacteria is then infected with a replication-defective helper phage that produces essential genes for the packaging
of the phagemid vector into bacteriophage GDVs. (C) The virus surface proteins are produced in cell culture and purified as capsid monomers. The
genetic cargo is then packaged into a virion as the monomers are transferred to a buffer that promotes assembly of the virion. (D) Erythrocytes
are harvested from the patient and lysed to produce erythrocyte ghosts. The ghosts are then loaded, usually through osmotic pressure, with the
genetic cargo before being reintroduced into the patient. (E) Patient-derived primary cells are first harvested and stimulated to produce exosomes,
which are then purified, and loaded, likely by electroporation, with the genetic cargo before being reintroduced into the patient. GDV, gene delivery
vehicle; MVB, multivesicular body.

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Biological Gene Delivery Vehicles
are designed to generate immune responses against foreign or
cancer antigens. For vaccination, cargoes delivered are typically
gene adjuvants such as interleukin-2 (refs. 15,81,82) that activate
the immune system against the VLP or genes for antigens targeted
to antigen presenting cells to induce a cytotoxic T-lymphocyte
(CTL) response.
72,83
Because VLPs were optimized for their abil-
ity to stimulate the immune system, they are typically unsuitable
for repeat administration because of the potentially high levels
of serum and mucosal antibodies generated.
85,88,91
e immune
response against the VLP also seems to adversely aect tolerance
to the transgene
88
which makes this delivery strategy problematic
if delivering neo-antigens for genetic loss-of-function diseases.
However, these properties also make VLPs good candidates for
cancer and vaccine gene therapy where the goal is to stimulate
recognition of tumor and foreign antigens transiently. If the same
strategies used for masking viral vectors, such as polyethylene
glycolation (reviewed in ref. 95), were similarly applied to VLPs,
repeat delivery may be feasible.
e development of VLPs for vaccination has resulted in
well-characterized, current good manufacturing practice–grade
production processes which can be utilized to scale up VLPs
designed for gene delivery. VLPs typically present the same pitfalls
as their parent viruses, such as immunostimulation, in addition to
the ineective loading during production and lower transfection
rates, but in return, enable the loading of alternate cargoes while
potentially reducing carryover contamination during produc-
tion in addition to the other benets oered by viral vectors. For
genetic vaccination and delivery of genetic adjuvants, VLPs are
proven workhorses, but novel uses involving the delivery of small
interfering RNAs and modied oligonucleotides deserve further
focus and development.
BIOLOGICAL LIPOSOMES
Biological liposomes are phospholipid-based spherical particles
derived from human cells. If derived from patients, these lipo-
somes are likely to be recognized as self and hence, would be
ideal ‘stealth’ GDVs, capable of evading host immune recogni-
tion and RES sequestration. Although conceptually appealing,
the cells used for production of these GDVs tend to be dicult
to harvest and maintain, requiring extraction of primary cells and
the costly use of growth factors. Hence, biological liposomes are
seldom explored as delivery options. Two populations of biologi-
cal liposomes—erythrocyte ghosts and secretory exosomes—are
currently being considered for gene delivery and although the
technologies remain relatively immature, both types of biological
liposome show promise.
Erythrocyte ghosts are red blood cells that have been broken
up into small spherical structures aer removal of most cytoplas-
mic content (Figure 2). e spherical structures are then dia-
lyzed in a hypotonic solution, sheared or electroporated to create
pores for the loading of the therapeutic cargo.
96–98
Erythrocyte
ghosts have been investigated as drug delivery vehicles for sev-
eral decades and more recently, they have been engineered to
carry cargoes from small molecules
97
to large plasmids,
96
includ-
ing modied oligonucleotides,
99,100
and should presumably be able
to incorporate RNA cargoes as well. e loading eciency for
plasmids appears to be relatively high if loaded by electropora-
tion rather than with hypotonic dialysis,
96
the loading eciency of
which is one of the main reasons erythrocyte ghosts are not used
more extensively for drug delivery. Erythrocyte ghosts are rela-
tively stable, retaining a small molecule cargo—uorescein—for at
least 3 weeks in saline at 4 °C,
101
although experiments pertaining
to the long-term stability of ghosts loaded with genetic cargo have
not been performed. Moreover, Magnani et al. have developed a
method that allows immediate reinfusion of erythrocyte ghosts
loaded in hypotonic solution aer red blood cell collection
102
circumventing the need for storage.
Several lines of evidence suggest that erythrocyte ghosts
derived from the patient are well-tolerated by the immune sys-
tem and are likely to be safe as GDVs. First, predeposit autolo-
gous blood transfusion, the use of a patient’s stored blood for
blood transfusion during operation, is believed to eliminate the
risk of hemolytic and allergic reactions mediated by the immune
system when compared to allogenic blood transfusion, the use
of someone else’s blood, partly because self erythrocytes are well
tolerated.
103
Also, Gressner et al. recently showed that >50% of
labeled human erythrocyte ghosts injected intravenously in rats
was lost from the blood within 20 minutes. Conversely, no loss
was detected for rat ghosts, suggesting that the RES and immune
system eectively recognized the rat erythrocyte ghosts as self as
opposed to structurally similar human erythrocyte ghosts.
104
In
humans, a pilot trial of erythrocyte-mediated intravenous sus-
tained drug delivery in cystic brosis patients registered no side
eects or adverse events aer a year of treatment and the ghosts
appeared ecacious for drug delivery, consistently releasing 0.1–
0.2 nmol of dexamethasone-21-phosphate per milliliter of plasma
for up to 1 month; however, the study yielded too few samples to
draw denitive conclusions.
105
Although the drug delivered was
an anti-inammatory corticosteroid, this trial nonetheless hints at
2 Delivery of cargo
1 Expression cassette
DNA plasmid
mRNA
shRNA
Protein
Short oligonucleotides
C. Biological
liposomes
D. Synthetic
liposomes
E. Viral vectors
B. Bacteriophage
and VLPs
A. Bacteria
Nucleus
Figure 3 Release of cargo intracellularly by delivery vehicles.
(A) Bacteria can deliver genetic cargoes in two distinct fashion after
endocytosis and endosomal release. First, short oligonucleotides and
DNA plasmids can be released directly into the host cells through the
lysis of the bacteria. Alternatively, intracellular bacteria can produce and
excrete therapeutic RNAs and proteins. (B–D) Bacteriophage, VLPs and
both types of liposomes are capable of delivering mRNAs, short oligonu-
cleotides and DNA plasmids. (E) Viral vectors are typically only capable
of delivering DNA or RNA vectors that ultimately end up in the nucleus
as DNA templates for transcription of mRNAs. shRNA, short hairpin RNA;
VLP, virus-like particle.

