Bacteriophages as vehicles for gene delivery into mammalian cells: prospects and problems.

Review

Bacteriophages as vehicles for gene delivery into mammalian cells: prospects and problems 1.

Introduction

2.

Bacteriophages: application as biotechnological tools and GDVs

3.

The interaction between

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phages and mammalian cells: an evolutionary chronicle 4.

Broad tropism as a double-edged sword

5.

Transduction of mammalian cells with phages: targeting strategy through phage display

6.

RME and phage-based gene delivery

7.

Mammalian intracellular barriers for phage-mediated gene expression

8.

Conclusion

9.

Expert opinion

Babak Bakhshinejad & Majid Sadeghizadeh† †

Tarbiat Modares University, Department of Genetics, Faculty of Biological Sciences, Tehran, Iran

Introduction: The identification of more efficient gene delivery vehicles (GDVs) is essential to fulfill the expectations of clinical gene therapy. Bacteriophages, due to their excellent safety profile, extreme stability under a variety of harsh environmental conditions and the capability for being genetically manipulated, have drawn a flurry of interest to be applied as a newly arisen category of gene delivery platforms. Areas covered: The incessant evolutionary interaction of bacteriophages with human cells has turned them into a part of our body’s natural ecosystem. However, these carriers represent several barriers to gene transduction of mammalian cells. The lack of evolvement of specialized machinery for targeted cellular internalization, endosomal, lysosomal and proteasomal escape, cytoplasmic entry, nuclear localization and intranuclear transcription poses major challenges to the expression of the phage-carried gene. In this review, we describe pros and cons of bacteriophages as GDVs, provide an insight into numerous barriers that bacteriophages face for entry into and subsequent trafficking inside mammalian cells and elaborate on the strategies used to bypass these barriers. Expert opinion: Tremendous genetic flexibility of bacteriophages to undergo numerous surface modifications through phage display technology has proven to be a turning point in the uncompromising efforts to surmount the limitations of phage-mediated gene expression. The revelatory outcomes of the studies undertaken within the recent years have been promising for phage-mediated gene delivery to move from concept to reality. Keywords: bacteriophage, gene delivery, gene therapy, intracellular barriers, phage display, targeting Expert Opin. Drug Deliv. [Early Online]

1.

Introduction

The fundamental ideology underlying gene therapy is the targeted delivery of a foreign genetic entity with therapeutic properties to specific cells and tissues. It has been suggested that the key to success of gene therapy is to introduce safe and efficient delivery vehicles [1]. Over the recent years, considerable efforts have been devoted to develop vectors ideally suited to application in the treatment of diseases. An ideal gene delivery vehicle (GDV) is characterized by some attributes including safety, low cost of production, efficiency and target cell specificity. Some of these characteristics are required to attain selective and long-lasting expression of the therapeutic gene in cells or tissues of interest without exerting toxic effects on normal parts of the body [2,3]. The strategies currently being used for the delivery and expression of genes in mammalian cells can be categorized into two main types: vectors derived from eukaryotic viruses and nonviral vehicles [4]. The viruses most frequently used as gene transfer carries are adenovirus [5], adeno-associated virus [6], gammaretrovirus [7] and lentivirus [8,9]. Also, numerous nonviral vehicles are used 10.1517/17425247.2014.927437 © 2014 Informa UK, Ltd. ISSN 1742-5247, e-ISSN 1744-7593 All rights reserved: reproduction in whole or in part not permitted

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B. Bakhshinejad & M. Sadeghizadeh

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Bacteriophages are emerging as a promising category of gene delivery vehicles for clinical purposes. The incessant and long-term evolutionary relationship between bacteriophages and mammals has turned phages into a biologically significant part of the natural microflora of the mammalian body. Phage display is a potent molecular methodology that can be applied to introduce a wide variety of surface modifications for molecularly targeting phage vectors into specialized cells and tissues. Receptor-mediated endocytosis can be used by targeted phage vectors for selective delivery of gene therapeutics into mammalian cells. When internalized into mammalian cells, bacteriophages are faced with numerous intracellular barriers for expressing their encoded therapeutic genes. Bacteriophage vectors can undergo surface modifications, thereby developing the capability of endosomal and lysosomal release, proteasomal escape, cytoplasmic entry, nuclear localization and intranuclear gene transcription for expressing their encoded therapeutic genes in mammalian cells.

