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Phage in the Era of Synthetic

E. Magda Barbu, Kyle C. Cady, and Bolyn Hubby

Synthetic Genomics, La Jolla, California 92037 Correspondence: [email protected]

For more than a century, (or phage) research has enabled some of the most important discoveries in biological sciences and has equipped scientists with many of the molecular biology tools that have advanced our understanding of replication, maintenance, andexpressionof genetic material. Phageshavealso beenrecognizedandexploitedasnatural agents and nanovectors for , but their potential as therapeutics has notbeenfullyexploitedinWesternmedicinebecauseofchallengessuchasnarrowhostrange, bacterial resistance, and unique pharmacokinetics. However, increasing concern related to the emergence of resistant to multiple has heightened interest in phage therapyand the development of strategiesto overcome hurdles associated with bacteri- ophage therapeutics. Recent progress in sequencing technologies, DNA manipulation, and allowed scientists to refactor the entire bacterial of Mycoplasma mycoides, thereby creating the first synthetic cell. These new strategies for engineering may have the potential to accelerate the construction of designer phage genomes with superior therapeutic potential. Here, we discuss the use of phage as therapeutics, as well as how synthetic biology can create bacteriophage with desirable attributes.

ysogenic (temperate) and lytic bacteriophag- ered for therapeutic use, as many have been Les are that specifically infect bacteria, shown to encode bacterial virulence factors or and have long been considered as natural anti- horizontally transfer virulence genes in a pro- microbial agents for treatment of bacterial infec- cess known as (Boyd and Brussow tions (d’Herelle 1931; Twort 1936). On binding 2002; Ptashne 2004; Tinsley et al. 2006). In con- to a bacterial surface moiety, phages inject their trast, virulent phages immediately begin replica- genetic material into the cell. In the case of tion and quickly subvert host-cell metabolism, temperate phage, the is initiated which results in the destruction of the host cell by viral genome integration into the bacterial within minutes to hours (Fig. 1) (Sulakvelidze chromosome () where it remains until et al. 2001; Hanlon 2007). Virulent phages do specific lytic genes are activated and phage viri- not enter a prophage state and have not shown ons are produced, eventually lysing the host cell. the ability to transfer virulence factors between Before the activation of the lytic cycle, the phage bacterial cells, making them suitable for phage genome remains repressed and can be trans- therapy (Hendrix 2003; Hatfull 2008). ferred to daughter cells during bacterial replica- The clinical potential of virulent bacterio- tion. Temperate phages are largely not consid- phages as antibacterial agents was first recog-

Editors: Daniel G. Gibson, Clyde A. Hutchison III, Hamilton O. Smith, and J. Additional Perspectives on Synthetic Biology available at www.cshperspectives.org Copyright # 2016 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a023879 Cite this article as Cold Spring Harb Perspect Biol 2016;8:a023879

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E.M. Barbu et al.

Lysis of bacterial cell and Phage binding to phage progeny release bacterial host cell

Virulent phage life cycle

Phage progeny assembly Phage DNA injection and capsid release

Protein synthesis and DNA replication

Figure 1. Lytic cycle of bacteriophage. Schematic representation of virulent phage , replication, and of bacterial host cells.

nized by Felix d’Herelle, one of the pioneers of controlled by specific receptor–ligand interac- the phage therapy field (d’Herelle et al. 1931). tions (Furukawa et al. 1983). Phage–host selec- Although early successes of personalized clini- tivity permits phage therapy to be directed at cal applications with tailored formulations were specific bacterial populations, preventing sec- reported, the use of phage therapy against bac- ondary by leaving nontarget bacteria terial in large patient populations unaffected (Sulakvelidze et al. 2001). Host dis- was never achieved in part because of its dis- cernment prevents phage therapy from enrich- placement by antibiotics (Merril et al. 2003, ing for resistance, a major problem associated 2006). The growing threat posed by highly an- with use. Historically, the diversity tibiotic-resistant bacteria combined with the and malleability of bacterial surface ligands, decreasing numbers of newly licensed drugs even at the species level, makes it difficult to has recently triggered interest in the develop- identify phage capable of treating a clinical ment of phage . However, several infection without prescreening against phage challenges associated with phage therapy limit libraries (Keen 2012). The specificity of viral– their therapeutic potential. host interactions has made phage therapy attractive, but has also required either the tar- geting of pathogens that are largely phylogenet- OVERCOMING PHAGE THERAPY ically constrained (clonal) or the use of large CHALLENGES IN THE ERA OF SYNTHETIC BIOLOGY phage cocktails capable of covering the diversity of a bacterial population (O’Flaherty et al. 2005; Host Specificity McVay et al. 2007). Viral attachment and genome injection are reg- The plummeting cost of DNA sequencing imented and stepwise processes that are tightly and new molecular biology techniques may al-