Molecular erapy vol. 17 no. 5 may 2009 773
© The American Society of Gene Therapy
Biological Gene Delivery Vehicles
the stealth properties of this treatment vehicle. e fact that eryth-
rocytes and their derivatives are tolerated immunologically and
are able to evade RES sequestration suggests that these delivery
vehicles are both safe and capable of readministration, properties
desirable in a GDV for systemic use. In contrast to the long half-
life in rats measured with radioactive labels by Gressner et al.,
104
the in vivo circulation half-life of plasmid-loaded allogenic eryth-
rocyte ghosts in mouse is ~10 minutes as measured by quantitative
PCR.
96
e widely diering reports relating to half-life suggest that
signicant variation occurs between species and further research
needs to be carried out to determine the pharmacokinetics of the
ghosts and their likely behavior and eects in human subjects
before future clinical application.
Erythrocytes are constantly recycled by the spleen and RES,
hence it is no surprise that untargeted erythrocyte ghosts are mainly
localized to the spleen and liver.
104,106
Nonetheless, targeting of
erythrocyte ghosts can be achieved by various means. Attachment
of targeting ligands, such as antibodies
104,107
and metabolites
98
by chemical conjugation can expand the cell- specicity of these
erythrocytes. Another interesting strategy lies in crosslinking of
major transmembrane (band 3) proteins with chemical reagents
aer stimulation of clustering with zinc chloride.
97,108,109
Clustering
Table 2 Advantages and limitations of biological delivery vehicles
Method Advantages Limitations Potential applications Key References
Bactofection Able to encapsulate shRNAs and DNA plasmids
Natural tropism
Auxotropism
Intrinsic targeting
Modication of delivery vehicle well-tolerated
Bacteria-mediated production of shRNA and proteins
may overcome limitations for nuclear delivery
Scale-able purication processes, potentially cheaper
than mammalian cells-based systems
Single step production of vehicle and cargo if using
plasmid
Easily controlled by antibiotics
Triggers B-cell and T-cell
response if administered
systemically
Potential toxicity
Nuclear transport and
subsequent expression of
plasmids inhibited by host
Gastrointestinal tract
gene therapy
Cancer gene therapy
23,24,33,34,37–39,47
Bacteriophage Able to encapsulate antisense oligonucleotides, RNA and
DNA
Resistant to nonenzymatic degradation, including high
temperatures, low pH
Targeting ligands easily engineered into capsid proteins
Well-characterized system of phage display to rapidly
discover novel peptides for targeting/cell penetration
Scale-able purication processes, potentially cheaper
than mammalian cells-based systems
Cell-free synthesis method available
Rapid eliminated by the
reticuloendothelium system
Very potent antigen
CGMP grade manufacture
dicult
Vaccine gene therapy
Single administration
gene therapy
Repeated administration at
immune-privileged sites
50,51,53,54,59–61,64
VLP Able to encapsulate antisense oligonucleotides, siRNAs,
long RNA and DNA
Natural tropism for targeting
Retargeting strategies (e.g. antibodies) for viruses
applicable to VLPs as well
Can be produced in dierent cellular systems including
bacteria, yeast and plants
Removal of residual cellular contaminants conceptually
simpler than for viruses
CGMP grade production processes well characterized
Benets from the large body of research on viral vectors
Limited packaging eciency
Heterogeneous population
of particles
As immunogenic as parental
viruses
Vaccine gene therapy
Single administration
gene therapy
Repeated administration at
immune-privileged sites
81–83,85,88,90
Erthrocyte ghosts Able to encapsulate antisense oligonucleotides,
RNA and DNA
Coencapsulation of drug molecules
Chemical modications of cell surface can change
properties
Minimal immunogenicity
Short circulatory half-life
of erthrocytes
Sequestration by the spleen
and liver
Diculty in genetic
engineering for the
production of targeting
ligands
Repeated delivery targeted
to the liver, spleen or blood
96,98,101,102,106
Exosomes Capable of transferring functional mRNA
Chemical modication of cell surface proteins can
change properties
Immunosuppressive and anti-inammatory Readily
taken up by cells
Minimal immunogenicity, may induce tolerance
Largely uncharacterized
Potentially expensive to
develop
Repeated delivery targeted
to neurons and immune cells
22,108–110
Abbreviations: shRNA, short hairpin RNA; siRNA, small interfering RNA; VLP, virus-like particle.

774 www.moleculartherapy.org vol. 17 no. 5 may 2009
© The American Society of Gene Therapy
Biological Gene Delivery Vehicles
of these proteins on the erythrocyte surface induces autologous
IgG binding and complement xation, hence favoring phagocy-
tosis by macrophages.
109
As erythrocytes are anucleate, targeting
ligands cannot be genetically engineered and expressed on these
ghosts, but the use of erythrocytes generated from stem cells
110
expressing targeting ligands can overcome this limitation. In
spite of the limitations in targeting to other organs, erythrocyte
ghosts are conceptually the best GDVs for persistent gene therapy
for cells of hematopoietic lineage. However, signicant hurdles,
namely the relatively low loading ecacy by hypotonic dialysis
and the diculty of cell targeting, need to be addressed before
therapeutic realization in the clinic.
A second class of biological liposome is the exosome, a
small membrane-bound microvesicle (30–90 nm) of endocytic
origin that is released into the extracellular environment fol-
lowing fusion of multivesicular bodies with the plasma mem-
brane (Figure 2). Exosomes have recently been demonstrated to
act as natural GDVs for a large number of functional mRNAs
and miRNA.
22
Murine mast cell-derived exosomes were found
to deliver functional mRNAs that produce murine proteins to
human mast cells, and therefore exosomes could potentially be
exploited to deliver other oligonucleotides, much like synthetic
liposomes. Exosomes can be engineered to be immunosuppres-
sive if the cells from which they are derived are genetically engi-
neered to express immunosuppressive ligands, such as FasL,
111
or
if they are treated with immunosuppressive cytokines, such as
interleukin-10 (ref. 112). is property makes them potentially
suitable for treatment regimens requiring repeat administration.