This box summarizes key points contained in the article.

for gene delivery purposes, the most prevalent of which include, but not limited to, polymers [10] and liposomes [11]. Both types of approaches mentioned represent some benefits and pitfalls to gene transfer. Nonviral vectors are low cost and less complicated, but their use is not without difficulties and their clinical application is challenging. These vehicles are inefficient transducers and, more importantly, their potential to undergo engineering for targeting is limited [12]. The exploitation of virus-based carriers as GDVs also imposes challenges that include time-consuming, laborintensive and costly procedure of their production in eukaryotic, especially mammalian, cells. This process necessitates the generation of vectors that are free of replication-competent viral particles. There exist as well some deep concerns about the inherent toxicity of viral vectors that result from their recognition by the immune system [13,14] and their oncogenic integration into the genome of host cells [15]. These complications have triggered attempts to find novel GDVs with desirable properties for gene therapy. Recently, bacteriophages----also abbreviated as phages----have received enormous amount of attention as a novel category of gene delivery vectors. The attraction of bacteriophages for gene delivery and therapy is because of their high safety profile. This safety results from their long-term residence in our body forming a part of the body’s natural ecosystem. Bacteriophages also provide the possibility of being manipulated----both genetically and chemically----to improve the efficiency of gene transfer. This review aims to yield an insight into the use of bacteriophages as gene delivery platforms. We discuss the 2

interaction of bacteriophages with mammalian cells from an evolutionary perspective. Furthermore, the pros and cons of bacteriophages for gene therapy are addressed in detail. The flexibility of bacteriophage vectors to undergo various surface modifications allows their targeted delivery towards specific cell types. This paves the way for taking advantage of receptor-mediated endocytosis (RME) as an efficient and powerful strategy for the entry of bacteriophages into mammalian cells. When internalized, the bacteriophages encounter a variety of barriers that interfere with the expression of the transferred gene. In this regard, we elaborate on the numerous intracellular barriers for the delivery and ultimate expression of the phage-encoded genes and different strategies that have been used to bypass these barriers.

Bacteriophages: application as biotechnological tools and GDVs

2.

Bacteriophages are viruses that specifically infect prokaryotic cells. The genetic makeup of these bacterial viruses is either DNA or RNA enclosed in a protein coat called capsid. They are metabolically inert, a feature that makes them dependent on their bacterial hosts for replication. To be propagated inside the bacterial cells, phages can follow two types of lifecycle: lytic or lysogenic. In the former, virulent (lytic) phages following replication are released from bacterial cells via actively degrading the cell wall and ultimately lysing the host cell. However, some phages are released from bacterial cells via budding from the cellular membrane. In the latter, temperate (lysogenic) phage particles integrate their genetic material into the host chromosome and, as a consequence, propagate together with the host bacterium [16]. These biological entities are regarded as the most abundant life forms in the biosphere and can be found in a wide spectrum of terrestrial and aquatic environments ranging from ocean’s depths and hot springs to arctic regions. Estimations have revealed that the approximate number of phages amount to 1030 particles in our planet outnumbering their bacterial hosts by 10-fold [16,17]. Interestingly, one of the habitats occupied by phages is the body of animals and human beings, particularly the gastrointestinal tract that is home to a large number of bacterial viruses. These gut-dwelling phages multiply by using symbiotic or pathogenic bacteria residing in the alimentary canal. Currently, this is a held view that phages, along with their specific bacterial hosts, form a major part of the gut flora [18]. Phages, since their landmark discovery by Ferederic Twort and Felix d’Herelle [19], have been used extensively in molecular biology especially as favorable cloning vectors. These prokaryotic viruses have enabled researchers to answer many fundamental questions and uncover numerous unresolved mysteries in molecular biology. Phages show astonishingly huge genetic diversity and it has been revealed that approximately half of newly identified phage genes do not have any known homologous counterpart deposited in genomic

Expert Opin. Drug Deliv. (2014) 11(10)

Bacteriophages as vehicles for gene delivery into mammalian cells

Diagnosis Prevention

Bacterial detection and typing Biosensor design Imaging

Vaccine development

Applications of bacteriophages in biotechnology and medicine


Treatment Gene delivery Drug delivery Phage therapy

Figure 1. Various potential applications represented by bacteriophages in biotechnology and medicine.