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Phage Therapy in the Era of Synthetic Biology

low scientists to leverage phenotypic screening, in vivo virulence (O’Flaherty et al. 2005; Ojala comparative genomics, and viral engineering to et al. 2013). However, bacteria can also acquire overcome the challenges associated with host resistance to phages through a diverse array of specificity. As bacterial phylogeny correlates restriction modification, toxin–antitoxin, and with phage host range, the ability to rapidly CRISPR/Cas systems (Labrie et al. 2010). Un- sequence clinical isolates will increase both the like purified small molecules, phages respond to accuracy and the speed at which lytic phage can selective pressure through evolution, which has be paired with potentially susceptible bacterial supplied scientists with a plethora of genetically infections (DeLappe et al. 2003). Last, newly encoded counter-resistance systems capable of developed viral engineering and DNA manipu- overcoming bacterial resistance mechanisms lation methods could be used to expand the host (Samson et al. 2013). In a clinical setting, it is range of through rational de- important to remember that phage and anti- sign, potentially decreasing the number of biotic resistance mechanisms do not overlap; phages needed to cover bacterial diversity. For therefore, loss of sensitivity to phage is unlikely example, fX174 genome (5.4 kb) was assem- to affect antibiotic susceptibility (Labrie et al. bled from a pool of synthetic and 2010; Blair et al. 2015). In fact, CRISPR/Cas- successfully launched in bacteria (Smith et al. mediated resistance to phage is associated with 2003). Furthermore, the entire decreased antibiotic resistance in enterococci of Mycoplasma mycoides was chemically synthe- (Palmer and Gilmore 2010). sized from DNA fragments (Gibson et al. 2009). The success of chemical cocktails, phage Other technologies, such as bacteriophage re- cocktails, and multivalent vaccines has shown combineering of electroporated DNA (BRED) that targeting multiple conserved but indepen- and via targetrons and recom- dent ligands drastically reduces the of binases (GETR), have been developed to edit evasion. Thus, endowing single bac- phage genomes (Marinelli et al. 2008; Enyeart teriophage with an independent bactericidal et al. 2013). Assembly of genomes in yeast arti- payload could prevent bacterial resistance to ficial chromosome and and clustered phages (Fig. 3). regularly interspaced short palindromic repeats (CRISPR)/Cas-editing systems in bacteria have Unique Pharmacological Properties of Viral also been recently used to manipulate phage Therapeutics DNA (Lu and Koeris 2011; Jaschke et al. 2012; Kiro et al. 2014; Martel and Moineau 2014; No- Biologics are large molecules with desirable brega et al. 2015). Together, these methods can therapeutic and prophylactic uses, which are be integrated to construct synthetic phages with very complex and are often 200 to more than desired characteristics and precise genomic con- 1000 times the size of small molecule drugs tent (Fig. 2). (Kingham et al. 2014). Similar to biologics, the large size of phage poses distinctive pharmaco- logical challenges when compared with chemi- Development of Resistance cal products. Additionally, therapeutic viruses, The ability of bacteria to rapidly acquire resis- such as oncolytic virions or bacteriophage, are tance to a single selective pressure hinders the capable of replicating in vivo, further differenti- success of both standard small molecule and ating them from static biologics or chemicals nonstandard antimicrobials, such as phages (Chiocca 2002). (Neu 1992; Labrie et al. 2010). Phage resistance The large size of biologics prevents them most commonly arises through the down-regu- from diffusing into cells or biofilms, crossing lation, mutation, or shielding of the viral recep- the blood–brain barrier, and makes administra- tor (Labrie et al. 2010). This simple fact has tion of large doses challenging (Baumann encouraged the use of phages that bind to re- 2006). Fortunately, bacteriophages are capable ceptors that are highly conserved or required for of density-dependent productive amplification

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E.M. Barbu et al.

In silico design of overlapping nucleotides based on phage DNA sequence

DNA fragment assembly

Pooling of DNA fragments Transformation in yeast

Gibson assembly DNA assembly into YAC

Transformation into bacterial host cells

Plaque formation and phage recovery

Figure 2. Synthetic assembly of bacteriophage genomes. Step-by-step construction of phage genomes starting from overlapping nucleotides and recovery of functional phage particles from host cells following transforma- tion. (Left) Chemical assembly of DNA fragments synthesized from overlapping nucleotides using Gibson assembly. (Right) Assembly of DNA fragments synthesized from overlapping nucleotides in yeast artificial chromosome (YAC, in purple).