More excitingly, because exosomes appear to have a dened set
of proteins and RNAs distinct from the parent cells, and are pro-
duced in numerous cell types, including hematopoietic, intestinal
epithelial, tumor, and neuronal cells, Smalheiser
113
has suggested
that they may have a natural role in intercellular communication.
As such, they may be readily taken up by cells without the need
for targeting ligands. Exosomes also readily express ligands trans-
fected into their parent cells, such as FasL mentioned above,
111
providing a means for targeted delivery to desired cell types if
required. Future studies will be needed to further characterize
these natural liposomes and dene their potential as GDVs for
specic gene therapy applications.
In future, biological liposome technology is likely to benet
from parallel advances in stem cell technologies. e production
of erythrocytes from human embryonic stem cells
110
should allow
for consistency in the production of erythrocyte ghosts. Patient-
derived erythrocytes cannot be manipulated genetically as they
lack a nucleus. However, with the use of stem cells, genes encoding
targeting antibodies can be genetically engineered and expressed
in the derived erythrocytes, augmenting the function of erythro-
cyte ghosts. Production of exosomes requires purication from
primary cells, which may be dicult to harvest. e ability to
generate multipotent or induced pluripotent stem cells, derived
from a patient’s dermal broblasts,
114
means that self-derived
exosomes could potentially be harvested without invasive proce-
dures, signicantly increasing their ease of application. Overall,
biological liposomes have the potential to rival conventional lipo-
somal systems as they can deliver multiple types of genetic cargo,
but with favorable immunological properties. However, the gene
delivery ecacies of biological liposomes have not yet been well
investigated by comparison against known liposomal delivery
methods. For delivery to blood-based targets, erythrocyte ghosts
and exosomes both appear to be suitable as they are well tolerated
in the blood stream and appear to be used naturally by cells of
hematopoietic lineage, such as mast cells and dendritic cells, for
communication. However, for organ delivery via local or systemic
administration, the development of surface modications to these
GDVs to increase epithelial permeability and permit tissue speci-
city will be necessary to make their use attractive.
OTHER STRATEGIES
Besides bacteriophages, nonmammalian pathogens currently
under investigation include baculoviruses which have insects as
natural host species. e extensively characterized Autographa
californica nuclear polyhedrosis virus is usually the baculovirus of
choice. Autographa californica nuclear polyhedrosis virus is able to
infect several mammalian cell types, but is incapable of replication
or integration in these cells.
115,116
However, transfection eciencies
in vivo have been poor despite impressive results in cell culture,
117
due to the activation of the classical complement system.
118
Although strategies to overcome the complement pathway have
been attempted,
119
the most eective route of delivery may be to sites
without complement components, such as the brain.
120
Autographa
californica nuclear polyhedrosis virus also appears to induce an
innate antiviral and antitumor immune response in vivo,
121–123
which is detrimental to repeated administration, but benecial for
vaccination strategies. Unfortunately, these baculoviruses do not
oer the ease-of-production of bacteriophages or VLPs, nor are
they more eective at gene delivery and as such are unlikely to be
more advantageous than other delivery systems.
eoretically, yeast particles could also be utilized for gene
delivery, although to date this possibility remains to be investigated.
Recombinant nonpathogenic yeast particles expressing tumor and
HIV antigens have been shown to strongly activate dendritic cells
and produce a cytotoxic T-lymphocyte response when introduced
subcutaneously,
124
demonstrating a high specicity toward den-
dritic cells. If engineered to release mammalian plasmids encoding
genes such as interleukin-12 or oligonucleotides that potently acti-
vate dendritic cells, the ecacy of vaccines could be signicantly
enhanced. Conversely, the strong targeting ability of recombinant
yeast could be used to promote antigenic tolerance via the delivery
of antigenic sequences to semimature dendritic cells
125
in order to
improve outcomes in organ transplantation and in autoimmune
disease. Given the relatively low costs of yeast production, this may
be an avenue of research worth pursuing.
CONCLUSIONS
e biological GDVs discussed above, although less well-estab-
lished than viral vectors and liposomal delivery agents, present
tantalizing opportunities for niche therapeutic applications given
further research and development (see Tab le 2). Besides their
intrinsic advantages, biological GDVs can also benet tremen-
dously from development of tangential technologies. For exam-
ple, VLP production can benet from the research and industrial
experience of vaccine production, while bacterial production and
delivery could build on the experience of the food industry with

Molecular erapy vol. 17 no. 5 may 2009 775
© The American Society of Gene Therapy
Biological Gene Delivery Vehicles
probiotics. Given the expertise already developed for nondelivery
applications, the barrier between bench and bedside applications
is probably not as high as perceived; hence the development of
biological GDVs probably deserves greater attention from the
gene therapy community.
It is clear that there is no one-size-ts-all solution to gene
delivery, which is why in spite of various developments in lipo-
some formulation and viral vector optimization, new compounds
and viruses are constantly being proposed. What is needed is an
arsenal of GDVs that can be utilized for specic diseases, routes,
or tissues and it is likely that biological GDVs will emerge as via-
ble options for repeat gene delivery for long-term treatment in
the case of chronic disease. What this review has aimed to achieve
is to summarize the progress in unconventional biological GDVs
and focus on how best to exploit their properties and associ-
ated tools. e common limitations of current systems appear
to be tractable if GDVs are chosen for their natural propensities
for certain niche targets rather than adopting the current one-
size-ts-all approach. Once the risks and limitations have been
minimized with such delivery strategies, gene therapy can then
be harnessed as a therapeutic for a wide variety of applications,
including pre-emptive gene therapy and therapy for diseases with
weaker genetic linkages, e.g., psychiatric illness. Hence, it is vital
that further research be undertaken into the development of
novel biological GDVs in order to diversify the delivery eld and
enable new gene therapy applications unimaginable today.
ACKNOWLEDGMENTS
We thank Graham McClorey and Marc Weinberg for comments and
critical reading of the manuscript. Y.S. acknowledges funding support
from the Agency for Science, Technology and Research (Singapore)
and M.J.A.W. from the UK MRC and Biotechnology and Biological
Sciences Research Council (BBSRC), The Wellcome Trust, the UK
Parkinson’s Disease Society, the Muscular Dystrophy Campaign and
Action Duchenne.
REFERENCES
1. Takakura, Y, Nishikawa, M, Yamashita, F and Hashida, M (2001). Development of
gene drug delivery systems based on pharmacokinetic studies. Eur J Pharm Sci 13:
71–76.