databases [20]. This extremely large gene pool as well as the flexibility of phages----indicated by their experimental tractability to undergo numerous genetic manipulations----has led them to gain widespread acceptability in the fast-growing field of modern biotechnology. Within the past years, there has been a resurgence of interest in making use of phages in a variety of biotechnological, pharmaceutical and clinical areas (Figure 1). These different areas range from prevention (development of the phage-delivered vaccines) and diagnosis (use of phages for bacterial detection and typing, biosensor design and imaging) to treatment (exploitation of phages as gene and drug carriers and also in phage therapy against pathogenic bacteria as alternatives to antibiotic compounds) [16,21]. One of the most fascinating aspects of phage studies is to use them as potential targeted GDVs for gene therapy of pathological conditions. It has been suggested that phages satisfy the requirements of an ideal gene therapy vector, thereby designated as future vehicles for gene delivery. In the light of the emergence of phage therapy concept and the practical exploitation of phages as an antibacterial remedy, there exists a long history of safe use of these biological agents in humans, even immunocompromised patients [22]. Furthermore, phage vectors have high stability under harsh environmental conditions such as exposure to nucleolytic and proteolytic enzymes and a diverse range of pH [23,24]. This extreme stability contributes to the viability of phages in the gastrointestinal tract putting forward the idea of their potential use for oral administration. In support of this notion, a number of studies have reported the transfer of phage particles from the digestive tract into the blood circulation after oral administration, both in animals and in human [25-27]. On the other hand, phage vehicles do not cause recombination events that can arise from the interchange of homologous sequences residing in the genomes of incoming vector and the host cell. As a matter of fact, the nonexistence of eukaryote-related sequences in the genome of phages functions as a barrier to these

recombination phenomena [28], thus clearing many doubts over their clinical safety. Nevertheless, with the development and evolvement of more efficient phage delivery vectors, questions such as the efficacy of these carriers for human gene therapy merit further consideration.

The interaction between phages and mammalian cells: an evolutionary chronicle

3.

A look at the large frequency of phages in a wide array of natural habitats, in particular the body of animals and human, reflects the fact that mammalian organisms have been extensively subjected to these viral particles. Historical evidence provides strong support to the notion of phage interaction with mammalian cells. The study conducted by Bloch in 1940 was presumably the first observation of this type, which indicated the accumulation of phages in cancer tissues and inhibition of tumor growth. In subsequent years, other investigations revealed the binding of phages to cancer cells in vitro and in vivo. Also, phage-neutralizing antibodies, the production of which is the outcome of eliciting humoral immunity by naturally occurring phages, have been detected in the sera of various species (including human), although these species had not been previously treated with phages [17]. These lines of evidence indicate that the natural contact between phages and mammalian cells is not incidental but rather strong and constant. This creates a long-standing and continuous evolutionary relationship between these particles and the cells of the body. Herein, it is important to note that one should distinguish between the concepts of interaction and infection because phages are able to interact with, but not infect and multiply in, mammalian cells. The isolation of long-circulating strains of lambda phage, Argo1 and Argo2, highlights the role played by capsid proteins in interaction with mammalian cells (in this case, the immune cells) [29]. These mutant strains were obtained in an


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attempt to select phage variants with more capacity for evading entrapment by the reticuloendothelial system (RES). This escaping property causes greater efficacy of phages for the treatment of bacterial infections. The long-circulating phenotype results from an amino acid substitution in the phage major head protein E at the solvent-exposed surface of phage particles. This alteration in the capsid protein diminishes phage rapid clearance by the defense system of the host animal and leads to the emergence of phage particles with much longer half-life in the circulatory system. In animals contaminated with pathogenic concentrations of bacteria, the mutant phage virions were demonstrated to be more potent antibacterial agents compared with the parental strain. On the other hand, recent findings have uncovered the fact that tailed dsDNA phages (Caudovirales) and mammalian viruses show considerable similarities at different levels including structural capsid proteins, DNA encapsidation mechanisms and packaging proteins [30,31]. These novel insights indicate a shared ancestry between prokaryote-infecting and eukaryoteinfecting viruses and trigger speculation that these very divergent lineages of viruses may have originated from a common primordial progenitor. The architectural similarities among the capsids of bacterial and mammalian viruses as well as the role suggested for phage capsid proteins----obtained from the study of Argo phages----provide compelling evidence about the potential capability of phages in making interaction with mammalian cells. It is interesting to note that deciphering the evolutionary connections between bacterial and eukaryotic viruses is currently a terra incognita requiring much further consideration. These deeply-rooted evolutionary links shed light on the modifications that primordial bacterial viruses underwent to gain the capacity of infecting new hosts and be converted into eukaryotic cell-specific viruses. In this process, the mammalian viruses have evidently evolved a novel machinery, different from those developed by bacterial viruses, to effectively infect their eukaryotic hosts. In addition to evolutionary implications, unraveling these different mechanisms can also have practical and clinical outcomes. Certainly, these data yield insightful information to develop efficient and targeted platforms for phage-based delivery of therapeutic genes into mammalian cells. 4.