within a target pathogen at the site of infection. effects have been reported in bacteremic pa- Whereas autoamplification may require a cer- tients successfully treated with phages (Kutter tain concentration of phage particles to accu- et al. 2014). mulate at the site of infection, it is believed that Using molecular biology, scientists have al- amplification can substantially lower the dose ready created bacteriophages coated in cell-per- required to treat an infection (Abedon 2011). As meable peptides, allowing them to diffuse into replication absolutely requires the presence of human cells or through the blood–brain barrier the target bacteria, phages are self-limiting (Staquicini et al. 2011; Rangel et al. 2012). and quickly cleared following pathogen eradi- Additionally, phages are known to encode and cation (Abedon 2011). Phage replication results can be engineered to produce , allowing in the lysis of target cells and potential release of them to degrade bacterial biofilms (Lu and Col- bacterial toxins, an important concern especial- lins 2007). Researchers have also created non- ly for endotoxin encoding Gram-negative path- replicating or lysis-deficient phages to deliver ogens (Hagens et al. 2004). However, no toxic bacterial static toxins, enabling inhibition of

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Engineering Engineering Engineering

Wild-type phage B Phage B V1.0 Phage B V2.0 Phage B V3.0 Biology Synthetic of Era the in Therapy Phage

Figure 3. Iterative engineering of phage genomes to create phages with superior therapeutic potential. Phage B V1.0 represents a phage with enhanced host range (includes the host range specific to phage A). Phage B V2.0 delineates iterative engineering of phage B V1.0 to include moieties aiding in biofilm disruption. Similarly, phage B V3.0 captures the properties of phage B V2.0 and is engineered to express antimicrobial payloads that may aid in elimination of bacterial subpopulation that lost susceptibility to phage. 5 Downloaded from http://cshperspectives.cshlp.org/ on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press

E.M. Barbu et al.

bacterial growth without release of toxins biologics than small molecule antibiotics. Like through lysis (Hagens et al. 2004; Matsuda et other biologics, phage are difficult to fully char- al. 2005; Fairhead 2009). Whereas viral size is acterize, and are sensitive to manufacturing and unlikely to be meaningfully altered, genetically handling conditions, making their production programing phages with the ability to produce much more complex than chemical drugs small peptides with useful properties could off- (Kingham et al. 2014). However, a handful of set many of the size constraints inherent to phage products have been approved by Ameri- classic biologics (Fig. 3). can regulatory agencies, including the U.S. Food and Drug Administration (FDA), granting mul- tiple bacteriophages generally recognized as safe Human Immune Response and Phage (GRAS) status when used as antimicrobial Clearance agents in human food (Soni and Nannapaneni The mammalian recog- 2010; Carter et al. 2012; Woolston et al. 2013). nizes and clears viral particles, creating a major Further, phage cocktails for human therapeutic hurdle for any viral-based therapeutic (Geier use have been produced for decades within the et al. 1973). Phagocytic cells located in the retic- former Soviet Republics of Eastern Europe, ular connective tissue play a key role in rapidly indicating some level of safety and efficacy (Su- clearing circulating phage particles through the lakvelidze et al. 2001). Scalable oncolytic innate immune mononuclear phagocyte system current good manufacturing practice (cGMP)- (MPS) (Merril et al. 1996). Whereas the MPS compliant processes provide significant insight may form the first major barrier to phage circu- into the methods and regulatory oversight lation, the adaptive immune response provides required for viral therapeutic production. Inter- the second. The adaptive response mediated by estingly, the substantially higher cost of produc- B/T cells takes 5–7 d to produce robust anti- ing biologics than chemical therapeutics has not body titers against phage in naı¨ve humans, pro- prevented their rapid ascension into the ranks of viding an ample window for phage therapeutic the best selling drugs in the United States. Un- activity (Ochs et al. 1971; Wedgwood et al. fortunately, phage therapeutic production costs 1975). However, it is hypothesized that the adap- relative to multidrug-resistant (MDR) infection tive memory response may prevent the reuse of rates may still provide the largest single barrier phage therapy vectors within the same patient, to phage therapy success. Many phage therapy as protective will already have been products currently being tested in clinical trials raised (Go´rski et al. 2012). or used in Eastern Europe constitute a cocktail Long-circulating phage harboring single of multiple independent types of phages mutations in the major capsid pro- (Wright et al. 2009; Abedon et al. 2011). Current tein have already been selected in vivo and Western medical standards would require each shown to provide an increased evasion of MPS independent phage to be produced and purified by more than 10,000-fold, drastically improving under cGMP-compliant processes, multiplying phage therapy efficacy (Merril et al. 1996; Vi- production costs to prohibitive levels (Kingham tiello et al. 2005). Thus, engineering of muta- et al. 2014). tions that enhance pharmacokinetics properties The ability to generate whole bacteriophage of phage capsids may improve the therapeutic genomes directly from synthetic oligonucleo- potential of multiple phage vectors for anti- tides will be attractive to regulators, as it de- microbial and phage-based therapeutic nano- creases contamination with adventitious agents. applications. Additionally, launching and amplifying bacter- iophage in cell-free reactions could significantly lower production costs and contamination Regulatory and Production Challenges (Kerr and Sadowski 1974; Gunther et al. 1993; Bacteriophages are large and complex therapeu- Shin et al. 2012). These synthetic biology ad- tics more similar to other genetically engineered vances coupled with our growing ability to en-