2. Soutschek, J, Akinc, A, Bramlage, B, Charisse, K, Constien, R, Donoghue, M et al.
(2004). Therapeutic silencing of an endogenous gene by systemic administration of
modified siRNAs. Nature 432: 173–178.
3. Kawabata, K, Takakura, Y and Hashida, M (1995). The fate of plasmid DNA after
intravenous injection in mice: involvement of scavenger receptors in its hepatic
uptake. Pharm Res 12: 825–830.
4. Liu, F, Shollenberger, LM and Huang, L (2004). Non-immunostimulatory nonviral
vectors. FASEB J 18: 1779–1781.
5. Gene therapy Clinical Trials Worldwide. <http://www.wiley.co.uk/genmed/clinical/>.
Accessed 9 September 2008.
6. Lowenstein, PR, Mandel, RJ, Xiong, WD, Kroeger, K and Castro, MG (2007). Immune
responses to adenovirus and adeno-associated vectors used for gene therapy of brain
diseases: the role of immunological synapses in understanding the cell biology of
neuroimmune interactions. Curr Gene Ther 7: 347–360.
7. Alexander, IE, Cunningham, SC, Logan, GJ and Christodoulou, J (2008).
Potential of AAV vectors in the treatment of metabolic disease. Gene Ther 15:
831–839.
8. Hasbrouck, NC and High, KA (2008). AAV-mediated gene transfer for the treatment
of hemophilia B: problems and prospects. Gene Ther 15: 870–875.
9. Daniel, R and Smith, JA (2008). Integration site selection by retroviral vectors:
molecular mechanism and clinical consequences. Hum Gene Ther 19: 557–568.
10. Zhang, JS, Liu, F and Huang, L (2005). Implications of pharmacokinetic behavior of
lipoplex for its inflammatory toxicity. Adv Drug Deliv Rev 57: 689–698.
11. Jiang, H, Couto, LB, Patarroyo-White, S, Liu, T, Nagy, D, Vargas, JA et al. (2006). Effects
of transient immunosuppression on adenoassociated, virus-mediated, liver-directed
gene transfer in rhesus macaques and implications for human gene therapy. Blood
108: 3321–3328.
12. Wang, Z, Kuhr, CS, Allen, JM, Blankinship, M, Gregorevic, P, Chamberlain, JS et al.
(2007). Sustained AAV-mediated dystrophin expression in a canine model of
Duchenne muscular dystrophy with a brief course of immunosuppression. Mol Ther
15: 1160–1166.
13. Li, JZ, Li, H, Hankins, GR, Dunford, B and Helm, GA (2005). Local immunomodulation
with CD4 and CD8 antibodies, but not cyclosporine A, improves osteogenesis
induced by ADhBMP9 gene therapy. Gene Ther 12: 1235–1241.
14. Zaiss, AK and Muruve, DA (2005). Immune responses to adeno-associated viral
vectors. Curr Gene Ther 5: 323–331.
15. Lufino, MM, Edser, PA and Wade-Martins, R (2008). Advances in high-capacity
extrachromosomal vector technology: episomal maintenance, vector delivery, and
transgene expression. Mol Ther 16: 1525–1538.
16. Taylor, N, Uribe, L, Smith, S, Jahn, T, Kohn, DB and Weinberg, K (1996). Correction of
interleukin-2 receptor function in X-SCID lymphoblastoid cells by retrovirally mediated
transfer of the γ-c gene. Blood 87: 3103–3107.
17. Smith, AJ, Schlichtenbrede, FC, Tschernutter, M, Bainbridge, JW, Thrasher, AJ and
Ali, RR (2003). AAV-mediated gene transfer slows photoreceptor loss in the RCS rat
model of retinitis pigmentosa. Mol Ther 8: 188–195.
18. Oh, YK, Sohn, T, Park, JS, Kang, MJ, Choi, HG, Kim, JA et al. (2004). Enhanced mucosal
and systemic immunogenicity of human papillomavirus-like particles encapsidating
interleukin-2 gene adjuvant. Virology 328: 266–273.
19. Williams, DA (2007). RAC reviews serious adverse event associated with AAV therapy
trial. Mol Ther 15: 2053–2054.
20. Irshad, M, Joshi, YK, Sharma, Y and Dhar, I (2006). Transfusion transmitted virus: a
review on its molecular characteristics and role in medicine. World J Gastroenterol 12:
5122–5134.
21. Fadok, VA, Voelker, DR, Campbell, PA, Cohen, JJ, Bratton, DL and Henson, PM (1992).
Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers
specific recognition and removal by macrophages. J Immunol 148: 2207–2216.
22. Valadi, H, Ekström, K, Bossios, A, Sjöstrand, M, Lee, JJ and Lötvall, JO (2007).
Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of
genetic exchange between cells. Nat Cell Biol 9: 654–659.
23. Castagliuolo, I, Beggiao, E, Brun, P, Barzon, L, Goussard, S, Manganelli, R et al. (2005).
Engineered E. coli delivers therapeutic genes to the colonic mucosa. Gene Ther 12:
1070–1078.
24. Grillot-Courvalin, C, Goussard, S, Huetz, F, Ojcius, DM and Courvalin, P (1998).
Functional gene transfer from intracellular bacteria to mammalian cells. Nat Biotechnol
16: 862–866.
25. Stritzker, J, Pilgrim, S, Szalay, AA and Goebel, W (2008). Prodrug converting enzyme
gene delivery by L. monocytogenes. BMC Cancer 8: 94.
26. Akin, D, Sturgis, J, Ragheb, K, Sherman, D, Burkholder, K, Robinson, JP et al. (2007).
Bacteria-mediated delivery of nanoparticles and cargo into cells. Nat Nanotechnol 2:
441–449.
27. Souders, NC, Verch, T and Paterson, Y (2006). In vivo bactofection: Listeria can
function as a DNA-cancer vaccine. DNA Cell Biol 25: 142–151.
28. Shen, H, Kanoh, M, Liu, F, Maruyama, S and Asano, Y (2004). Modulation of the
immune system by Listeria monocytogenes-mediated gene transfer into mammalian
cells. Microbiol Immunol 48: 329–
337.
29. Abdul-Wahid, A and Faubert, G (2007). Mucosal delivery of a transmission-blocking
DNA vaccine encoding Giardia lamblia CWP2 by Salmonella typhimurium bactofection
vehicle. Vaccine 25: 8372–8383.
30. Zhao, M, Geller, J, Ma, H, Yang, M, Penman, S and Hoffman, RM (2007). Monotherapy
with a tumor-targeting mutant of Salmonella typhimurium cures orthotopic metastatic
mouse models of human prostate cancer. Proc Natl Acad Sci USA 104: 10170–10174.
31. Pawelek, JM, Low, KB and Bermudes, D (1997). Tumor-targeted Salmonella as a novel
anticancer vector. Cancer Res 57: 4537–4544.
32. Toso, JF, Gill, VJ, Hwu, P, Marincola, FM, Restifo, NP, Schwartzentruber, DJ et al. (2002).
Phase I study of the intravenous administration of attenuated Salmonella typhimurium
to patients with metastatic melanoma. J Clin Oncol 20: 142–152.
33. Nemunaitis, J, Cunningham, C, Senzer, N, Kuhn, J, Cramm, J, Litz, C et al. (2003).
Pilot trial of genetically modified, attenuated Salmonella expressing the E. coli cytosine
deaminase gene in refractory cancer patients. Cancer Gene Ther 10: 737–744.
34. Shiau, AL, Chen, CC, Yo, YT, Chu, CY, Wang, SY and Wu, CL (2005). Enhancement
of humoral and cellular immune responses by an oral Salmonella choleraesuis vaccine
expressing porcine prothymosin α. Vaccine 23: 5563–5571.
35. Hamaji, Y, Fujimori, M, Sasaki, T, Matsuhashi, H, Matsui-Seki, K, Shimatani-Shibata, Y
et al. (2007). Strong enhancement of recombinant cytosine deaminase activity in
Bifidobacterium longum for tumor-targeting enzyme/prodrug therapy. Biosci Biotechnol
Biochem 71: 874–883.
36. Larsen, MD, Griesenbach, U, Goussard, S, Gruenert, DC, Geddes, DM, Scheule, RK
et al. (2008). Bactofection of lung epithelial cells in vitro and in vivo using a genetically
modified Escherichia coli. Gene Ther 15: 434–442.
37. Xiang, S, Fruehauf, J and Li, CJ (2006). Short hairpin RNA-expressing bacteria elicit
RNA interference in mammals. Nat Biotechnol 24: 697–702.
38. Celec, P, Gardlík, R, Pálffy, R, Hodosy, J, Stuchlík, S, Drahovská, H et al. (2005). The use
of transformed Escherichia coli for experimental angiogenesis induced by regulated
in situ production of vascular endothelial growth factor—an alternative gene therapy.
Med Hypotheses 64: 505–511.
39. Schoen, C, Kolb-Mäurer, A, Geginat, G, Löffler, D, Bergmann, B, Stritzker, J et al.
(2005). Bacterial delivery of functional messenger RNA to mammalian cells. Cell
Microbiol 7: 709–724.
40. Scheule, RK (2000). The role of CpG motifs in immunostimulation and gene therapy.
Adv Drug Deliv Rev 44: 119–134.
41. Pannell, D and Ellis, J (2001). Silencing of gene expression: implications for design of
retroviral vectors. Rev Med Virol 11: 205–2017.
42. Birmingham, CL, Canadien, V, Kaniuk, NA, Steinberg, BE, Higgins, DE and Brumell, JH
(2008). Listeriolysin O allows Listeria monocytogenes replication in macrophage
vacuoles. Nature 451: 350–354.
43. Yu, YA, Shabahang, S, Timiryasova, TM, Zhang, Q, Beltz, R, Gentschev, I et al. (2004).
Visualization of tumors and metastases in live animals with bacteria and vaccinia virus
encoding light-emitting proteins. Nat Biotechnol 22: 313–320.
44. Zelmer, A, Krusch, S, Koschinski, A, Rohde, M, Repp, H, Chakraborty, T et al. (2005).
Functional transfer of eukaryotic expression plasmids to mammalian cells by Listeria
monocytogenes: a mechanistic approach. J Gene Med 7: 1097–1112.

776 www.moleculartherapy.org vol. 17 no. 5 may 2009
© The American Society of Gene Therapy
Biological Gene Delivery Vehicles
45. Hyde, SC, Pringle, IA, Abdullah, S, Lawton, AE, Davies, LA, Varathalingam, A et al.
(2008). CpG-free plasmids confer reduced inflammation and sustained pulmonary
gene expression. Nat Biotechnol 26: 549–551.
46. Artis, D (2008). Epithelial-cell recognition of commensal bacteria and maintenance of
immune homeostasis in the gut. Nat Rev Immunol 8: 411–420.
47. Chatel, JM, Pothelune, L, Ah-Leung, S, Corthier, G, Wal, JM and Langella, P (2008).
In vivo transfer of plasmid from food-grade transiting lactococci to murine epithelial
cells. Gene Ther 15: 1184–1190.
48. Tlaskalová-Hogenová, H, Stepánková, R, Hudcovic, T, Tucková, L, Cukrowska, B,
Lodinová-Zádníková, R et al. (2004). Commensal bacteria (normal microflora),
mucosal immunity and chronic inflammatory and autoimmune diseases. Immunol Lett
93: 97–108.
49. Brüssow, H, Fremont, M, Bruttin, A, Sidoti, J, Constable, A and Fryder, V (1994).
Detection and classification of Streptococcus thermophilus bacteriophages isolated
from industrial milk fermentation. Appl Environ Microbiol 60: 4537–4543.
50. Jepson, CD and March, JB (2004). Bacteriophage lambda is a highly stable DNA
vaccine delivery vehicle. Vaccine 22: 2413–2419.
51. Larocca, D, Jensen-Pergakes, K, Burg, MA and Baird, A (2001). Receptor-targeted
gene delivery using multivalent phagemid particles. Mol Ther 3: 476–484.
52. Lundstrom, K (2003). Latest development in viral vectors for gene therapy. Trends
Biotechnol 21: 117–122.
53. Chauthaiwale, VM, Therwath, A and Deshpande, VV (1992). Bacteriophage lambda as
a cloning vector. Microbiol Rev 56: 577–591.
54. Greenstein, D and Brent, R (2001). Introduction to vectors derived from filamentous
phages. Curr Protoc Mol Biol. Chapter 1:Unit1.14.
55. Kaneda, Y, Saeki, Y, Nakabayashi, M, Zhou, WZ, Kaneda, MW and Morishita, R (2000).
Enhancement of transgene expression by cotransfection of oriP plasmid with EBNA-1
expression vector. Hum Gene Ther 11: 471–479.
56. Burg, MA, Jensen-Pergakes, K, Gonzalez, AM, Ravey, P, Baird, A and Larocca, D
(2002). Enhanced phagemid particle gene transfer in camptothecin-treated carcinoma
cells. Cancer Res 62: 977–981.
57. Poul, MA and Marks, JD (1999). Targeted gene delivery to mammalian cells by
filamentous bacteriophage. J Mol Biol 288: 203–211.
58. Larocca, D, Witte, A, Johnson, W, Pierce, GF and Baird, A (1998). Targeting
bacteriophage to mammalian cell surface receptors for gene delivery. Hum Gene Ther
9: 2393–2399.
59. Mount, JD, Samoylova, TI, Morrison, NE, Cox, NR, Baker, HJ and Petrenko, VA (2004).
Cell targeted phagemid rescued by preselected landscape phage. Gene 341: 59–65.
60. Piersanti, S, Cherubini, G, Martina, Y, Salone, B, Avitabile, D, Grosso, F et al. (2004).
Mammalian cell transduction and internalization properties of lambda phages
displaying the full-length adenoviral penton base or its central domain. J Mol Med 82:
467–476.
61. Molenaar, TJ, Michon, I, de Haas, SA, van Berkel, TJ, Kuiper, J and Biessen, EA (2002).
Uptake and processing of modified bacteriophage M13 in mice: implications for
phage display. Virology 293: 182–191.
62. Chen, Y, Shen, Y, Guo, X, Zhang, C, Yang, W, Ma, M et al. (2006). Transdermal protein
delivery by a coadministered peptide identified via phage display. Nat Biotechnol 24:
455–460.
63. Geier, MR, Trigg, ME and Merril, CR (1973). Fate of bacteriophage lambda in non-
immune germ-free mice. Nature 246: 221–223.
64. Merril, CR, Biswas, B, Carlton, R, Jensen, NC, Creed, GJ, Zullo, S et al. (1996).
Long-circulating bacteriophage as antibacterial agents. Proc Natl Acad Sci USA 93:
3188–3192.
65. Reynaud, A, Cloastre, L, Bernard, J, Laveran, H, Ackermann, HW, Licois, D et al.
(1992). Characteristics and diffusion in the rabbit of a phage for Escherichia coli 0103.
Attempts to use this phage for therapy. Vet Microbiol 30: 203–212.
66. Shearer, WT, Lugg, DJ, Rosenblatt, HM, Nickolls, PM, Sharp, RM, Reuben, JM et al.
(2001). Antibody responses to bacteriophage ϕX-174 in human subjects exposed to
the Antarctic winter-over model of spaceflight. J Allergy Clin Immunol 107: 160–164.
67. Ochs, HD, Davis, SD and Wedgwood, RJ (1971). Immunologic responses to
bacteriophage ΦX174 in immunodeficiency diseases. J Clin Invest 50: 2559–2568.
68. Bundy, BC, Franciszkowicz, MJ and Swartz, JR (2008). Escherichia coli-based cell-free
synthesis of virus-like particles. Biotechnol Bioeng 100: 28–37.
69. Wu, M, Sherwin, T, Brown, WL and Stockley, PG (2005). Delivery of antisense
oligonucleotides to leukemia cells by RNA bacteriophage capsids. Nanomedicine 1:
67–76.
70. Chen, XS, Casini, G, Harrison, SC and Garcea, RL (2001). Papillomavirus capsid
protein expression in Escherichia coli: purification and assembly of HPV11 and HPV16
L1. J Mol Biol 307: 173–182.
71. Huang, Z, Elkin, G, Maloney, BJ, Beuhner, N, Arntzen, CJ, Thanavala, Y et al. (2005).
Virus-like particle expression and assembly in plants: hepatitis B and Norwalk viruses.
Vaccine 23: 1851–1858.
72. Garnier, L, Ravallec, M, Blanchard, P, Chaabihi, H, Bossy, JP, Devauchelle, G et al.
(1995). Incorporation of pseudorabies virus gD into human immunodeficiency
virus type 1 gag particles produced in baculovirus-infected cells. J Virol 69:
4060–4068.
73. O’Neal, CM, Crawford, SE, Estes, MK and Conner, ME (1997). Rotavirus virus-like
particles administered mucosally induce protective immunity. J Virol 71: 8707–8717.
74. Sedlik, C, Saron, M-F, Sarraseca, J, Casal, I and Leclerc, C (1997). Recombinant
parvovirus-like particles as an antigen carrier: a novel nonreplicative exogenous
antigen to elicit protective antiviral cytotoxic T cells. Proc Natl Acad Sci USA 94:
7503–7508.
75. Guerrero, RA, Ball, JM, Krater, SS, Pacheco, SE, Clements, JD and Estes, MK (2001).
Recombinant Norwalk virus-like particles administered intranasally to mice induce
systemic and mucosa (fecal and vaginal) immune responses. J Virol 75: 9713–9722.
76. Zhang, H, Fayad, R, Wang, X, Quinn, D and Qiao, L (2004). Human
immunodeficiency virus type 1 gag-specific mucosal immunity after oral immunization
with papillomavirus pseudoviruses encoding gag. J Virol 78: 10249–10257.
77. Touzé, A, Bousarghin, L, Ster, C, Combita, AL, Roingeard, P and Coursaget, P (2001).
Gene transfer using human polyomavirus BK virus-like particles expressed in insect
cells. J Gen Virol 82: 3005–3009.
78. Yamada, T, Iwasaki, Y, Tada, H, Iwabuki, H, Chuah, MK, VandenDriessche, T et al.
(2003). Nanoparticles for the delivery of genes and drugs to human hepatocytes. Nat
Biotechnol 21: 885–90.
79. Iwasaki, Y, Ueda, M, Yamada, T, Kondo, A, Seno, M, Tanizawa, K et al. (2007). Gene
therapy of liver tumors with human liver-specific nanoparticles. Cancer Gene Ther 14:
74–81.
80. Sakuragi, S, Goto, T, Sano, K and Morikawa, Y (2002). HIV type 1 Gag virus-like
particle budding from spheroplasts of Saccharomyces cerevisiae. Proc Natl Acad Sci
USA 99: 7956–7961.