Broad tropism as a double-edged sword

Perhaps, at the first glance, the broad tropism of eukaryotic viruses is viewed as an absolute advantage with entirely positive aspects for gene therapy. However, if we delve deeper into this issue, it becomes clear that this property acts as a double-edged sword and besides opportunities can also provide challenges for targeted gene therapy. Viral vectors can achieve high efficiency in the transduction of mammalian cells being recognized as the most efficient vectors for gene delivery [32]. This high level of gene transfer efficiency in eukaryotic viruses lies in the fact that they have evolved 4

essential and pivotal machinery for cell entry, trafficking inside the cell and ultimate expression of the delivered transgene. These efficiency-increasing traits have emerged within a long evolutionary course of interaction between viruses and their eukaryotic host cells and enabled the viral particles to transport their genetic material across plasma membrane, through cytoplasmic environment and finally the nuclear envelope of eukaryotic cells. Collectively, these evolutionarily acquired features confer broad natural tropism for mammalian cells on viral vectors. But, it is not the end of targeted delivery scenario. In addition to facilitation of gene delivery, this wide tropism for multiple cell types has negative aspects and raises concerns with regard to the selectivity of gene transfer; hence, this other side of viral-based gene delivery coin can pose major limitations to the specific delivery of therapeutic transgene into the target cell [32]. On the contrary, phage-based vectors are much less likely to enter eukaryotic cells. In the context of gene delivery, this lack of intrinsic tropism dramatically lowers the likelihood of nontargeted delivery and its consequent adverse effects [33-35]. For this reason, phage vectors show an inherent biological safety profile in mammalian organisms [36], making these vectors suitable for clinical uses. Hence, it is not necessary to modify phages for eliminating the possibility of nonselective tropism into mammalian cells; whereas for eukaryotic viruses homing to nontarget cells is the cause of major concern, thus emphasizing the need to introduce changes in the viral structure to remove this concern.

Transduction of mammalian cells with phages: targeting strategy through phage display

5.

The first reports on the capacity of bacteriophages to transduce mammalian cells date back to the early 1970s in which lambda phage was utilized to infect human fibroblast cells [37,38]. These pioneering studies provided the first evidence of practicality of using bacterial viruses for internalization and subsequent gene expression in mammalian cells as their atypical hosts. These findings prompted following attempts for phage particle-mediated gene transfer into cultured mammalian cells through chemical transfection techniques. In these chemical methods, transduction of mammalian cells with both double-stranded lambda phage and single-stranded filamentous phage was described [39-42]. Furthermore, cell transfection of phage particles was evaluated in the presence of different chemical agents including diethylaminoethanol dextran (DEAE) and lipopolyamine [41,42]. Interestingly, introducing foreign DNA into mammalian cells using f1 phage particles further revealed that the single-stranded DNA of a filamentous phage can be converted into the double-stranded form. This observation favored the idea that filamentous phages could serve as a carrier for the delivery and expression of foreign genetic cargoes in mammalian cells.



Cell surface receptors

pVIII

Bacteriophage genome

pIII


Eukaryotic cell

Targeting ligand

Filamentous bacteriophage

Cell membrane

Figure 2. The targeting of a phage virion towards a eukaryotic cell through phage display. Display of a targeting ligand as fusion to one of phage surface proteins (such as pIII) guides the phage virion towards a specific receptor on the surface of target eukaryotic cell, thereby directing the phage particle to a specialized cell type. There are different receptor molecules on the cell surface (indicated in various shapes). However, only the receptor able to attach to the phage-displayed moiety makes interaction with the targeting ligand.