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Phage Therapy in the Era of Synthetic Biology

gineer bacteriophage with expanded host range, (Tanji et al. 2005). Similar studies found that antiresistance mechanisms, and immune eva- a threshold of an orally administered phage sion capabilities may lower the number of cocktail was required to reduce the colonization phages and cost of production to a level attrac- of mice infected with pediatric diarrhea E. coli tive to regulators and commercial entities (Mer- clinical isolates (Chibani-Chennoufi et al. ril et al. 1996; Tetart et al. 1996, 1998; Samson 2004). The effect of phage treatment on fecal et al. 2013). Last, the decreasing cost of sequenc- counts was negligible, which led the investiga- ing will greatly aid the speed and accuracy of tors to hypothesize that either phages lose infec- matching a particular therapeutic phage with tivity because of exposure to acid during gastric a susceptible , further limiting the known passage or that once in the gut E. coli becomes challenge of phage host range. resistant to phage infection. Although subse- quent investigations showed that intestinal tran- sit has no impact on phage infectivity, protec- PHAGES AS ANTI-INFECTIVES tion from gastric acidity by microencapsulation Numerous strategies have been used to show or coadministration with may enhance that virulent phages are realistic alternatives to their efficacy (Stanford et al. 2010). Thus, acid standard antibiotics. However, insight into the sensitivity must be characterized for individual potential clinical and therapeutic relevance of phage before oral administration. preclinical efficacy models of phage treatment The preventative and therapeutic efficacy is often obscured by a lack of standardized tech- of oral administration of phage was also exam- niques and protocols and, as a consequence, vast ined in a chicken model of jejuni differences in therapeutic outcomes. Whereas decolonization (Wagenaar et al. 2005). To show some studies report superior therapeutic value therapeutic effect, poultry received an oral dose 5 (McVay et al. 2007; Oliveira et al. 2010; Gu et al. of 10 CFU C. jejuni, followed by inoculation 10 2012; Hall et al. 2012; Jaiswal et al. 2013), others with a single phage (10 PFU) for six successive note marginal reduction in bacterial loads days starting 5 d after the bacterial challenge. (Rozema et al. 2009). Nonetheless, results stem- Initially, a 30-fold decrease in C. jejuni viable ming from recent attempts to show efficacy of counts was observed in the therapeutic group. phage therapy exemplify how delivery routes, However, after 5 d, phage and bacteria reached single or multiple treatments, monophage ver- equilibrium most likely because of the presence sus polyphage therapy, and dosage/timing of of a resistant subpopulation of microbes. administration contribute to therapeutic suc- Hence, using a cocktail or an engineered phage cess in preclinical models of infection. with awide host range rather than a single phage may overcome bacterial resistance. The timing of treatment also affects the suc- Lessons Learned from Preclinical Phage cess of phage therapy. Prophylaxis with phage Therapy KPP1 in a model of – induced gut-derived septicemia had little to no Oral Administration of Phages effect on mice survival, whereas concomitant Per os administration of phage has been found administration of phage and bacteria induced suitable for treatment of gastrointestinal infec- significant protection against infection. tions. For example, a cocktail of three bacterio- phages administered orally in a single dose Parenteral Administration of Phages (108 PFU/mL) resulted in significant reduction of coli O157:H7 in the feces of mice Intramuscular injection (i.m.) of different con- (99%). Further examination revealed that the centrations of phage R in chickens and calves most successful administration, as determined suffering from E. coli–derived septicemia by reduction in E. coli load, was daily oral in- showed that significant protection is conferred gestion of high amounts of phage (1010 PFU/mL) by a high concentration of phages (Barrow et al.

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E.M. Barbu et al.