81. Wang, M, Tsou, TH, Chen, LS, Ou, WC, Chen, PL, Chang, CF et al. (2004). Inhibition
of simian virus 40 large tumor antigen expression in human fetal glial cells by an
antisense oligodeoxynucleotide delivered by the JC virus-like particle. Hum Gene Ther
15: 1077–1090.
82. Touze, A and Coursaget, P (1998). In vitro gene transfer using human papillomavirus-
like particles. Nucleic Acids Res 26: 1317–1323.
83. Pattenden, LK, Middelberg, AP, Niebert, M and Lipin, DI (2005). Towards the
preparative and large-scale precision manufacture of virus-like particles. Trends
Biotechnol 23: 523–529.
84. Fayad, R, Zhang, H, Quinn, D, Huang, Y and Qiao, L (2004). Oral administration with
papillomavirus pseudovirus encoding IL-2 fully restores mucosal and systemic immune
responses to vaccinations in aged mice. J Immunol 173: 2692–2698.
85. Shi, W, Liu, J, Huang, Y and Qiao, L (2001). Papillomavirus pseudovirus: a novel
vaccine to induce mucosal and systemic cytotoxic T-lymphocyte responses. J Virol 75:
10139–10148.
86. Malboeuf, CM, Simon, DA, Lee, YE, Lankes, HA, Dewhurst, S, Frelinger, JG et al.
(2007). Human papillomavirus-like particles mediate functional delivery of plasmid
DNA to antigen presenting cells in vivo. Vaccine 25: 3270–3276.
87. Brandenburg, B, Stockl, L, Gutzeit, C, Roos, M, Lupberger, J, Schwartlander, R et al.
(2005). A novel system for efficient gene transfer into primary human hepatocytes via
cell-permeable hepatitis B virus-like particle. Hepatology 42: 1300–1309.
88. Takamura, S, Niikura, M, Li, TC, Takeda, N, Kusagawa, S, Takebe, Y et al. (2004). DNA
vaccine-encapsulated virus-like particles derived from an orally transmissible virus
stimulate mucosal and systemic immune responses by oral administration. Gene Ther
11: 628–635.
89. Gleiter, S and Lilie, H (2003). Cell-type specific targeting and gene expression using a
variant of polyoma VP1 virus-like particles. Biol Chem 384: 247–255.
90. May, T, Gleiter, S and Lilie, H (2002). Assessment of cell type specific gene transfer of
polyoma virus like particles presenting a tumor specific antibody Fv fragment. J Virol
Methods 105: 147–157.
91. Clark, B, Caparrós-Wanderley, W, Musselwhite, G, Kotecha, M and Griffin, BE (2001).
Immunity against both polyomavirus VP1 and a transgene product induced following
intranasal delivery of VP1 pseudocapsid-DNA complexes. J Gen Virol 82: 2791–2797.
92. Krauzewicz, N, Stokrová, J, Jenkins, C, Elliott, M, Higgins, CF and Griffin, BE (2000).
Virus-like gene transfer into cells mediated by polyoma virus pseudocapsids. Gene Ther
7: 2122–2131.
93. Krauzewicz, N, Cox, C, Soeda, E, Clark, B, Rayner, S and Griffin, BE (2000). Sustained
ex vivo and in vivo transfer of a reporter gene using polyoma virus pseudocapsids.
Gene Ther 7: 1094–1102.
94. Luo, L, Li, Y, Cannon, PM, Kim, S and Kang, CY (1992). Chimeric gag-V3 virus-like
particles of human immunodeficiency virus induce virus-neutralizing antibodies. Proc
Natl Acad Sci USA 89: 10527–10531.
95. Eto, Y, Yoshioka, Y, Mukai, Y, Okada, N and Nakagawa, S (2008). Development of
PEGylated adenoviral vector with targeting ligand. Int J Pharm 354: 3–8.
96. Byun, HM, Suh, D, Yoon, H, Kim, JM, Choi, HG, Kim, WK et al. (2004). Erythrocyte
ghost-mediated gene delivery for prolonged and blood-targeted expression. Gene
Ther 11: 492–496.
97. Magnani, M, Casabianca, A, Fraternale, A, Brandi, G, Gessani, S, Williams, R et al.
(1996). Synthesis and targeted delivery of an azidothymidine homodinucleotide
conferring protection to macrophages against retroviral infection. Proc Natl Acad Sci
USA 93: 4403–4408.
98. Mishra, PR and Jain, NK (2003). Folate conjugated doxorubicin-loaded membrane
vesicles for improved cancer therapy. Drug Deliv 10: 277–282.
99. Magnani, M, Rossi, L, Fraternale, A, Bianchi, M, Antonelli, A, Crinelli, R et al. (2002).
Erythrocyte-mediated delivery of drugs, peptides and modified oligonucleotides. Gene
Ther 9: 749–751.
100. Chiarantini, L, Cerasi, A, Fraternale, A, Andreoni, F, Scarí, S, Giovine, M et al. (2002).
Inhibition of macrophage iNOS by selective targeting of antisense PNA. Biochemistry
41: 8471–8477.
101. Doberstein, SK, Wiegand, G, Machesky, LM and Pollard, TD (1995). Fluorescent
erythrocyte ghosts as standards for quantitative flow cytometry. Cytometry 20: 14–18.
102. Magnani, M, Rossi, L, D’ascenzo, M, Panzani, I, Bigi, L and Zanella, A (1998).
Erythrocyte engineering for drug delivery and targeting. Biotechnol Appl Biochem 28
(Pt 1): 1–6.
103. Vanderlinde, ES, Heal, JM and Blumberg, N (2002). Autologous transfusion. BMJ 324:
772–775.
104. Gressner, OA, Lahme, B, Koch, M and Gressner, AM (2008). Evaluation of
hepatotropic targeting properties of allogenic and xenogenic erythrocyte ghosts in
normal and liver-injured rats. Liver Int 28: 220–
232.
105. Lucidi, V, Tozzi, AE, Bella, S and Turchetta, A (2006). A pilot trial on safety and efficacy
of erythrocyte-mediated steroid treatment in CF patients. BMC Pediatr 6: 17.
106. Updike, SJ and Wakamiya, RT (1982). Infusion of red blood cell-loaded asparaginase in
monkey. Immunologic, metabolic, and toxicologic consequences. J Lab Clin Med 101:
679–691.