These experiments extended the concept of phage-based gene delivery. However, these studies did not make any stride towards the development of targeted phage-based delivery platforms and the prospect of phages for targeted gene therapy was not deciphered. One of the desirable attributes of phage carriers is their genetic flexibility to undergo diverse surface manipulations. This capacity, known as phage display, was originally presented in the seminal and innovative paper of Smith [43]. The relatively simple genetics of bacteriophages makes them amenable to a huge variety of genetic manipulations showing itself in a plethora of structural modifications of capsid proteins. These surface modifications allow phages to be selectively targeted to mammalian cell surface receptors. In this procedure, a targeting ligand is displayed as fusion to one of the phage surface proteins. This homing ligand is able to bind to a membrane receptor of the target cell, thereby guiding the phage virion towards a specialized cell type (Figure 2). Furthermore, directed evolution can be conducted on large populations of phage variants that results in selecting phages with improved binding ability to a given target and specifically transducing desired cells and tissues [23]. Introduction of specific modifications, through display of targeting moieties, on the surface of phage particles is an approach to direct phages carriers towards a specific cell of interest. The study of Larocca et al., by genetically targeting filamentous phages into mammalian cells through a specific ligand-receptor binding provided a new perspective to the little-known territory of phage-based gene delivery [34,44].

They demonstrated that M13 phage displaying fibroblast growth factor 2 (FGF2) on its surface and harboring a cytomegalovirus promoter-driven green fluorescent protein (GFP) reporter gene can undergo RME and transduce COS-1 cells in a FGF2-specific manner. This ultimately led to the reporter gene delivery and expression in the cells. Within the past years, a variety of efforts have been focused to explore the potential of tailed and filamentous bacteriophage particles for targeted gene delivery and expression in vitro [45,46] and in vivo [36,47,48]. Different phages can be used as candidates for transgene delivery. However, lambda and, more importantly, filamentous bacteriophages take a special place in this area [49,50]. Various molecules including growth factors, antibodies and viral capsid proteins have been surface displayed and applied to phage targeting [34,44,51-54]. These findings show that the development of phage-based targeted gene delivery platforms has changed the portrait of gene delivery and brought new opportunities to the establishment of novel strategies for the targeted therapy of numerous pathological conditions including cancer. 6.

RME and phage-based gene delivery

Appropriate targeting is fundamentally critical to reach the expression of transgene in cells of interest as the ultimate goal of gene therapy. In targeting of phages, RME is an important issue requiring considerable attention. The ability of targeted phages to deliver genes to cells via RME is a valuable opportunity that provides a new breadth to the use of


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phage vectors in gene therapy [35]. RME is a typical strategy of eukaryotic viruses for the infection of host cells. In this scenario, specific compounds are selectively transported from the extracellular milieu into the cell cytosol [55]. This natural mechanism can be applied to targeted internalization of phage-borne therapeutic cargoes into mammalian cells. To this end, there is a need to couple, chemically or genetically, a ligand with high affinity for a cell surface receptor to the phage vector. This triggers binding of the ligand to its specific receptor and clustering of ligand-receptor complexes into clathrin-coated pits. Invagination of the cell membrane translocates the clathrin-coated endocytic vesicles containing the phage particles into specific intracellular compartments to exert their therapeutic effects. The relatively small diameter renders it possible for phage particles to be packaged into clathrin-coated vesicles. The impact caused by chemical inhibitors of clathrin-mediated endocytosis such as chlorpromazine in the decrease of phage-mediated gene transfer highlights the fact that cells can take up phage particles through endocytic pathway [56]. Receptor clustering is another factor contributing to the process of phage internalization via RME. The phenomenon of receptor clustering indicates the involvement of zipper mechanism through which cells can internalize relatively large particles such as phage virions. The zipper model creates multiple contact points between the phage-linked ligand and the cell surface receptor and these numerous contacts are crucial for maximizing the reaction avidity. This shows the significance of multivalent and high-density display of targeting moieties on the surface of phage particles to enhance the rate of phage internalization [57,58]. These targeting ligands can be natural (growth factors such as EGF, FGF2, etc., and their modified variants) or synthetic (short peptides). On the other hand, studies have demonstrated that inhibitors of actin polymerization such as latrunculin A led to a sizable decrease in the cellular uptake of phage particles and subsequent phage-mediated gene expression [56]. This favors the model that cytoskeleton components, including actin filaments, seem to be implicated in clathrin-coated vesicle-mediated endocytosis of ligands bound to the cell surface. In addition to RME, phagocytosis has also been proposed as another route for phage uptake into mammalian cells. This approach is used by viral entities such as dengue virus. However, the huge depletion of phagocytic cells via pretreatment of mice with clodronate-containing liposomes did not induce a considerable reduction in phage-mediated gene transfer implying that phagocytosis only contributes to a minor portion of gene transfer by phages [36,48].