1998). Similarly, intraperitoneal (i.p.) injection eliminated when the multiplicity of infection of ENB6 phage in a vancomycin-resistant En- (MOI) of viral particles to bacteria was above terococcus faecium mouse bacteremia model 100. However, the minimum levels of phage within an hour of bacterial challenge rescued particles necessary to significantly reduce colo- all experimental animals (Biswas et al. 2002). nization were specific for each within Several other reports highlighted the rapid dis- a species (Goode et al. 2003). tribution and bioavailability of phage on i.m. Recent reports highlighted the successful or i.p. injection. For instance, phage FMR11 treatment of –induced appeared in the blood shortly after i.p. injection burn wound infection in mice with the Klebsiel- into a aureus mouse bacteremia la-specific phage Kpn5 incorporated in a 3% model, and high levels of circulating phage hydroxypropylmethylcellulose hydrogel (Ku- could be detected until bacteria were eliminated mari et al. 2010). On bacterial challenge, mice (Matsuzaki et al. 2003). Most importantly, were treated once with low and high doses of McVay et al. (2007) showed that, in the case of phage hydrogel (MOI of 1 or 100, respectively). a P. aeruginosa–specific phage cocktail, i.p. in- Significant levels of protection against invasive jection of phage has enhanced their therapeutic K. pneumonia infection were achieved only potential. In the absence of phage administra- by high titers of phage. These results indicate tion, almost all wounded mice (94%) suc- that the dosage of phage is important for effec- cumbed to the infection within 3 d. In contrast, tive phage therapy. However, whether applica- the rate of mortality was reduced to 12% when tion of high titers of phage triggers bacterial the phage cocktail was delivered by i.p. in- death because of a productive infection or lysis jection. A low percentage of animals (28% or from without remains to be determined. It is 22%, respectively) were rescued by i.m. or sub- possible that the need for excessive MOIs may cutaneous phage delivery. In summary, these be overcome by multiple applications of phage results suggest that the route of parenteral ad- following an empirically determined treatment ministration must be carefully chosen based on schedule. empirical phage pharmacokinetics data and Otic administration of a phage cocktail in tailored to the type of infection requiring treat- dogs suffering from P. aeruginosa otitis has ment. At the same time, the dosage levels appear shown great promise. Two days after instillation to directly correlate with the success of phage of a single dose of phage into the auditory canal therapy. of one ear, bacterial counts decreased signifi- cantly. Concomitantly, phage titers increased and no adverse effects were registered. Although Local Application of Phage the cocktail formulation was not disclosed, otic Successful topical administration of phage has phage therapy may be a suitable alternative to been widely reported. Although early studies antibiotics. Moreover, amplification of phage at focused almost solely on wound healing, more the site of infection indicates that the infectious recent investigations also include otic and ocu- agent was susceptible to at least one phage ad- lar applications (Kumari et al. 2009; Hawkins ministered and the dose applied was amenable et al. 2010; Fukuda et al. 2012). For certain mi- to a productive viral infection (Hawkins et al. crobes, skin and mucosal colonization repre- 2010). sents a prerequisite for infection (Casadevall and Pirofski 2000). Therefore, effective decon- Inhalation of Phage tamination with an antimicrobial that has no effect on normal microbiota significantly de- In vivo efficacy of phage therapy against Burk- creases the risk of disease. Early attempts to holderia cepacia (isolated from the sputum of use phage for decolonization of chicken skin patients with cystic fibrosis) respiratory tract indicated that enterica, C. jejuni, infections was evaluated in a mouse model of and Campylobacter spp. could be effectively acute lung infection (Carmody et al. 2010).

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Phage Therapy in the Era of Synthetic Biology

Twenty-four hours postintratracheal bacterial has been achieved by pretreatment with bacter- challenge, mice were treated by i.p. injection or iophage (Curtin and Donlan 2006). In similar intranasal inhalation with a single phage at an studies, Fu et al. (2010) investigated the effect of MOI of 100. No significant differences in lung pre-, post-, and recurring treatment of Foley bacterial load or inflammatory levels catheters with a cocktail of phage on P. aerugi- were found between untreated mice and rodents nosa biofilm formation. Significant reduction of treated intranasally with phage. In contrast to bacterial density was observed regardless of the inhalation, systemic administration of phages timing of treatment (Fu et al. 2010). However, was moderatelyeffective, suggesting that the sys- regrowth of biofilm occurred between 24 and temic application of phages may facilitate acces- 48 h. Importantly, sequential treatments had sibility to bacteria present in the lungs. triggered mature biofilm dissolution. Another Debarbieux et al. (2010) reported multiple study reported prevention of biofilm formation studies of successful intranasal phage treatment on Foley catheters following impregnation of of P.aeruginosa pulmonary infection in a mouse neutral hydrogel (Lubri-Sil, Bard, Covington, model. In a first study, phage were administered GA) loaded with lytic bacteriophages (Carson either 24 h before or 2 h after bacterial challenge et al. 2010). The densities of E. coli and Proteus with a bioluminescent strain of P. aeruginosa. mirabilis on the silicone tubing were reduced No significant difference in survival of untreat- by 90% after 24 h of exposure to the gel. ed animals and mice treated with the lowest dose These results highlight the potential use of of phage (MOI of 0.1) was observed. A sub- phage cocktails for mitigation of biofilm forma- stantially higher dose of phage (MOI of 10) tion by clinically relevant bacteria on the sur- facilitated complete recovery of infected mice. faces of indwelling medical devices. Moreover, Further analysis revealed that phage propagated wide host range phage engineered to express within lungs, underscoring the importance of biofilm disruptive enzymes may become the dosage and timing in phage therapy. Given that next-generation coating materials for indwell- treatment was delivered shortly after bacterial ing devices. infection, it is difficult to predict whether inha- lation of phage would be successful for treating Anti-Infective Phage Therapy in Humans lower pulmonary tract or chronic infections. These data indicate that inhalation of phage Before the late 1930s, phage therapy was used may confer therapeutic and prophylactic pro- worldwide to treat infections. In the United tection against pulmonary infection. States, phage therapy was of interest mainly be- tween the 1920s and the 1930s. However, the advent of antibiotics and a couple of controver- Prevention of Biofilm Formation and Medical sial opinions regarding the preparation and ef- Device–Associated Infections ficacyof phage lysates led to decreased interest in Indwelling medical devices are often used to re- phage therapy (Eaton and Bayne-Jones 1934). store function or regenerate tissue. However, the Nonetheless, seven phage products for human propensity to become colonized by bacteria and use were produced by Eli Lilly (Indianapolis, serve as abiotic support for biofilm formation IN). Another product, Staph Phage Lysate, was leading to medical device–associated infections developed by Delmont Laboratories (Swarth- undermines their use in medical practice. Over more, PA) and licensed for human use in 1959 the past couple of decades, therapeutic manage- and veterinary use in 1987 for treatment of ca- ment of implant-associated infections has been nine pyoderma. Phage therapy continued to be further complicated by the emergence of antibi- used in France until the 1990s when phage prep- otic-resistant bacteria (Arias and Murray 2009; arations were discontinued. Ever since then, Boucher et al. 2013). phage therapists have continued their practice Mitigation of Staphylococcus epidermidis with products obtained from Poland and the biofilm formation on hydrogel-coated catheters United States, where phage therapy is included

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E.M. Barbu et al.

in the current health care standard (Abedon affects wound healing. Although outside of the et al. 2011). scope of the , efficacy could not be Many studies have been conducted to eval- established. Currently, several other clinical uate the efficacy and safety of phage therapy and studies are being conducted, which highlights much clinical experience has been accumulated, the interest of the scientific community in de- especially in Eastern Europe (reviewed in Alisky veloping bacteriophage as antimicrobials (Kut- et al. 1998; Summers 2001; Sulakvelidze et al. ter et al. 2014). However, the development of 2001; Go´rski et al. 2009; Kutter et al. 2010; Abe- phage for contemporary clinical use will likely don et al. 2011; Burrowes et al. 2011; Harper rely on the synthesis of phage with built-in char- et al. 2011; Chanishvili 2012). However, these acteristics (such as wide host range, biofilm-de- trials did not follow current Western rigorous grading enzymes, and secondary antimicrobial standards. The first double-blind, randomized, payloads) that can be evaluated in rigorous clin- placebo-controlled phase I trial to show the ical trials (Fig. 4). safety of phage treatment was performed by Nestle Research Center (Lausanne, Switzerland) PHAGE IN THE (Bruttin and Brussow 2005). Investigators AND DEGENERATIVE DISEASES showed no significant side effects following administration of phage and also showed that Natural phages have been long considered poor oral administration of T4 did not disturb the gene delivery vehicles because they only infect natural gut E. coli population. In subsequent bacteria (Ivanenkovet al. 1999). Recognizing the studies, the group performed a detailed meta- potential of phages for gene therapy, Hajitou genomics analysis of their entire T4-related et al. (2006) engineered a new generation of antidiarrheagenic phage collection and assessed hybrid prokaryotic–eukaryotic nanovectors as the clinical risk of a subset of phages following a chimera between eukaryotic adeno-associated oral administration in healthy adults (Sarker virus (AAV) and the filamentous M13 bacterio- et al. 2012). phage, referred to as adeno-associated virus/ The first reported randomized controlled phage (AAVP).This phage particle expresses the trial evaluated both safety and efficacy of phage cyclic peptide RGD4C (CDCRGDCFC) ligand therapy against chronic otitis caused by antibi- on the phage pIII minor coat , allowing otic-resistant P. aeruginosa (Wright et al. 2009). systemic and specific targeting to the avb3- No treatment-related adverse events were re- integrin receptor, which is present primarily ported, and the results showed that topical on tumor vasculature and tumor cells, and is administration of a therapeutic cocktail (Bio- expressed at barely detectable levels in normal phage-PA, Biocontrol, UK) in the ear results endothelium and tissues (Folkman 1997). The in an improvement of clinical manifestation of hybrid nanovector genome was also modified by the infection. However, the patients were select- inserting an engineered AAV(recombinant ad- ed based on being infected with a P. aeruginosa eno-associated virus [rAAV]) cassette strain susceptible to one or more of the phage into an intergenomic region of the phage included in the cocktail, which shows that, sim- genome, under the regulation of the cytomega- ilar to antibiotics, a phage antibiogram is nec- lovirus (CMV) and flanked by full- essary before treatment. length inverted terminal repeats (ITRs) from The first FDA-approved phase I clinical trial AAVserotype 2. The ITRs improve transduction of phage therapy was performed in 2007 at the efficiency and enhance transgene expression by Southwest Regional Wound Care Center in Lub- maintaining and forming concatemers of the bock, Texas to evaluate local administration of a eukaryotic transgene cassette (Hajitou et al. small set of well-characterized phage in patients 2006; Trepel et al. 2009). Further, this vector with chronic venous leg ulcers (Rhoads et al. has been engineered to carry the herpes simplex 2009). The study showed that topical phage ad- gene cassette suitable for tu- ministration neither had any adverse effects nor mor treatment with ganciclovir and positron

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Phage Therapy in the Era of Synthetic Biology

Classical Synthetic phage therapy phage therapy

Identification Cocktail of synthetic phage with: of etiological agent - Wide host range - Enhanced biofilm penetration Phage library screening - Secondary antimicrobial payloads - Resistant to clearing mechanisms

Therapeutic phage formulation In vitro and preclinical Single of multiple phage dose application cGMP product manufacturing

Clinical assessment Clinical efficacy and Reformulation to address FDA approval resistant bacteria or biofilm

First-line/adjuvant or First-line/adjuvant or last-line therapy last-line therapy

Figure 4. Phage therapy approaches. Classical (or Eastern European approach) relies on extensive banks of phages that can be used for treatment on a case-by-case basis by a physician specializing in phage therapy and adjusted based on clinical assessment. The synthetic phage therapy approach aims to develop phage with drug- like properties that can be used, much like antibiotics, in standardized Western treatment approaches. cGMP, Current good manufacturing practice.

emission tomography imaging for theranostic Recent studies have shown that the linear approaches (Soghomonyan et al. 2007; Hajitou structure of M13 bacteriophage permits bind- et al. 2007, 2008). The RGD4C-AAVPhas been ing to b-amyloid and a-synuclein , used to deliver tumor necrosis factor a (TNF-a) leading to plaque disaggregation in models of to the angiogenic vasculature of human mela- Alzheimer’s and Parkinson’s diseases. Convec- noma xenografts in nude mice (Tandle et al. tion-enhanced delivery of M13 phage to the 2009). Following systemic administration of brain of nonhuman primates confirmed distri- phage, the TNF-a expression was shown to be bution into both white and gray matter, which specifically localized in tumors, leading to apo- makes filamentous phage very attractive thera- ptosis in tumor blood vessels and significant nostics for direct plaque dissolution, as well as inhibition of tumor growth, while remaining delivery of therapeutic and imaging agents into virtually undetectable in all other tissues, nota- the brain (Frenkel and Solomon 2002; Ksend- bly the and spleen. The efficacy of targeted zovsky et al. 2012). RGD4C-AAVPexpressing the TNF-a has been also shown in dogs with soft tissue sarcoma PHAGE-BASED VACCINOLOGY (Paoloni et al. 2009). This vector was modified even further to replace the CMV promoter with Recombinant DNA technology allows synthesis the tumor-specific promoter Grp78, which of vaccine candidates that are subunits of path- drives expression only in the targeted tumor ogens. However, such vaccines have limited im- vasculature (Kia et al. 2012). munogenic features and require adjuvants or

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effective delivery systems for proper activation CONCLUDING REMARKS of the immune system (Petrovsky and Aguilar The global escalation of bacterial resistance to 2004; Barrett and Stanberry 2008). Thus, antibiotics raises the possibility of returning to phages are now being evaluated as vaccine de- the clinical equivalent of the preantibiotic era livery agents because of their inherited ability to and requires the development of new classes of stimulate humoral and cell-mediated immunity antimicrobials that could strengthen the effec- (Burdin et al. 2004). Two strategies are often tiveness of current use drugs. Phage therapy is a combined to produce phage vaccines: (1) phage promising, yet challenging, strategy for the de- display, when virions are decorated with pep- velopment of new antimicrobial that tides selected for their ability to bind - can enable more strategic treatment approaches presenting cells; and (2) phage DNA vaccines, that do not affect beneficial normal microbiota. when viral DNA is engineered to carry a foreign Another major benefit of phage therapy is that antigen gene under the control of a strong eu- the mode of action of bacteriophage is distinct karyotic promoter and has the ability to deliver from those of antibiotics; thus, bacterial strains this element to mammalian cells (Clark and that are resistant to antibiotics are nevertheless March 2004a; Zanghi et al. 2007). susceptible to phage infection. However, limi- Filamentous phage M13 has been the first tations such as narrow host range, bacterial re- virus manipulated to express a - sistance to phage, cost of manufacturing, and specific tumor antigen fragment and has been challenging dose finding/delivery methods successfully used to raise an immune response need to be addressed. Recent advances in syn- capable of reducing tumor growth in animal thetic genome assembly and viral genome engi- models (Benhar 2001; Fang et al. 2005). Cur- neeringcanbeusedtocreatephagewithsuperior rently, several vaccines for infectious diseases therapeuticproperties.Designersyntheticphage are prepared by using the T4 sys- may enable new intellectual property and attract tem, which has shown promising results in an- interest from large pharmaceutical companies in imal models (Jiang et al. 1997). Similarly, phage these products. Moreover, these techniques can T7 has been engineered to display vascular en- also be applied to engineering novel theranostic dothelial growth factor (VEGF) and has been nanovectors with improved tissue penetration successfully used to break immunologic toler- and payload carrying potential for treatment of ance and produce a strong immune response cancer and other diseases. against Lewis lung cell carcinoma (Li et al. 2006). Alternatively, phage can be exploited to transfer genes into mammalian cells. In these REFERENCES vectors, the , under the control of a eu- Abedon S. 2011. Phage therapy pharmacology: Calculating karyotic promoter, are cloned inside of a nones- phage dosing. Adv Appl Microbiol 77: 1–40. sential region of a phage genome. When injected Alisky J, Iczkowski K, Rapoport A, Troitsky N. 1998. Bacte- in a mammalian system, these phage particles, riophages show promise as antimicrobial agents. J Infect acting as a DNAvaccine, can induce potent im- 36: 5–15. Arias CA, Murray BE. 2009. Antibiotic-resistant bugs in the mune response by expressing foreign antigen in- 21st century—A clinical super-challenge. N Engl J Med side of anaphase-promoting complexes (APCs) 360: 439–443. or other cells (Clark and March 2006). Several Barrett ADT, Stanberry LR. 2008. Vaccines for biodefence l-based DNA vaccines for infectious diseases and emerging and neglected diseases. Academic, Waltham, MA. have been prepared that have shown promising Barrow P, Lovell M, Berchieri A Jr. 1998. Use of lytic bacte- results in animal models (Clark and March riophage for control of experimental sep- 2004b; March et al. 2004). It should be noted ticemia and meningitis in chickens and calves. Clin Diag that these viruses are manipulated to penetrate Lab Immun 5: 294–298. Baumann A. 2006. Early development of therapeutic bio- mammalian cells and, in the absence of engi- logics—Pharmacokinetics. Curr Drug Metab 7: 15–21. neering, phages are only capable of infecting Benhar I. 2001. Biotechnological applications of phage and bacteria. cell display. Biotech Adv 19: 1–33.

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Phage Therapy in the Era of Synthetic Biology

E. Magda Barbu, Kyle C. Cady and Bolyn Hubby

Cold Spring Harb Perspect Biol 2016; doi: 10.1101/cshperspect.a023879 originally published online August 1, 2016

Subject Collection Synthetic Biology

Minimal Cells−−Real and Imagined Synthetic DNA Synthesis and Assembly: Putting John I. Glass, Chuck Merryman, Kim S. Wise, et al. the Synthetic in Synthetic Biology Randall A. Hughes and Andrew D. Ellington Synthetic Botany Design Automation in Synthetic Biology Christian R. Boehm, Bernardo Pollak, Nuri Evan Appleton, Curtis Madsen, Nicholas Roehner, Purswani, et al. et al. Synthetic Biology in Cell and Organ Cell-Free Synthetic Biology: Engineering Beyond Transplantation the Cell Sean Stevens Jessica G. Perez, Jessica C. Stark and Michael C. Jewett Genome-Editing Technologies: Principles and The Need for Integrated Approaches in Metabolic Applications Engineering Thomas Gaj, Shannon J. Sirk, Sai-lan Shui, et al. Anna Lechner, Elizabeth Brunk and Jay D. Keasling Alternative Watson−Crick Synthetic Genetic Synthetic Biology of Natural Products Systems Rainer Breitling and Eriko Takano Steven A. Benner, Nilesh B. Karalkar, Shuichi Hoshika, et al. Phage Therapy in the Era of Synthetic Biology At the Interface of Chemical and Biological E. Magda Barbu, Kyle C. Cady and Bolyn Hubby Synthesis: An Han Xiao and Peter G. Schultz Synthetic Morphogenesis Building Spatial Synthetic Biology with Brian P. Teague, Patrick Guye and Ron Weiss Compartments, Scaffolds, and Communities Jessica K. Polka, Stephanie G. Hays and Pamela A. Silver Engineering Gene Circuits for Mammalian Cell− Based Applications Simon Ausländer and Martin Fussenegger

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