107. Erchler, HG, Gasic, S, Bauer, K, Korn, A and Bacher, S (1986). In vivo clearance of
antibody-sensitized human drug carrier erythrocytes. Clin Pharmacol Ther 40: 300–303.

Molecular erapy vol. 17 no. 5 may 2009 777
© The American Society of Gene Therapy
Biological Gene Delivery Vehicles
108. Magnani, M, Rossi, L, Brandi, G, Schiavano, GF, Montroni, M and Piedimonte, G
(1992). Targeting antiretroviral nucleoside analogues in phosphorylated form to
macrophages: in vitro and in vivo studies. Proc Natl Acad Sci USA 89: 6477–6481.
109. Chiarantini, L, Rossi, L, Fraternale, A and Magnani, M (1995). Modulated red blood
cell survival by membrane protein clustering. Mol Cel. Biochem 144: 53–59.
110. Lu, SJ, Feng, Q, Park, JS, Vida, L, Lee, BS, Strausbauch, M et al. (2008). Biological
properties and enucleation of red blood cells from human embryonic stem cells. Blood
112: 4475–4484.
111. Kim, SH, Bianco, N, Menon, R, Lechman, ER, Shufesky, WJ, Morelli, AE et al. (2006).
Exosomes derived from genetically modified DC expressing FasL are anti-inflammatory
and immunosuppressive. Mol Ther 13: 289–300.
112. Kim, SH, Lechman, ER, Bianco, N, Menon, R, Keravala, A, Nash, J et al. (2005).
Exosomes derived from IL-10-treated dendritic cells can suppress inflammation and
collagen-induced arthritis. J Immunol 174: 6440–6448.
113. Smalheiser, NR (2007). Exosomal transfer of proteins and RNAs at synapses in the
nervous system. Biol Direct 2: 35.
114. Park, IH, Arora, N, Huo, H, Maherali, N, Ahfeldt, T, Shimamura, A et al. (2008).
Disease-specific induced pluripotent stem cells. Cell 134: 877–886.
115. Tjia, ST, zu Altenschildesche GM and Doerfler, W (1983). Autographa californica
nuclear polyhedrosis virus (AcNPV) DNA does not persist in mass cultures of
mammalian cells. Virology 125: 107–117.
116. Shoji, I, Aizaki, H, Tani, H, Ishii, K, Chiba, T, Saito, I et al. (1997). Efficient gene transfer
into various mammalian cells, including non-hepatic cells, by baculoviral vectors.
J Gen Virol 78: 2657–2664.
117. Hofmann, C and Strauss, M (1998). Baculovirus-mediated gene transfer in the
presence of human serum or blood facilitated by inhibition of the complement
system. Gene Ther 5: 531–536.
118. Hofmann, C, Hüser, A, Lehnert, W and Strauss, M (1999). Protection of baculovirus-
vectors against complement-mediated inactivation by recombinant soluble
complement receptor type 1. Biol Chem 380: 393–395.
119. Hüser, A, Rudolph, M and Hofmann, C (2001). Incorporation of decay-accelerating
factor into the baculovirus envelope generates complement-resistant gene transfer
vectors. Nat Biotechnol 19: 451–455.
120. Sarkis, C, Serguera, C, Petres, S, Buchet, D, Ridet, JL, Edelman, L et al. (2000). Efficient
transduction of neural cells in vitro and in vivo by a baculovirus-derived vector. Proc
Natl Acad Sci USA 97: 14638–14643.
121. Abe, T, Takahashi, H, Hamazaki, H, Miyano-Kurosaki, N, Matsuura, Y and Takaku, H
(2003). Baculovirus induces an innate immune response and confers protection from
lethal influenza virus infection in mice. J Immunol 171: 1133–1139.
122. Gronowski, AM, Hilbert, DM, Sheehan, KC, Garotta, G and Schreiber, RD (1999).
Baculovirus stimulates antiviral effects in mammalian cells. J Virol 73: 9944–9951.
123. Kitajima, M, Abe, T, Miyano-Kurosaki, N, Taniguchi, M, Nakayama, T and
Takaku, H (2008). Induction of natural killer cell-dependent antitumor immunity
by the Autographa californica multiple nuclear polyhedrosis virus. Mol Ther 16:
261–268.
124. Stubbs, AC, Martin, KS, Coeshott, C, Skaates, SV, Kuritzkes, DR, Bellgrau, D et al.
(2001). Whole recombinant yeast vaccine activates dendritic cells and elicits
protective cell-mediated immunity. Nat Med 7: 625–629.
125. Lutz, MB and Schuler, G (2002). Immature, semi-mature and fully mature
dendritic cells: which signals induce tolerance or immunity? Trends Immunol 23:
445–449.
126. Coura Rdos, S and Nardi, NB (2007). The state of the art of adeno-associated virus-
based vectors in gene therapy. Virol J 4: 99.
127. Rivière, C, Danos, O and Douar, AM (2006). Long-term expression and repeated
administration of AAV type 1, 2 and 5 vectors in skeletal muscle of immunocompetent
adult mice. Gene Ther 13: 1300–1308.
128. Hacein-Bey-Abina, S, Garrigue, A, Wang, GP, Soulier, J, Lim, A, Morillon, E et al.
(2008). Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of
SCID-X1. J Clin Invest 118: 3132–3142.
129. Ellis, J (2005). Silencing and variegation of gammaretrovirus and lentivirus vectors.
Hum Gene Ther 16: 1241–1246.
130. Hartman, ZC, Appledorn, DM and Amalfitano, A (2008). Adenovirus vector induced
innate immune responses: impact upon efficacy and toxicity in gene therapy and
vaccine applications. Virus Res 132: 1–14.
131. Ishida, T, Masuda, K, Ichikawa, T, Ichihara, M, Irimura, K and Kiwada, H (2003).
Accelerated clearance of a second injection of PEGylated liposomes in mice. Int J
Pharm 255: 167–174.
132. Sakurai, H, Kawabata, K, Sakurai, F, Nakagawa, S and Mizuguchi, H (2008). Innate
immune response induced by gene delivery vectors. Int J Pharm 354: 9–15.
133. Sellins, K, Fradkin, L, Liggitt, D and Dow, S (2005). Type I interferons potently
suppress gene expression following gene delivery using liposome-DNA complexes.
Mol Ther 12: 451–459.