Mammalian intracellular barriers for phage-mediated gene expression 7.

In the traditional viewpoint, targeting simply means binding and then internalization of the delivery vehicle of interest into the target cell. For this reason, to increase the efficiency of gene delivery through phages, initial efforts primarily 6

aimed to improve the binding capacity of phage particles to mammalian cells. But, it is not the whole story. The tale of targeting can extend beyond internalization and go further to gene expression. It has been well established that mammalian cells represent multiple barriers to phage-mediated gene expression and some postuptake events impose limitations on the expression of the delivered gene [56]. As a consequence, the efficiency of expression is less than the efficiency of internalization. This considerably low level of phage-based gene expression is due to the fact that phage particles have not developed the specialized tools (such as protein sequences), comparable with those of eukaryotic viruses, required for intracellular trafficking in mammalian cells. In the medium inside mammalian cells, phage particles face numerous obstacles to express their genetic cargo. These obstacles act as innate defense mechanisms for the cell to create a multilayered resistance against invasion of viral pathogens. In this context, phages are subjected to multiple degradative pathways resulting in their elimination from mammalian cells. When internalized, phages should survive the harsh environment of endosome and lysosome, be released from these compartments, escape proteasomal degradation, enter the cytoplasm and transfer their genomic payload to the nucleus, giving rise to the expression of the phage-encoded gene(s) [35,59]. Failure in each of these essential steps impedes the ultimate expression of the phage-delivered gene (Figure 3). Gaining insight into the rate-limiting steps, which affect phage-driven gene transfer and expression, is of particular importance for the development of sophisticated gene therapy vectors. Endosomal barrier The acidic milieu and proteolytic enzymes of endosome remarkably cause the degradation of capsid proteins present on the surface of phage virions. Studies have demonstrated the low capacity of phages to survive under the harsh environment of endosome [44,60]. This limited survivability is not sufficient for effectual trafficking through the endosomal pathway and does not meet the expectations for an efficient gene therapy vector. In contrast to phages, eukaryotic viruses have evolved specialized mechanisms to avoid undergoing intracellular degradation by the endosomal compartment [61,62]. To efficiently infect the host cell, different mammalian viruses have devised their characteristic means including endosomal escape sequences and endosome-disrupting factors [63-65]. Mimicking the strategies used by mammalian viruses has received attention for the development of vectors based on prokaryotic viruses. It has been shown that the surface display of endosome-disrupting and endosome-escaping proteins used by mammalian viruses can serve to potentiate phages for exit from the endosomal compartment, thereby promoting the efficiency of phage-based expression of foreign genes [52]. Adenovirus capsid protein Pb and influenza virus envelope protein hemagglutinin HA-2 are examples of the tools that mammalian viruses exploit to evade endosome-mediated degradation. Protein Pb of adenovirus underlies several biological functions including attachment to 7.1



Cell surface receptor

Cell membrane

No expression No expression

Proteasome Lack of NLS Degradation

Lysosome

Endosome

H+ H+ H+

H+


Nuclear pore complex Degradation

No expression

Degradation

No expression

Phage genome

Nuclear envelope Expression of phage-encoded genes

Figure 3. The intracellular barriers for expression of phage-encoded gene. A phage virion is attached to a specific receptor on the membrane of target cell. When this virion faces numerous intracellular barriers in order to express its genetic cargo. Incapacity to escape phage-eliminating elements including the acidic milieu of endosome, proteolytic enzymes of lysosome, degradative machinery of proteasome and also the lack of NLS sequences to cross the nuclear membrane leads to the degradation of phage virion and consequently the phage-carried therapeutic gene is not expressed. If able to bypass all of these intracellular obstacles, the phage virion containing the NLS peptide is delivered to the nucleus through nuclear pore complex that ultimately results in the expression of phage-encoded gene. NLS: Nuclear localization signal.

the cell surface receptor, internalization and, particularly, the escape of viral particles from endosome [52,63]. This protein has been indicated to be a promising candidate for targeting of phages to a specific trafficking route in mammalian cells and is able to confer an intracellular behavior similar to that of adenovirus on phage particles. Furthermore, the influenza virus exploits its envelope protein hemagglutinin HA-2 as an endosome-disrupting factor. The HA peptide derived from the N-terminal amino acid sequence of this protein triggers viral delivery via destabilizing the membrane of endosome under the acidic environment of this organelle [66]. The endosmolytic property of this peptide can be used to disrupt the endosomal compartment, leading to the elevation of phage-encoded gene expression. Lysosomal barrier The protein-degrading enzymes existing in lysosome account for breaking down the exogenous proteins (e.g., the 7.2

proteinaceous components of incoming viruses) internalized into the cell via endosomal route. Interestingly, some mammalian viruses in an evolutionary tit for tat tactic have seized these enzymes and use these proteases to their own advantage. In this condition, the activity of lysosomal proteases is exploited to promote the cytosolic entry and/or uncoating of viral particles and also activating viral fusion proteins. Adeno-associated virus [67], coronavirus [68], filovirus [69,70], reovirus [71,72] and retrovirus [73] apply this evolutionarily acquired strategy to find protection against the degradative machinery of the lysosomal compartment. Keeping this in mind, experiments have been undertaken to explore the role played by lysosomal proteases in the control of phage-mediated gene expression in mammalian cells. These investigations have demonstrated that inhibitors of cathepsin B and cathepsin L, the chief proteolytic enzymes of lysosome, give rise to a marked enhancement of the expression of luciferase gene present in the phage genome.


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Furthermore, simultaneous inhibition of both cathepsins elicited a more potent increase in reporter gene expression in comparison with single inhibition by either cathepsin B or cathepsin L. It has been shown that cathepsin proteins target the components of phage capsid for degradation. Due to this degradative activity on the structural proteins of capsid and as a result of loss of phage integrity, direct exposure of phage particles to cathepsin induces a robust decrease in phage viability [74]. These findings provide strong support to the notion that lysosomal proteases degrade internalized phage particles that are incapable of escaping the endolysosomal compartment. The negative regulation of these proteases on gene transfer of incoming phage represents limitation to phage-mediated gene expression. Prevention of the proteolytic activity of lysosome is viewed as a potential means to improve the expression of phage-encoded therapeutic gene.

expression via elevating both the mean level of gene expression per transduced cell and the overall number of transduced cells. Furthermore, experiments performed by incubating HEK 293A and COS-7 cells with luciferase-encoding phage particles in the presence or absence of bortezomib, MG132 and lactacystin (another pharmacological inhibitor of proteasome activity that irreversibly inhibits the 26S-proteasome complex) resulted in the augmentation of phage-mediated gene expression [74]. Taken together, these observations imply that the inhibition of proteasomal function leads to the survival of a significantly higher number of phage particles in the internal environment of mammalian cells with subsequent cytoplasmic escape and ultimately nuclear entry. Based on this notion, preventing the activity of proteasome complex can be exploited as a helpful approach for improving phagemediated gene expression.

Proteasomal barrier The proteasome complex plays an active role in many aspects of cellular function, including protein degradation, protein turnover, cell cycle progression and receptor signaling [75-77]. Proteasome has also been shown to be involved in regulating viral infection of mammalian cells. This regulatory function can be both positive and negative. The activity of proteasome promotes the efficiency of viruses such as minute virus of mice and reovirus in infecting their target cells [78-80]. In contrast, in HIV type-1 and adeno-associated virus, ubiquitination and proteasomal degradation fulfill a negative role and act as inhibitory factors for viral infection process [81-87]. In this context, proteasome can form an inherent defense mechanism that protects mammalian cells from attack by many viral agents capable of passing the barrier of plasma membrane. This also holds true for gene transfer by phage particles. Several lines of evidence have revealed that proteasome-mediated degradative activity can limit the efficiency of phage-mediated gene expression in mammalian cells. This reflects the fact that there exist similar mechanisms in the postinternalization events of mammalian viruses and bacterial viruses. The commonalities in the intracellular fate of these two types of viral particles further highlight their shared ancestry. The reductive effect of proteasome on the efficiency of phage-mediated gene transduction leads us to advance the hypothesis that selective antagonizing of proteasome function can enhance the intracellular survival of phage particles. Several pharmacological agents have been used to test this hypothesis. 293 cells were incubated with GFP-encoding lambda phage in the presence or absence of bortezomib and MG132 (reversible inhibitors of the 26S-proteasome complex). The treatment of cells with MG132 significantly raised both the percentage and the mean fluorescence intensity (MFI) of GFP positive cells. Bortezomib also induced a significant enhancement in the MFI of GFP expression, but a limited increase in the percentage of GFP-positive cells. Consistent with these observations, it has been suggested that proteasome inhibitors promote phage-mediated gene

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Nuclear membrane barrier The nuclear membrane is one of the major barriers of mammalian viruses for phage-mediated gene expression. This membrane acts as a selective barrier to impede the free movement of different macromolecules inside mammalian cells between the cytoplasm and the nucleus. In dividing cells, disintegration of the nuclear envelope during mitosis largely eliminates this limitation. However, many of target cells for gene therapy are nondividing or slow-dividing [88]. Hence, overcoming the barrier of nuclear membrane is a prerequisite for successful gene transfer and paves the way for the establishment of more effective phage-based gene therapy approaches. In the transport of macromolecules including GDVs between the cytoplasm and the nucleoplasm, the nuclear pore complex (NPC) plays a major role [89,90]. In most of the large karyophilic proteins, there exists a domain called the nuclear localization signal (NLS). By creating a binding site for cytoplasmic carrier proteins, NLS makes essential contribution to the active transport of cargoes----transferred by carrier proteins----into the nucleus through NPC [91]. The modification of large DNA molecules with NLS has previously been shown to facilitate DNA delivery across the nuclear envelope [92,93]. This highlights the potential of NLS-encoded peptides for improved targeting of GDVs to the nucleus. With regard to a karyophilic virus, the viral capsid proteins tightly associated with the viral genome seem to be implicated in the transport of virus to the nucleus [94,95]. This stems from the involvement of NLS domains of viral structural proteins in the transport of viral genome. Through engineering of phage particles and incorporation of a NLS domain, it is possible to use the attributes conferred by the domain to localize phages to the nucleus. In line with this scenario, Akuta et al. engineered lambda phages to display on their surface a 32-mer NLS peptide derived from the simian virus 40 (SV40) antigen [96]. When delivered into the cytoplasm of human embryonic lung fibroblast cells, these NLS-containing phages were monitored to evaluate their nuclear targeting capacity. They detected the presence of phage particles in the nucleoplasmin within 30 min after




injection into the cells. The number of phage particles was significantly higher (40-fold) in the nuclear extract prepared from the cells injected with NLS-phage compared with wild-type phage-injected cells. Furthermore, they found a significant enhancement in the expression of phage-encoded GFP (as a reporter gene) in mammalian cells exposed to NLS-phage in comparison with those treated with control phage. These observations exhibited that display of NLS peptide on the surface of phage particles creates an increased affinity to the nucleus and higher efficiency in inducing marker gene expression, highlighting the role of NLS in nuclear targeting of phage virions. In addition to the NLS of SV40 antigen, the NLS of the human immunodeficiency virus Tat protein has also been exploited to raise the targeting of phage particles (lambda phage) to the nucleus [97]. Although NLS-based manipulation of phages has proven to be an instrumental means for nuclear targeting of phage vectors, it is interesting to note that recent findings have demonstrated the presence of intrinsic NLSs in some proteins of phage origin. In this respect, Redrejo-Rodriguez et al. through in silico analysis identified putative NLSs in terminal proteins (TPs) of bacteriophages from different families [98]. Bacteriophage TPs are covalently attached to the genome ends and fulfill the role of priming DNA replication. After this initial bioinformatics-based finding, this research group experimentally examined the subcellular localization of double yellow fluorescent protein fusions to these phage-specific TPs when expressed in COS-7 mammalian cells. They unexpectedly observed that TPs belonging to bacteriophages F29, Nf, PRD1, Bam35 and Cp-1 lead to the localization of fluorescent fusion proteins in the nucleus of COS-7 cells. