ArticlePDF AvailableLiterature Review

Abstract

Background: Bacteriophages (bacterial viruses) have long been under investigation as vectors for gene therapy. Similar to other viral vectors, the phage coat proteins have evolved over millions of years to protect the viral genome from degradation post injection, offering protection for the valuable therapeutic sequence. Materials and methods: However, what sets phage apart from other viral gene delivery vectors is their safety for human use and the relative ease by which foreign molecules can be expressed on the phage outer surface, enabling highly targeted gene delivery. The latter property also makes phage a popular choice for gene therapy target discovery through directed evolution. Although promising, phage-mediated gene therapy faces several outstanding challenges, the most notable being lower gene delivery efficiency compared to animal viruses, vector stability, and nondesirable immune stimulation. Result: This review presents a critical review of promises and challenges of employing phage as gene delivery vehicles as well as an introduction to the concept of phage-based microbiome therapy as the new frontier and perhaps the most promising application of phage-based gene therapy.
Send Orders for Reprints to reprints@benthamscience.ae
Current Gene Therapy, 2017, 17, 000-000 1
REVIEW ARTICLE
1566-5232/17 $58.00+.00 © 2017 Bentham Science Publishers
Phage-Mediated Gene Therapy
Zeinab Hosseinidoust*
Department of Chemical Engineering, Faculty of Engineering, McMaster University, Hamilton, Canada
A R T I C L E H I S T O R Y
Received: April 04, 2017
Revised: May 02, 2017
Accepted: May 02, 2017
DOI:
10.2174/1566523217666170510151940
Abstract: Background: Bacteriophages (bacterial viruses) have long been under investigation as vec-
tors for gene therapy. Similar to other viral vectors, the phage coat proteins have evolved over millions
of years to protect the viral genome from degradation post injection, offering protection for the valu-
able therapeutic sequence.
Material and Methods: However, what sets phage apart from other viral gene delivery vectors is their
safety for human use and the relative ease by which foreign molecules can be expressed on the phage
outer surface, enabling highly targeted gene delivery. The latter property also makes phage a popular
choice for gene therapy target discovery through directed evolution. Although promising, phage-
mediated gene therapy faces several outstanding challenges, the most notable being lower gene deliv-
ery efficiency compared to animal viruses, vector stability, and nondesirable immune stimulation.
Result: This review presents a critical review of promises and challenges of employing phage as gene
delivery vehicles as well as an introduction to the concept of phage-based microbiome therapy as the
new frontier and perhaps the most promising application of phage-based for gene therapy.
Keywords: Microbiome therapy, DNA vaccines, Phage therapy, Bionanoparticles, Lysogeny.
1. INTRODUCTION ADD MORE CITATIONS
Gene therapy, the use of genetic material (DNA or RNA)
for treating or prevention of disease, holds great promise for
treating genetic disorders, [1] cardiovascular disease, [2] and
cancer [3]. More than 60% of clinical trials of gene therapy
to date have focused on cancer treatment, followed by
monogenic disease, infectious disease and cardiovascular
disease, each holding a share of close to 10% [4]. Further-
more, in the light of the new discoveries connecting various
aspect of our physical and mental health to the balance in the
collection of microorganisms that reside in our bodies (i.e.,
our microbiota), gene therapy is the center of attention for a
new therapeutic approach that targets dysbiosis in human
microbiota, namely microbiome therapy [5].
The efficacy of gene therapy depends to a large extent on
the efficiency of the vehicle used to introduce the desired
genetic materials into cells. Development of gene carriers or
vectors that can protect the gene of interest against harsh
environmental factors in the body (pH, nucleases), efficiently
transport the gene across all barriers external and internal to
the cell nucleus, and do so in a selective and targeted man-
ner, is the most important challenge of gene therapy.
Most applications of gene therapy to date have involved
the introduction of a therapeutic genetic payload into mam-
malian cells. The vectors utilized for introducing genetic
*Address correspondence to this author at the Department of Chemical
Engineering, Faculty of Engineering, McMaster University, Hamilton, On-
tario, Canada; Tel/Fax: +1 (905)5259140; E-mail: doust@mcmaster.ca
material into mammalian cells can be broadly categorized
into viral and non-viral.
Viral vectors mainly employ replication-deficient hu-
man/animal viruses such as those derived from adenoviruses,
adeno-associated viruses (AAV), [6] retroviruses, [7] lentivi-
ruses, [8] and herpes simplex viruses (HSV) [9]. Viruses
have been customized by evolution over millions of years to
effectively and selectively transfer their genes into their host
cells. Therefore, utilizing eukaryotic viruses is the most sure-
fire way to introduce high loads of therapeutic nucleic acid
into mammalian cells, which explains why close to 70% of
all clinical trials to date have utilized viral vectors [4]. Viral
vectors further offer the advantage of being customizable
through recombinant DNA technology, which means that
their surfaces can be decorated with the desired pro-
teins/peptides that add to the therapeutic function or allow
for more selectivity/targeted action [10]. The use of hu-
man/animal viruses as vectors for therapeutic gene delivery,
however, is hindered by serious concerns about immuno-
genicity and mutagenesis [11-13] with major concerns re-
garding oncogenic outcomes [14]. Furthermore, most viral
vectors are replication deficient and thus require the use of
helper viruses for propagation; the helper virus can thus con-
taminate the vector preparations, further adding to the safety
concerns of viral vectors [15]. Although methods have been
proposed for the production of self-inactivating helper vi-
ruses, [16] these methods require additional steps that are
lengthy and laborious and will still require purification of the
final vector stock.
2 Current Gene Therapy, 2017, Vol. 17, No. 1 Zeinab Hosseinidoust
The outstanding limitations of viral vectors can be allevi-
ated by using non-viral means of gene delivery, e.g.,
nanoparticles, polyelectrolytes, or transfections agents. Non-
viral methods can be designed to be non-immunogenic and
there are no outstanding concerns regarding oncogenic out-
comes [17]. Moreover, using non-viral vehicles partially
bypasses the limitation with gene size (one of the limitations
with viral vectors [18]). However, the efficiency of such
systems is limited in vivo compared to viral vectors, which
have been designed by nature to deliver genes into eukary-
otic cells, and thus large quantities of DNA are required to
achieve high efficiencies.
Bacteriophage-based gene delivery offers the best of both
worlds: harmless vectors that offer gene protection and are
customizable to enable targeted delivery. Bacteriophages (or
phages) are virus that infects bacteria and are the most abun-
dant biological entities on earth. Phages naturally exist in all
niches of the biosphere, including the human body, and rep-
licate by infecting their host bacterial cell. As bacteria’s
natural predators, bacteriophages have been customized by
evolution to infect bacterial cells, which they can do so with
high specificity, and lack the machinery to infect eukaryotic
cells. This characteristic is advantageous for gene delivery
because it ensures that the delivery vector is safe for human
use. The same property also significantly limits the effi-
ciency of gene delivery by phage, because they are not
armed with the means to invade and deliver genes to mam-
malian cells. In the following, the advantages and challenges
of using phage for gene delivery are outlined.
2. EXPLOITABLE CHARACTERISTICS OF PHAGE
FOR GENE THERAPY
2.1. History of Safe Human Use
The chief advantage of bacteriophages in the context of
gene delivery is their safety for human use. The discovery of
phage in 1915 played an instrumental role in understanding
the origins of life and the structure of DNA and phage con-
tinues to play an important role in molecular biology to this
day, as exemplified by the recent development of the revolu-
tionary CRISPER/Cas9 technology [19, 20]. Furthermore,
being discovered before penicillin, phage was employed as
the sole antibacterial treatment against infectious disease for
almost 25 years before being overshadowed by antibiotics
[21]. Bacteriophages have a curious history, rich with poli-
tics, personal feuds, and conflicts [22]. Phage therapy, the
use of phage to treat and control bacterial infections, was
abandoned in the West after the 1940s when it was com-
pletely overshadowed by antibiotics, mainly affected by ini-
tial exaggerated claims, lack of understanding of infection
microbiology, and lack of rigour in experimental design and
documentation in the early attempts of phage therapy. How-
ever, for decades after antibiotics were widespread, patients
behind the Iron Curtain were denied access to some of the
best antibiotics developed in the West. As a result, the Soviet
Union invested heavily in the use of bacteriophages to treat
infections. Phage therapy is still widely used in Russia,
Georgia and Poland. Georgia’s Eliava Institute, in particular,
has been the global center of phage expertise for over 80
years and attracts patients from around the world [23].
The dwindling effectiveness of antibiotics against infec-
tious diseases, caused by the development of drug resistance,
is one of the most pressing public health issues of the early
21st century. Mounting concerns about drug resistant patho-
genic bacteria have rekindled the interest in bacteriophages
long after their abandonment in the 1940’s. In the past 30
years, numerous animal case studies [24-29] and human
clinical trials [30-32] have been conducted to modern stan-
dards in both the European Medicines Agency and the
United States Food and Drug Administration (FDA) jurisdic-
tions. One of the most notable recent attempts is a clinical
report form University of California San Diego School of
medicine and the US Navy where intravenous phage injec-
tion was used to treat a complex case of multidrug resistant
infection and save the life of a comatose patient [33]. An-
other notable recent attempt is Phagoburn. Funded by the
European Commission, Phagoburn is the first large, multi-
center clinical trial of phage therapy for human infections
and is currently in the phase II clinical trial stage [34]. It
should be noted that like any external agent, bacteriophage
introduction into the body might stimulate the immune sys-
tem. This immunogenicity, however, has proven to be highly
context dependent, depending strongly on the route of ad-
ministration and they type of phage [35, 36]. Many instances
of phage administration have been reported with negligible
immunogenicity [37]. All in all, bacteriophages have been
used in a therapeutic capacity for a century. Despite the
many unknowns and outstanding challenges in the field of
phage therapy, [38] the long-term use of phages as human
therapeutics provides strong evidence for safety for human
use. Furthermore, the FDA has approved use of phage for
food decontamination, as dietary supplements, and for envi-
ronmental prophylaxis [39]. Adding to all this, the fact that,
in addition to having a rich collection of phages that live
inside our body as part of our microbiota, we consume
phages in our food on a daily basis, provides further proof of
the safety of phage vectors for human use.
2.2. DNA and RNA Protection and Stability
A bacteriophage virion consists of a protein coat that en-
cases a DNA or RNA genome. The protein coat protects the
genetic material (single or double stranded DNA or RNA
molecule) and can further serve as a scaffold for both genetic
modification and chemical modification, adding more func-
tionality to the virion. Bacteriophage virions can be found in
various shapes and sizes, including tailed, filamentous, and
icosahedral (Fig. 1) [40]. The shape and size of the virion
may be a limiting factor for the size of the gene insertion,
which is a limitation phage vectors share with viral vectors.
This concept is again highly dependent on the phage type.
Some virions, such as the filamentous M13, can accommo-
date larger insertions by increasing the virion size (up to a
certain extent). Other, less flexible virions limit the maxi-
mum size of the insertion because they cannot pack the DNA
above a certain limit. This is rooted in the mechanism of
viral assembly. M13 proteins assemble around its genome,
whereas T phages pack the DNA inside the head after the
head has formed, so there is no room for accommodating
oversized genomes [41, 42]. Three phages that have been
well characterized and broadly employed for cargo delivery
are M13 (filamentous phage, 1 µm in length 6-8 nm in di-
Phage-Mediated Ge ne Therapy Current Gene Therapy, 2017, Vol. 17, No. 1 3
ameter, ssDNA genome), [43] lambda (tailed phage, 200 nm
long head diameter 55 nm, dsDNA genome), [44] and MS2
(icosahedral phage, 60 nm in diameter, RNA genome) [45].
Reports also exist of gene delivery using T phages (T7 and
T4, both tailed, dsDNA genome) [46].
The marked advantage of phage compared to other non-
viral gene delivery vectors is its inherent ability to effec-
tively protect RNA. Ribonucleic acids, including antisense
oligonucleotides, small interfering RNA (siRNA), aptamers,
and ribozymes, can interfere with the flow of genetic infor-
mation from DNA to protein and are thus important thera-
peutic tools [47]. RNA, however, is very unstable and must
be protected effectively to lead to efficient therapeutic re-
sults. Using RNA phage such as MS2 has been shown to be
an effective method for RNA delivery [47-49].
Bacteriophages face a range of extra-and intracellular
barriers following administration in to the body, many of
which require them to tolerate relatively harsh nvironemntal
conditions (pH changes and degrading enzyme) [50, 51]
Most phage capsids are stable over a range of pH values and
are able to protect their genome against nuclease degradation
as long as they maintain their integrity. Virion stability under
in vitro intracellular and extracellular environmental condi-
tions, however, is an important design parameter that must
be taken into account and investigated when designing phage
therapeutics.
From the point of view of mass production, storage, and
handling, the stability of bacteriophage vectors has been the
subject of many investigations. Phage Lambda was found to
be stable at 20°C, unaffected by freezing or thawing over a
six month period at 4 and 70°C, whereas freeze-drying
without stabilizers led to 520% residual viability [52]. The
ability of bacteriophages to survive under unfavorable condi-
tions is highly diversified. The reader is referred to the com-
prehensive review by Jończyk et al. for the influence of dif-
ferent external physical and chemical factors, such as tem-
perature, acidity, and ions, on phage persistence [53]. The
stability of phage-based vectors must therefore be investi-
gated on a case-by-case basis with regard to the process that
is used for mass production and purification.
2.3. Targeted Delivery, Target Discovery and Phage Dis-
play
Bacteriophages lack the natural ability to target and trig-
ger uptake in eukaryotic cells. Specific recognition could,
however, be engineered into bacteriophages. Using recombi-
nant DNA technology and novel synthetic biology tools,
bacteriophages can be genetically engineering to express the
desired specific recognition/uptake protein/peptide on its
capsid that will enable targeted recognition and uptake by
eukaryotic cells..
If the sequence of disease biomarker or cell internaliza-
tion triggers is known, it is possible to express the peptide or
protein sequences as fusions to the coat proteins of a bacte-
riophage by cloning the sequence into a suitable region of a
phage surface protein. Sequences such as RGD (specific to
certain integrins overexpressed on cancer cells) [54, 55] or
peptide/protein transduction domains (small cationic pep-
tides called TAT) [56-58] have been extensively used for
designing targeted phage vectors. In another example, M13
phage that was engineered to display fibroblast growth factor
(FGF2) on their surface coat as a fusion to the minor coat
protein, pIII [59]. The phage vector was then successfully
employed to transfect COS-1 cells. It is noteworthy that (1)
the cloned recognition agent must be expressed on the sur-
face of the virion to be available as a recognition moiety, and
(2) the size and crystalline structure of the chosen phage coat
protein will lead to limitations in the size and conformation
of protein/peptide to be expressed on the capsid.
When the target (disease) biomarker is unknown, how-
ever, a discovery step usually precedes designing phage gene
delivery vectors. Bacteriophages are also the star of the show
for the biomarker discovery step, as part of the phage display
technology [60]. Phage display technology is a powerful in
vitro screening technique employed for investigating protein-
protein, protein-peptide, protein-DNA, and protein-mineral
Fig. (1). Schematic representation of major phage groups [22]. RightLink number.
4 Current Gene Therapy, 2017, Vol. 17, No. 1 Zeinab Hosseinidoust
interactions in various contexts, one of which is discovering
disease biomarkers [61]. Libraries of phage-displayed pep-
tides or proteins are created by inserting random DNA se-
quences in the phage genome, inside a sequence that ex-
presses a surface protein. The resulting phage library will
thus express random fusion proteins that are then linked to
their encoding nucleic acid by iterative rounds of in vitro
panning and amplification, followed by DNA sequencing.
Phage display technology has been used since 1990s to dis-
cover disease biomarkers and is a strong tool for designing
targeted gene and drug delivery vehicles using bacterio-
phages [62-65].
Although bacteriophages can be engineered to trigger up-
take by eukaryotic cells, there is no clear consensus on their
fate after uptake. It is believed that some phages, like M13,
degrade in the lysosomes. Others that survive are believed to
degrade in the ribosomes [66]. I have added explanations as
well as references to the text. This is another shortfall com-
pared to viral vectors and one of the reasons for low gene
delivery efficiency of phage vectors; viral vectors can effec-
tively deliver the genetic cargo to the cell nucleolus, but with
phage vectors, the precious genetic payload is not guaranteed
protection after uptake by the eukaryotic cell.
2.4. Modular, Multifunctional Vehicles
Bacteriophages possess a large cloning capacity [67].
Lambda phages, for example, have a cloning capacity of
around 20 kilobase pairs (kbp) of DNA, which is much
higher than the 5 kbp maximum for plasmid DNA vaccines
[68]. This large cloning capacity, combined with the rela-
tively simple phage genome, enables multiple insertions so
that different therapeutic applications plus targeting and pos-
sibly imaging functions to be packed on the same particle.
Multiple insertions are, however, limited by the availability
of surface proteins, the different capacities of each surface
protein for accommodating a foreign peptide, and the pres-
ence of overlapping genes, amongst others. (Fig. 2) presents
a notable example, in which the genome of M13 bacterio-
phage was redesigned to physically separate overlapping
genetic elements, leading to a genome that was especially
amenable to incorporating multiple insertions. Overlaps of
key genetic elements directly couple the coding sequence of
one gene to the coding or regulatory sequence of another,
making it difficult to alter one gene without disrupting the
other. The utility of this refactored genome was further dem-
onstrated by developing a multifunction nanoparticle for
targeted imaging of and drug delivery to prostate cancer cells
[69]. This example demonstrates the significance of techno-
logical and scientific advancements in the past decade, par-
ticularly our ability to make large synthetic DNA molecules,
in realizing the promise of bacteriophages as suitable drug
and gene delivery vectors. In addition to genetic modifica-
tions, chemical modification of the viral capsid can further
add to the toolbox of techniques available for converting
viral capsids into modular carrier systems for drugs/genes
and imaging agents [70, 71]. For example, MS2 vectors have
been reported that carry 5070 copies of a fluorescent mole-
cule in their capsid and are covalently covered with polyeth-
ylene glycol (PEG) chains (to reduce immunogenicity) [72]
or with DNA for targeting [73].
2.5. Facile Mass-Production
Bacteriophages can be mass-produced by infecting their
host bacterial cell. Compared to the method for the produc-
tion of viral vectors, the method for mass production of
phage is simple, fast, and cost effective [74]. Purity of phage
vector preparations from bacterial debris and endotoxins
(lipopolysaccharides, LPS) is critical for biomedical applica-
tions of phage such as, but not limited to, phage therapy.
Methods such as chromatography on Cellulofine Sulfate,
ultrafiltration, and gel filtration have been shown to elimi-
nate traces of endotoxins from final preparations and yield
phage preparations suitable for intravenous administration
[75, 76].
3. PHAGE FOR MICROBIOME THERAPY
One of the most revolutionary recent advances in the bio-
logical sciences has been the realization that the rich micro-
bial communities that occupy our body (i.e., our microbiota)
play a central role in the initiation and progression of human
diseases such as diabetes, [77] cancer, [78] chronic intestinal
conditions, [79] neuronal degeneration, [80] and various
mental disorders [81]. We have further realized that many
medications that save our lives also cause significant pertur-
bations to our microbiota, increasing our susceptibility to
physical and mental diseases [82-85]. Antibiotics, in particu-
lar, are notorious for the long-term damage they can cause to
our microbiota because they blindly wipeout the beneficial
bacteria along with the infection [86, 87]. Microbiome ther-
apy (approaches that aim to restore balance of the micro-
biome for treating a variety of ailments) is a new frontier for
gene therapy with bacteriophages playing the central role.
Efforts to understand and characterize the human microbiota
have shed a new light on the importance of phage, both as a
Fig. (2). Multifunctional phage delivery vector, [47]. RightLink number.
Phage-Mediated Ge ne Therapy Current Gene Therapy, 2017, Vol. 17, No. 1 5
critical component of the microbiota, and as a powerful tool
for engineering the microbiota [88]. As bacteria’s natural
predators, bacteriophages offer a critical advantage over an-
tibiotics, namely that they can be highly specific, targeting
only their host bacteria. This means that phage therapeutics
can be designed to target only the infectious agent(s) and/or
the species that are out of balance, without causing harm to
the microbiota. Bacteriophages possess the inherent ability to
effectively deliver genetic material into bacterial cells and
have evolved over millions of years (and continue to evolve)
to escape the notorious bacterial resistance and restriction
mechanisms. Bacteriophages are broadly categorized into
two groups of lytic or virulent and temperate phage. Virulent
phage delivers its genetic payload to the host cell and leads
to the lysis of the host, whereas temperate phage incorpo-
rates its genome into the host genome, replicating with the
host as a prophage, and existing in a silenced life cycle
within bacteria. The combination of specificity, natural bac-
terial targeting, and (in the case of temperate phage) the abil-
ity to incorporate their genome into the bacterial genome
presents phage as the ideal tool for engineering the microbi-
ota. Both lytic and temperate phage offer promise for micro-
biome therapy; virulent phage can be used to specifically
target and destroy infectious agents or commensal microor-
ganisms that that have out competed their neighbors causing
dysbiosis, and temperate phage can deliver therapeutic genes
that offer select beneficial strains a competitive advantage
over others, thus offering a strong tool for microbiome based
gene therapy [89]. Phages have been used as DNA delivery
agents to reverse antibiotic resistance, [90, 91] or to exert
sequence-specific antimicrobial activity [92, 93]. To be able
to design successful phage-based microbiome therapies, we
need to understand the determining factors that lead to suc-
cessful therapies in the complex ecosystem of our body.
Therefore, in depth knowledge attained from research into
the behavioral ecology of phages (effect of phage on the be-
havior of its bacterial host) in each niche in the body will be
critical for designing effective therapies.
CONCLUSION
In summary, bacteriophages offer several distinct advan-
tages over viral and other non-viral gene delivery vectors,
namely safety for human use, ability to engineer for surface
customization, modularity and multifunctionality. Their
shortfall compared to viral vectors, however, is their rela-
tively low efficiency for gene delivery into eukaryotic cells.
Bacteriophages are designed by nature to deliver their ge-
netic payload to prokaryotic cells and lack the machinery to
internalize eukaryotic cells. This shortfall has been alleviated
to a certain extend through genetic modification. The inherent
ability of bacteriophages to target bacteria, however, is a distinct
advantage for microbiome therapy. Microbiome therapy targets
the collection of microorganisms that live inside the human
body. The natural ability of bacteriophages to target bacteria
with high specificity and to effectively deliver their genetic pay-
load to a bacterial host has enables bacteriophage to outshine
every other agent for microbiome therapy. Microbiome therapy
is thus the new frontier for bacteriophage-based gene therapy.
CONSENT FOR PUBLICATION
Not applicable.
CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flict of interest.
ACKNOWLEDGEMENTS
The author would like to acknowledge NSERC and
McMaster University for funding the research.
REFERENCES
[1] Cavazzana-Calvo, M.; Hacein-Bey, S.; Basile, G.d.S.; Gross, F.;
Yvon, E.; Nusbaum, P.; Selz, F.; Hue, C.; Certain, S.; Casanova, J.-
L.; Bousso, P.; Deist, F.L.; Fischer, A., Gene Therapy of Human
Severe Combined Immunodeficiency (SCID)-X1 Disease. Science,
2000, 288, (5466), 669-672.
[2] Morishita, R.; Higaki, J.; Tomita, N.; Ogihara, T., Application of
Transcription Factor “Decoy” Strategy as Means of Gene Therapy
and Study of Gene Expression in Cardiovascular Disease. Circul.
Res., 1998, 82, (10), 1023-1028.
[3] Cross, D.; Burmester, J.K., Gene Therapy for Cancer Treatment:
Past, Present and Future. Clin. Med. Res., 2006, 4, (3), 218-227.
[4] The Journal of Gene Medicine Clinical Trial site. http://
www.wiley.com/legacy/wileychi/genmed/clinical/, access date:
March 2017
[5] Scarpellini, E.; Ianiro, G.; Attili, F.; Bassanelli, C.; De Santis, A.;
Gasbarrini, A., The human gut microbiota and virome: Potential
therapeutic implications. Dig. Liver Dis., 47, (12), 1007-1012.
[6] Lipshutz, G.S.; Gruber, C.A.; Cao, Y.-a.; Hardy, J.; Contag, C.H.;
Gaensler, K.M.L., In Utero Delivery of Adeno-Associated Viral
Vectors: Intraperitoneal Gene Transfer Produces Long-Term Ex-
pression. Mo l. Ther., 2001, 3, (3), 284-292.
[7] Deregowski, V.; Canalis, E., Gene delivery by retroviruses. Meth-
ods Mol. Biol., 2008, 455, 157-162.
[8] Cockrell, A.S.; Kafri, T., Gene delivery by lentivirus vectors. Mol.
Biotechnol., 2007, 36, (3), 184-204.
[9] Breakefield, X.O.; DeLuca, N.A., Herpes simplex virus for gene
delivery to neurons. The New biologist, 1991, 3, (3), 203-218.
[10] Mateu, M.G., Virus engineering: functionalization and stabiliza-
tion. Protein Eng. Des. Sel., 2011, 24, (1-2), 53-63.
[11] Muruve, D.A.; Barnes, M.J.; Stillman, I.E.; Libermann, T.A., Ade-
noviral gene therapy leads to rapid induction of multiple chemoki-
nes and acute neutrophil-dependent hepatic injury in vivo. Hum.
Gene Ther., 1999, 10, (6), 965-976.
[12] Bowen, G.P.; Borgland, S.L.; Lam, M.; Libermann, T.A.; Wong,
N.C.; Muruve, D.A., Adenovirus vector-induced inflammation:
capsid-dependent induction of the C-C chemokine RANTES re-
quires NF-kappa B. Hum. Gene Ther., 2002, 13, (3), 367-379.
[13] Thaci, B.; Ulasov, I.V.; Wainwright, D.A.; Lesniak, M.S., The
challenge for gene therapy: innate immune response to adenovi-
ruses. Oncotarget, 2011, 2, (3), 113-121.
[14] Sadelain, M., Insertional oncogenesis in gene therapy: how much
of a risk? Gene Ther., 2004, 11, (7), 569-573.
[15] Vetrini, F.; Ng, P., Gene Therapy with Helper-Dependent Adenovi-
ral Vectors: Current Advances and Future Perspectives. Viruses,
2010, 2, (9), 1886-1917.
[16] Gonzalez-Aparicio, M.; Mauleon, I.; Alzuguren, P.; Bunuales, M.;
Gonzalez-Aseguinolaza, G.; San Martin, C.; Prieto, J.; Hernandez-
Alcoceba, R., Self-inactivating helper virus for the production of
high-capacity adenoviral vectors. Gene Ther., 2011, 18, (11), 1025-
1033.
[17] Mishra, S.; Webster, P.; Davis, M.E., PEGylation significantly
affects cellular uptake and intracellular trafficking of non-viral
gene delivery particles. Eur. J. Cell Biol., 2004, 83, (3), 97-111.
[18] Cotten, M.; Wagner, E.; Zatloukal, K.; Phillips, S.; Curiel, D.T.;
Birnstiel, M.L., High-efficiency receptor-mediated delivery of
small and large (48 kilobase gene constructs using the endosome-
disruption activity of defective or chemically inactivated adenovi-
rus particles. Proceedings of the National Academy of Sciences,
1992, 89, (13), 6094-6098.
[19] Barrangou, R.; Fremaux, C.; Deveau, H.; Richards, M.; Boyaval,
P.; Moineau, S.; Romero, D.A.; Horvath, P., CRISPR Provides Ac-
quired Resistance Against Viruses in Prokaryotes. Science, 2007,
315, (5819), 1709-1712.
6 Current Gene Therapy, 2017, Vol. 17, No. 1 Zeinab Hosseinidoust
[20] Horvath, P.; Barrangou, R., CRISPR/Cas, the Immune System of
Bacteria and Archaea. Science, 2010, 327, (5962), 167-170.
[21] Chanishvili, N., 2012; Vol. 83, pp 3-40.
[22] Summers, W.C., The strange history of phage therapy. Bacterio-
phage, 2012, 2, (2), 130-133.
[23] Kutateladze, M., Experience of the Eliava Institute in bacterio-
phage therapy. Virol. Sin., 2015, 30, (1), 80-81.
[24] Hawkins, C.; Harper, D.; Burch, D.; Änggård, E.; Soothill, J.,
Topical treatment of Pseudomonas aeruginosa otitis of dogs with a
bacteriophage mixture: A before/after clinical trial. Vet. Microbiol.,
2010, 146, (34), 309-313.
[25] Heo, Y.J.; Lee, Y.R.; Jung, H.H.; Lee, J.; Ko, G.; Cho, Y.H., Anti-
bacterial efficacy of phages against Pseudomonas aeruginosa in-
fections in mice and Drosophila melanogaster. Antimicrob. Agents
Chemother., 2009, 53, (6), 2469-2474.
[26] McVay, C.S.; Velásquez, M.; Fralick, J.A., Phage therapy of Pseu-
domonas aeruginosa infection in a mouse burn wound model. An-
timicrob. Agents Chemother., 1934, 51, (6), 2007.
[27] Soothill, J.; Hawkins, C.; Anggard, E.; Harper, D., Therapeutic use
of bacteriophages. Lancet Infect. Dis., 2004, 4, (9), 544-545.
[28] Debarbieux, L.; Leduc, D.; Maura, D.; Morello, E.; Criscuolo, A.;
Grossi, O.; Balloy, V.; Touqui, L., Bacteriophages Can Treat and
Prevent Pseudomonas aeruginosa Lung Infections. J. Infect. Dis.,
2010, 201, (7), 1096-1104.
[29] Marza, J.A.; Soothill, J.S.; Boydell, P.; Collyns, T.A., Multiplica-
tion of therapeutically administered bacteriophages in Pseudo-
monas aeruginosa infected patients. Burns, 2006, 32, (5), 644-646.
[30] Wright, A.; Hawkins, C.H.; Änggård, E.E.; Harper, D.R., A con-
trolled clinical trial of a therapeutic bacteriophage preparation in
chronic otitis due to antibiotic-resistant Pseudomonas aeruginosa; a
preliminary report of efficacy. Clin. Otolaryngol., 2009, 34, (4),
349-357.
[31] University Hospital, M. ClinicalTrials.gov [Internet]. Bethesda
(MD): National Library of Medicine (US); Set 2016, Sept 2013.
[32] Center, S.R.W.C. A Prospective, Randomized, Double-Blind Con-
trolled Study of WPP-201 for the Safety and Efficacy of Treatment
of Venous Leg Ulcers (WPP-201); Sept 2016, Sept 2011.
[33] LaFee, S.; Buschman, H. Novel Phage Therapy Saves Patient with
Multidrug-Resistant Bacterial Infection. http://ucsdnews.ucsd.edu/
pressrelease, April 25 2017, access date: March 2017
[34] Commission, E. Phagoburn: the first large, multi-centre clinical
trial of phage therapy for human infections, funded by the Euro-
pean Commission.; Sept 2016, 2014.
[35] Jerne, N.K.; Avegno, P., The Development of the Phage-
Inactivating Properties of Serum During the Course of Specific
Immunization of an Animal: Reversible and Irreversible Inactiva-
tion. The Journal of Immunology, 1956, 76, (3), 200-208.
[36] Khan Mirzaei, M.; Haileselassie, Y.; Navis, M.; Cooper, C.; Sver-
remark-Ekström, E.; Nilsson, A.S., Morphologically Distinct Es-
cherichia coli Bacteriophages Differ in Their Efficacy and Ability
to Stimulate Cytokine Release In Vitro. Frontiers in Microbiology,
2016, 7, 437.
[37] Kaur, T.; Nafissi, N.; Wasfi, O.; Sheldon, K.; Wettig, S.; Slavcev,
R., Immunocompatibility of Bacteriophages as Nanomedicines.
Journal of Nanotechno logy , 2012, 2012, 13.
[38] Salmond, G.P.C.; Fineran, P.C., A century of the phage: past, pre-
sent and future. Nat Rev Micro, 2015, 13, (12), 777-786.
[39] Sarhan, W.A.; Azzazy, H.M.E., Phage approved in food, why not
as a therapeutic? Expert Rev. Anti Infect. Ther., 2015, 13, (1), 91-
101.
[40] Ackermann, H.W. In The Bacteriophages, second ed. Calendar,
R.L., Ed.; Oxford University press: New York, 2006.
[41] Laemmli, U.K., Cleavage of Structural Proteins during the Assem-
bly of the Head of Bacteriophage T4. Nature, 1970, 227, (5259),
680-685.
[42] Smeal, S.W.; Schmitt, M.A.; Pereira, R.R.; Prasad, A.; Fisk, J.D.,
Simulation of the M13 life cycle I: Assembly of a genetically-
structured deterministic chemical kinetic simulation. Virology,
2017, 500, 259-274.
[43] Bernheim, A.G.; Libis, V.K.; Lindner, A.B.; Wintermute, E.H.,
Phage-mediated Delivery of Targeted sRNA Constructs to Knock
Down Gene Expression in E. coli. 2016, (109), e53618.
[44] Ghaemi, A.; Soleimanjahi, H.; Gill, P.; Hassan, Z.; Jahromi,
S.R.M.; Roohvand, F., Recombinant λ-phage nanobioparticles for
tumor therapy in mice models. Genet. Vaccines Ther., 2010, 8, (1),
3.
[45] Wu, M.; Sherwin, T.; Brown, W.L.; Stockley, P.G., Delivery of
antisense oligonucleotides to leukemia cells by RNA bacteriophage
capsids. Nanomedicine, 2005, 1, (1), 67-76.
[46] Tao, P.; Mahalingam, M.; Marasa, B.S.; Zhang, Z.; Chopra, A.K.;
Rao, V.B., In vitro and in vivo delivery of genes and proteins using
the bacteriophage T4 DNA packaging machine. Proceedings of the
National Academy of Sciences, 2013, 110, (15), 5846-5851.
[47] Bedi, D.; Gillespie, J.W.; Petrenko, V.A., Jr.; Ebner, A.; Leitner,
M.; Hinterdorfer, P.; Petrenko, V.A., Targeted delivery of siRNA
into breast cancer cells via phage fusion proteins. Mol. Pharm.,
2013, 10, (2), 551-559.
[48] Kim, D.-H.; Longo, M.; Han, Y.; Lundberg, P.; Cantin, E.; Rossi,
J.J., Interferon induction by siRNAs and ssRNAs synthesized by
phage polymerase. Nat Biotech, 2004, 22, (3), 321-325.
[49] Bedi, D.; Musacchio, T.; Fagbohun, O.A.; Gillespie, J.W.; Deinno-
centes, P.; Bird, R.C.; Bookbinder, L.; Torchilin, V.P.; Petrenko,
V.A., Delivery of siRNA into breast cancer cells via phage fusion
protein-targeted liposomes. Nanomedicine, 2011, 7, (3), 315-323.
[50] Pecot, C.V.; Calin, G.A.; Coleman, R.L.; Lopez-Berestein, G.;
Sood, A.K., RNA interference in the clinic: challenges and future
directions. Nat. Rev. Cancer, 2011, 11, (1), 59-67.
[51] Bakhshinejad, B.; Sadeghizadeh, M., Bacteriophages as vehicles
for gene delivery into mammalian cells: prospects and problems.
Expert Opinion on Drug Delivery, 2014, 11, (10), 1561-1574.
[52] Jepson, C.D.; March, J.B., Bacteriophage lambda is a highly stable
DNA vaccine delivery vehicle. Vaccine, 2004, 22, (19), 2413-2419.
[53] Jończyk, E.; Kłak, M.; Międzybrodzki, R.; Górski, A., The influ-
ence of external factors on bacteriophagesreview. Folia Micro-
biol., 2011, 56, (3), 191-200.
[54] Koivunen, E.; Wang, B.; Ruoslahti, E., Isolation of a highly spe-
cific ligand for the alpha 5 beta 1 integrin from a phage display li-
brary. The Journal of Cell Biology, 1994, 124, (3), 373-380.
[55] Harbottle, R.P.; Cooper, R.G.; Hart, S.L.; Ladhoff, A.; McKay, T.;
Knight, A.M.; Wagner, E.; Miller, A.D.; Coutelle, C., An RGD-
oligolysine peptide: a prototype construct for integrin-mediated
gene delivery. Hum. Gene Ther., 1998, 9, (7), 1037-1047.
[56] Debaisieux, S.; Rayne, F.; Yezid, H.; Beaumelle, B., The Ins and
Outs of HIV-1 Tat. Traffic, 2012, 13, (3), 355-363.
[57] Paschke, M.; Höhne, W., A twin-arginine translocation (Tat)-
mediated phage display system. Gene, 2005, 350, (1), 79-88.
[58] Wei, B.; Wei, Y.; Zhang, K.; Wang, J.; Xu, R.; Zhan, S.; Lin, G.;
Wang, W.; Liu, M.; Wang, L.; Zhang, R.; Li, J., Development of an
antisense RNA delivery system using conjugates of the MS2 bacte-
riophage capsids and HIV-1 TAT cell penetrating peptide. Biomed.
Pharmacother., 2009, 63, (4), 313-318.
[59] LAROCCA, D.; KASSNER, P.D.; WITTE, A.; LADNER, R.C.;
PIERCE, G.F.; BAIRD, A., Gene transfer to mammalian cells us-
ing genetically targeted filamentous bacteriophage. The FASEB
Journal, 1999, 13, (6), 727-734.
[60] Dobroff, A.S.; Rangel, R.; Guzman-Roja, L.; Salmeron, C.C.;
Gelovani, J.G.; Sidman, R.L.; Bologa, C.G.; Oprea, T.I.; Brinker,
C.J.; Pasqualini, R.; Arap, W., Ligand-directed profiling of organ-
elles with internalizing phage libraries. Current protocols in pro-
tein science, 2015, 79, 30.34.31-30.
[61] Martins, I.M.; Reis, R.L.; Azevedo, H.S., Phage Display Technol-
ogy in Biomaterials Engineering: Progress and Opportunities for
Applications in Regenerative Medicine. ACS Chem. Biol., 2016,
11, (11), 2962-2980.
[62] Scott, J.; Smith, G., Searching for peptide ligands with an epitope
library. Science, 1990, 249, (4967), 386-390.
[63] Azzazy, H.M.E.; Highsmith Jr, W.E., Phage display technology:
clinical applications and recent innovations. Clin. Biochem., 2002,
35, (6), 425-445.
[64] Sergeeva, A.; Kolonin, M.G.; Molldrem, J.J.; Pasqualini, R.; Arap,
W., Display technologies: Application for the discovery of drug
and gene delivery agents. Adv. Drug Del. Rev., 2006, 58, (15),
1622-1654.
[65] Sidhu, S.S., Phage display in pharmaceutical biotechnology. Curr.
Opin. Biotechnol., 2000, 11, (6), 610-616.
[66] Hohlweg, U.; Doerfler, W., On the fate of plant or other foreign
genes upon the uptake in food or after intramuscular injection in
mice. Mol. Gen et. Genomics, 2001, 265, (2), 225-233.
[67] Sunderland, K.S.; Yang, M.; Mao, C., Phage-Enabled Nanomedi-
cine: From Probes to Therapeutics in Precision Medicine. Angew.
Chem. Int. Ed., 2017, 56, (8), 1964-1992.
Phage-Mediated Ge ne Therapy Current Gene Therapy, 2017, Vol. 17, No. 1 7
[68] Chauthaiwale, V.; Therwath, A.; Deshpande, V.V., Bacteriophage
lambda as a cloning vector. Microbiol. Rev., 1992, 56.
[69] Ghosh, D.; Kohli, A.G.; Moser, F.; Endy, D.; Belcher, A.M.,
Refactored M13 bacteriophage as a platform for tumor cell imag-
ing and drug delivery. ACS Synthetic Biology, 2012, 1, (12), 576-
582.
[70] Bernard, J.M.L.; Francis, M.B., Chemical strategies for the cova-
lent modification of filamentous phage. Frontiers in Microbiology,
2014, 5, (DEC).
[71] Kwak, E.A.; Jaworski, J., Controlled surface immobilization of
viruses via site-specific enzymatic modification. Journal of Mate-
rials Chemistry B, 2013, 1, (28), 3486-3493.
[72] Kovacs, E.W.; Hooker, J.M.; Romanini, D.W.; Holder, P.G.; Berry,
K.E.; Francis, M.B., Dual-Surface-Modified Bacteriophage MS2 as
an Ideal Scaffold for a Viral Capsid-Based Drug Delivery System.
Bioconj. Chem., 2007, 18, (4), 1140-1147.
[73] Tong, G.J.; Hsiao, S.C.; Carrico, Z.M.; Francis, M.B., Viral Capsid
DNA Aptamer Conjugates as Multivalent Cell-Targeting Vehicles.
J. Am. Chem. Soc., 2009, 131, (31), 11174-11178.
[74] Warner, C.M.; Barker, N.; Lee, S.W.; Perkins, E.J., M13 bacterio-
phage production for large-scale applications. Bioprocess Biosyst
Eng, 2014, 37, (10), 2067-2072.
[75] Gill, J.J.; Hyman, P., Phage choice, isolation, and preparation for
phage therapy. Curr. Pharm. Biotechnol., 2010, 11, (1), 2-14.
[76] Kramberger, P.; Honour, R.C.; Herman, R.E.; Smrekar, F.; Peterka,
M., Purification of the Staphylococcus aureus bacteriophages
VDX-10 on methacrylate monoliths. J. Virol. Methods, 2010, 166,
(12), 60-64.
[77] Qin, J.; Li, Y.; Cai, Z.; Li, S.; Zhu, J.; Zhang, F.; Liang, S.; Zhang,
W.; Guan, Y.; Shen, D.; Peng, Y.; Zhang, D.; Jie, Z.; Wu, W.; Qin,
Y.; Xue, W.; Li, J.; Han, L.; Lu, D.; Wu, P.; Dai, Y.; Sun, X.; Li,
Z.; Tang, A.; Zhong, S.; Li, X.; Chen, W.; Xu, R.; Wang, M.; Feng,
Q.; Gong, M.; Yu, J.; Zhang, Y.; Zhang, M.; Hansen, T.; Sanchez,
G.; Raes, J.; Falony, G.; Okuda, S.; Almeida, M.; LeChatelier, E.;
Renault, P.; Pons, N.; Batto, J.-M.; Zhang, Z.; Chen, H.; Yang, R.;
Zheng, W.; Li, S.; Yang, H.; Wang, J.; Ehrlich, S.D.; Nielsen, R.;
Pedersen, O.; Kristiansen, K.; Wang, J., A metagenome-wide asso-
ciation study of gut microbiota in type 2 diabetes. Nature, 2012,
490, (7418), 55-60.
[78] Schwabe, R.F.; Jobin, C., The microbiome and cancer. Nat. Rev.
Cancer, 2013, 13, (11), 800-812.
[79] Jostins, L.; Ripke, S.; Weersma, R.K.; Duerr, R.H.; McGovern,
D.P.; Hui, K.Y.; Lee, J.C.; Philip Schumm, L.; Sharma, Y.; Ander-
son, C.A.; Essers, J.; Mitrovic, M.; Ning, K.; Cleynen, I.; Theatre,
E.; Spain, S.L.; Raychaudhuri, S.; Goyette, P.; Wei, Z.; Abraham,
C.; Achkar, J.-P.; Ahmad, T.; Amininejad, L.; Ananthakrishnan,
A.N.; Andersen, V.; Andrews, J.M.; Baidoo, L.; Balschun, T.;
Bampton, P.A.; Bitton, A.; Boucher, G.; Brand, S.; Buning, C.;
Cohain, A.; Cichon, S.; D/'Amato, M.; De Jong, D.; Devaney, K.L.;
Dubinsky, M.; Edwards, C.; Ellinghaus, D.; Ferguson, L.R.; Fran-
chimont, D.; Fransen, K.; Gearry, R.; Georges, M.; Gieger, C.;
Glas, J.; Haritunians, T.; Hart, A.; Hawkey, C.; Hedl, M.; Hu, X.;
Karlsen, T.H.; Kupcinskas, L.; Kugathasan, S.; Latiano, A.;
Laukens, D.; Lawrance, I.C.; Lees, C.W.; Louis, E.; Mahy, G.;
Mansfield, J.; Morgan, A.R.; Mowat, C.; Newman, W.; Palmier i,
O.; Ponsioen, C.Y.; Potocnik, U.; Prescott, N.J.; Regueiro, M.; Rot-
ter, J.I.; Russell, R.K.; Sanderson, J.D.; Sans, M.; Satsangi, J.;
Schreiber, S.; Simms, L.A.; Sventoraityte, J.; Targan, S.R.; Taylor,
K.D.; Tremelling, M.; Verspaget, H.W.; De Vos, M.; Wijmenga,
C.; Wilson, D.C.; Winkelmann, J.; Xavier, R.J.; Zeissig, S.; Zhang,
B.; Zhang, C.K.; Zhao, H.; Silverberg, M.S.; Annese, V.; Hakonar-
son, H.; Brant, S.R.; Radford-Smith, G.; Mathew, C.G.; Rioux,
J.D.; Schadt, E.E.; Daly, M.J.; Franke, A.; Parkes, M.; Vermeire,
S.; Barrett, J.C.; Cho, J.H., Host-microbe interactions have shaped
the genetic architecture of inflammatory bowel disease. Nature,
2012, 491, (7422), 119-124.
[80] Berer, K.; Mues, M.; Koutrolos, M.; Rasbi, Z.A.; Boziki, M.; Joh-
ner, C.; Wekerle, H.; Krishnamoorthy, G., Commensal microbiota
and myelin autoantigen cooperate to trigger autoimmune demyeli-
nation. Nature, 2011, 479, (7374), 538-541.
[81] Cryan, J.F.; Dinan, T.G., Mind-altering microorganisms: the impact
of the gut microbiota on brain and behaviour. Nat. Rev. Neurosci.,
2012, 13, (10), 701-712.
[82] Dethlefsen, L.; Relman, D.A., Incomplete recovery and individual-
ized responses of the human distal gut microbiota to repeated anti-
biotic perturbation. Proceedings of the National Academy of Sci-
ences, 2011, 108, (Supplement 1), 4554-4561.
[83] Devkota, S., Prescription drugs obscure microbiome analyses.
Science, 2016, 351, (6272), 452-453.
[84] Maurice, Corinne F.; Haiser, Henry J.; Turnbaugh, Peter J., Xeno-
biotics Shape the Physiology and Gene Expression of the Active
Human Gut Microbiome. Cell, 2013, 152, (12), 39-50.
[85] Langdon, A.; Crook, N.; Dantas, G., The effects of antibiotics on
the microbiome throughout development and alternative ap-
proaches for therapeutic modulation. Genome Med., 2016, 8, 39.
[86] Blaser, M., Antibiotic overuse: Stop the killing of beneficial bacte-
ria. Nature, 2011, 476, (7361), 393-394.
[87] Sjolund, M.; Wreiber, K.; Andersson, D.I.; Blaser, M.J.; Engstrand,
L., Long-term persistence of resistant Enterococcus species after
antibiotics to eradicate Helicobacter pylori. Ann. Intern. Med.,
2003, 139, (6), 483-487.
[88] Mimee, M.; Citorik, R.J.; Lu, T.K., Microbiome therapeutics -
Advances and challenges. Adv Drug Deliv Rev, 2016, 105, (Pt A),
44-54.
[89] Lu, T.K.; Koeris, M.S., The next generation of bacteriophage ther-
apy. Curr. Opin. Microbiol., 2011, 14, (5), 524-531.
[90] Lu, T.K.; Collins, J.J., Engineered bacteriophage targeting gene
networks as adjuvants for antibiotic therapy. Proc. Natl. Acad. Sci.
U. S. A., 2009, 106, (12), 4629-4634.
[91] Edgar, R.; Friedman, N.; Molshanski-Mor, S.; Qimron, U., Revers-
ing bacterial resistance to antibiotics by phage-mediated delivery of
dominant sensitive genes. Appl. Environ. Microbiol., 2012, 78, (3),
744-751.
[92] Bikard, D.; Euler, C.W.; Jiang, W.; Nussenzweig, P.M.; Goldberg,
G.W.; Duportet, X.; Fischetti, V.A.; Marraffini, L.A., Exploiting
CRISPR-Cas nucleases to produce sequence-specific antimicrobi-
als. Nat. Biotechnol., 2014, 32, (11), 1146-1150.
[93] Citorik, R.J.; Mimee, M.; Lu, T.K., Sequence-specific antimicrobi-
als using efficiently delivered RNA-guided nucleases. Nat. Bio-
technol., 2014, 32, (11), 1141-1145.
... The local intratumoral administration of natural infectious agents of mammalian cells facilitates the efficient delivery and expression of therapeutic genes into cancer cells. However, the systemic administration of these vectors is accompanied by a decrease in the selectivity of delivery, in connection with the wide tropism of eukaryotic viruses to the cells and tissues of host organisms, the absorption of their particles by the liver and the reticuloendothelial system of the body and the triggering of a powerful immune response, which complicates repeated injections [4,5]. ...
... Currently, bacteriophages are considered as promising vectors for cancer gene therapy [5]. They are believed to have a number of advantages over non-viral and eukaryotic virus-based vectors. ...
... In addition, the use of vectors based on these viruses is limited by the immunogenicity of their systemic administration and the absorption by unwanted tissues and organs, such as the liver [6]. Other serious problems associated with the utilization of therapeutic drugs based on eukaryotic viruses include: oncogenicity, extremely limited capacity of the viral genome for transgene cloning, weak resistance of the capsid structure during its modification and the need for helper viruses [5]. ...
Article
Full-text available
Bacteriophages have long been considered only as infectious agents that affect bacterial hosts. However, recent studies provide compelling evidence that these viruses are able to successfully interact with eukaryotic cells at the levels of the binding, entry and expression of their own genes. Currently, bacteriophages are widely used in various areas of biotechnology and medicine, but the most intriguing of them is cancer therapy. There are increasing studies confirming the efficacy and safety of using phage-based vectors as a systemic delivery vehicle of therapeutic genes and drugs in cancer therapy. Engineered bacteriophages, as well as eukaryotic viruses, demonstrate a much greater efficiency of transgene delivery and expression in cancer cells compared to non-viral gene transfer methods. At the same time, phage-based vectors, in contrast to eukaryotic viruses-based vectors, have no natural tropism to mammalian cells and, as a result, provide more selective delivery of therapeutic cargos to target cells. Moreover, numerous data indicate the presence of more complex molecular mechanisms of interaction between bacteriophages and eukaryotic cells, the further study of which is necessary both for the development of gene therapy methods and for understanding the cancer nature. In this review, we summarize the key results of research into aspects of phage–eukaryotic cell interaction and, in particular, the use of phage-based vectors for highly selective and effective systemic cancer gene therapy.
... Repression efficiency of sRNA is dependent on the energy needed for binding with the target mRNA. The binding energy should be in the range of -20 to −30 kcal/mol for the maximum repression [38,39]. The free energy/binding energy of csgA mRNA and sRNA were found to be -29.60 kcal/mole, which lied in the significant repression range. ...
... Phagemids can be easily modified through single PCR to target nearly any gene and can be produced within a day before phagemid transformation. The sRNA expression cassette on a standard plasmid vector is found to work on a wide range of metabolic targets and exhibits greater than 90% of typical repression level, which correlates with findings from our study [38]. In a previous study, the repression of bacterial target genes through sRNA via phagemid infection was observed to be about 80%, with mKate fluoresce gene as a measuring standard [26]. ...
Article
Full-text available
Curli fimbriae, a virulent factor of the Avian Pathogenic Escherichia coli (APEC), is responsible for adhesion, biofilm formation, and colonization of pathogen. Major curli fimbriae protein is encoded by csgA gene. APEC is one of the leading causes of colibacillosis in poultry flocks and due to excessive use of antibiotics and vaccines in poultry, the emergence of various multi-drug resistant (MDR) bacterial strainsare is frequently reported. The growing concern of MDR bacterial strains necessitate novel antibacterial approaches to combat colibacillosis in poultry. RNA-based gene silencing is a very specific and robust strategy to target specific bacterial factors involved in pathogenicity and virulence. In this study, a phagemid-mediated sRNA expression system to target a vital gene, csgA, is employed. This comprises an M13 phagemid harboring a sRNA expression cassette and a pre-designed GUIDE sequences for the csgA target gene. To target the csgA gene at the mRNA level, a GUIDE sequence was computationally designed for pre-designed sRNA expression cassette. Online web tools were used to predict the binding energy, secondary structure, and off-target binding potential of the sRNA to optimize its expression. Results showed that the designed sRNA has a binding energy of − 29.60 kcal/mol with zero off-targets. After expression of the sRNA in the APEC cells, ̴ 45% reduction in the csgA level was observed via RT-PCR in the CS-APEC-O1 strains compared to the wt-APEC-O1. Similarly, the biofilm forming ability decreased by 40% in the CS-APEC-O1 strains. The swarming motility and hemagglutination efficiency were not affected by the sRNA expression. Future studies investigating the in vivo efficiency of M13 phagemid delivery are required to evaluate its candidacy in phage therapy.
... Conventional gene therapy approaches have typically relied on eukaryotic viral vectors, but interest in using phage vectors or vectors that are hybrids of eukaryotic and phage viruses is increasing due to key benefits they offer regarding targeting, cargo capacity, and safety (for reviews see [89,90]). Bacteriophages have a large cargo capacity relative to most mammalian viruses and, as discussed above, many can be easily engineered to express eukaryotic cell targeting motifs that also mediate entry via receptor mediated endocytosis. Bacteriophages might also prove safer than mammalian viral vectors given their lack of natural tropism for eukaryotic cells and their history of safe use in humans. ...
Article
Full-text available
Since their independent discovery by Frederick Twort in 1915 and Felix d’Herelle in 1917, bacteriophages have captured the attention of scientists for more than a century. They are the most abundant organisms on the planet, often outnumbering their bacterial hosts by tenfold in a given environment, and they constitute a vast reservoir of unexplored genetic information. The increased prevalence of antibiotic resistant pathogens has renewed interest in the use of naturally obtained phages to combat bacterial infections, aka phage therapy. The development of tools to modify phages, genetically or chemically, combined with their structural flexibility, cargo capacity, ease of propagation, and overall safety in humans has opened the door to a myriad of applications. This review article will introduce readers to many of the varied and ingenious ways in which researchers are modifying phages to move them well beyond their innate ability to target and kill bacteria.
... [65]. However, literature review also suggest lower efficiency in gene delivery via use of bacteriophages in comparison other viral and non-viral modes of gene carrying vectors [66]. ...
Article
Full-text available
CRISPR genome editing technologies have been improving by every passing day. The initial CRISPR/Cas9 technologies, though emerged an improved version of genome editing in competition with TALENS and ZFNs, was nevertheless not free from technical and off-target effects. Technological improvements overtime start addressing issues with original CRISPR/Cas9 technology. The major areas of improvement targeted nucleases and delivery methods. Overtime the nuclease like Cas9 had some modifications like FokI-dCas9, Truncated guide RNAs (tru-gRNAs), Paired Cas9 nickase, Cpf1, Cas6 with Csm/Csr complex and chemically treated Cas9. In terms of delivery methods the improvements came along after almost all methods including viral methods like Recombinant Adeno Associated Viruses (rAAV), Lentivirus (LV), and bacteriophages. The review summarizes various non-viral gene delivery modes including physical methods like electroporation and chemical methods like nano particles, cell-derived membrane vesicles (CMVs) with upgraded developments. The review also compares various modes of delivering CRISPR gene editing machinery.
... Phage particles can be engineered, or a gene of interest can be inserted into the phage genome and its protein will be expressed on the phage surface. These properties can be exploited to achieve targeted delivery of oncolytic gene cassettes against cancer (Hosseinidoust, 2017;Petrenko and Gillespie, 2017). Shoae-Hassani et al. (2013) designed Apoptin expressing λ Phage nanobioparticles and observed significant inhibition of growth in hBC cells in vitro and suppression of xenograft tumor growth in vivo in mice. ...
Article
Full-text available
Cancer remains one of the leading causes of death worldwide in humans and animals. Conventional treatment regimens often fail to produce the desired outcome due to disturbances in cell physiology that arise during the process of transformation. Additionally, development of treatment regimens with no or minimum side-effects is one of the thrust areas of modern cancer research. Oncolytic viral gene therapy employs certain viral genes which on ectopic expression find and selectively destroy malignant cells, thereby achieving tumor cell death without harming the normal cells in the neighborhood. Apoptin, encoded by Chicken Infectious Anemia Virus’ VP3 gene, is a proline-rich protein capable of inducing apoptosis in cancer cells in a selective manner. In normal cells, the filamentous Apoptin becomes aggregated toward the cell margins, but is eventually degraded by proteasomes without harming the cells. In malignant cells, after activation by phosphorylation by a cancer cell-specific kinase whose identity is disputed, Apoptin accumulates in the nucleus, undergoes aggregation to form multimers, and prevents the dividing cancer cells from repairing their DNA lesions, thereby forcing them to undergo apoptosis. In this review, we discuss the present knowledge about the structure of Apoptin protein, elaborate on its mechanism of action, and summarize various strategies that have been used to deliver it as an anticancer drug in various cancer models.
Chapter
Bacteriophage, being the most abundant organism on earth, has provided a virtually unlimited resource to develop various novel and exciting biotechnological and medical strategies. Although phage therapy was conceived nearly a century ago, its real power was only realized with the global emergence of multidrug-resistant bacterial pathogens. With tremendous progress in various technologies like high-throughput next-generation sequencing, genome editing, and synthetic biology, new avenues have opened up to explore phages. In this review, we tried to explore the various phage engineering strategy which has been used to develop mutant phages having multivalent applications. Phage engineering promises to develop mutant phages, which can be used to deliver cargos into cells/tissues that can enhance the immune response and prevent infection. These engineering approaches have the potential to accelerate basic biotechnological research as well as have the ability to develop into new therapeutics in the future.
Article
In this communication, we will review the problems caused by cell-mediated gene therapy, taking skeletal muscle as a physiological model. In particular we have utilised vectors transferring telomerase under the control of retroviral promoters into human satellite cells. The set of results presented here has several implications regarding gene therapy trials. Nevertheless, more experiments will be required to fully validate this cellular model and to use telomerase to safely extend the lifespan of putative gene therapy vectors.
Article
Full-text available
The widespread use of antibiotics in the past 80 years has saved millions of human lives, facilitated technological progress and killed incalculable numbers of microbes, both pathogenic and commensal. Human-associated microbes perform an array of important functions, and we are now just beginning to understand the ways in which antibiotics have reshaped their ecology and the functional consequences of these changes. Mounting evidence shows that antibiotics influence the function of the immune system, our ability to resist infection, and our capacity for processing food. Therefore, it is now more important than ever to revisit how we use antibiotics. This review summarizes current research on the short-term and long-term consequences of antibiotic use on the human microbiome, from early life to adulthood, and its effect on diseases such as malnutrition, obesity, diabetes, and Clostridium difficile infection. Motivated by the consequences of inappropriate antibiotic use, we explore recent progress in the development of antivirulence approaches for resisting infection while minimizing resistance to therapy. We close the article by discussing probiotics and fecal microbiota transplants, which promise to restore the microbiota after damage of the microbiome. Together, the results of studies in this field emphasize the importance of developing a mechanistic understanding of gut ecology to enable the development of new therapeutic strategies and to rationally limit the use of antibiotic compounds.
Article
Full-text available
Due to a global increase in the range and number of infections caused by multi-resistant bacteria, phage therapy is currently experiencing a resurgence of interest. However, there are a number of well-known concerns over the use of phages to treat bacterial infections. In order to address concerns over safety and the poorly understood pharmacokinetics of phages and their associated cocktails, immunological characterization is required. In the current investigation, the immunogenicity of four distinct phages (taken from the main families that comprise the Caudovirales order) and their interaction with donor derived peripheral blood mononuclear cells and immortalized cell lines (HT-29 and Caco-2 intestinal epithelial cells) were investigated using standard immunological techniques. When exposed to high phage concentrations (109 PFU/well), cytokine driven inflammatory responses were induced from all cell types. Although phages appeared to inhibit the growth of intestinal epithelial cell lines, they also appear to be non-cytotoxic. Despite co-incubation with different cell types, phages maintained a high killing efficiency, reducing extended-spectrum beta-lactamase-producing Escherichia coli numbers by 1–4 log10 compared to untreated controls. When provided with a suitable bacterial host, phages were also able to actively reproduce in the presence of human cells resulting in an approximately 2 log10 increase in phage titer compared to the initial inoculum. Through an increased understanding of the complex pharmacokinetics of phages, it may be possible to address some of the safety concerns surrounding phage preparations prior to creating new therapeutic strategies.
Article
Full-text available
RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.
Article
The field of regenerative medicine has been gaining momentum steadily over the past few years. The emphasis in regenerative medicine is to use various in-vitro and in-vivo approaches that leverage on the intrinsic healing mechanisms of the body to treat patients with disabling injuries and chronic diseases such as diabetes, osteoarthritis, and degenerative disorders of the cardiovascular and central nervous system. Phage display has been successfully employed to identify peptide ligands for a wide variety of targets, ranging from relatively small molecules (enzymes, cell receptors) to inorganic, organic, and biological (tissues) materials. Over the last two decades, phage display technology has advanced tremendously and has become a powerful tool in the most varied fields of research, including biotechnology, materials science, cell biology, pharmacology, and diagnostics. The growing interest in and success of phage display libraries is largely due its incredible versatility and practical use. This review discusses the potential of phage display technology in biomaterials engineering for applications in regenerative medicine.
Article
To expand the quantitative, systems level understanding and foster the expansion of the biotechnological applications of the filamentous bacteriophage M13, we have unified the accumulated quantitative information on M13 biology into a genetically-structured, experimentally-based computational simulation of the entire phage life cycle. The deterministic chemical kinetic simulation explicitly includes the molecular details of DNA replication, mRNA transcription, protein translation and particle assembly, as well as the competing protein-protein and protein-nucleic acid interactions that control the timing and extent of phage production. The simulation reproduces the holistic behavior of M13, closely matching experimentally reported values of the intracellular levels of phage species and the timing of events in the M13 life cycle. The computational model provides a quantitative description of phage biology, highlights gaps in the present understanding of M13, and offers a framework for exploring alternative mechanisms of regulation in the context of the complete M13 life cycle.
Article
Nanomedicine is the application of nanotechnology in medicine. This review is focused on the use of both lytic and temperate bacteriophages (phages) nanoparticles in nanomedicine, in particular, in the context of developing nano-probes for precise disease diagnosis and nano-therapeutics for targeted disease treatment. Phages as bacteria-specific viruses do not naturally infect eukaryotic cells and are not toxic to them. They not only can be genetically engineered to bear the capability of targeting nanoparticles, cells, tissues, and organs, but also can be integrated with functional abiotic nanomaterials to gain physical properties that enable disease diagnosis and treatment. Therefore, they are ideal for many applications in precision nanomedicine. This review will summarize the current use of the great diversity of phage structures in many aspects of precision nanomedicine including ultrasensitive biomarker detection, enhanced bioimaging for disease diagnosis, targeted drug and gene delivery, directed stem cell differentiation, accelerated tissue formation, effective vaccination, and nano-therapeutics for targeted disease treatment. It will also propose future directions in the area of phage-based nanomedicines as well as the state of phage-based clinical trials.
Article
The microbial community that lives on and in the human body exerts a major impact on human health, from metabolism to immunity. In order to leverage the close associations between microbes and their host, development of therapeutics targeting the microbiota has surged in recent years. Here, we discuss current additive and subtractive strategies to manipulate the microbiota, focusing on bacteria engineered to produce therapeutic payloads, consortia of natural organisms and selective antimicrobials. Further, we present challenges faced by the community in the development of microbiome therapeutics, including designing microbial therapies that are adapted for specific geographies in the body, stable colonization with microbial therapies, discovery of clinically relevant biosensors, robustness of engineered synthetic gene circuits and addressing safety and biocontainment concerns. Moving forward, collaboration between basic and applied researchers and clinicians to address these challenges will poise the field to herald an age of next-generation, cellular therapies that draw on novel findings in basic research to inform directed augmentation of the human microbiota.
Article
Although observations linking members of the gut microbiome to human disease have been plentiful, some are fraught with complex and confounding variables, emphasizing the need for vetting such associations with greater computational and mechanistic rigor. A recent study by Forslund et al. ( 1 ) adds another dimension for consideration by illustrating how medications may adversely affect the microbiome—an interaction often overlooked in post hoc analyses of disease-microbe relationships.
Article
Viruses that infect bacteria (bacteriophages; also known as phages) were discovered 100 years ago. Since then, phage research has transformed fundamental and translational biosciences. For example, phages were crucial in establishing the central dogma of molecular biology - information is sequentially passed from DNA to RNA to proteins - and they have been shown to have major roles in ecosystems, and help drive bacterial evolution and virulence. Furthermore, phage research has provided many techniques and reagents that underpin modern biology - from sequencing and genome engineering to the recent discovery and exploitation of CRISPR-Cas phage resistance systems. In this Timeline, we discuss a century of phage research and its impact on basic and applied biology.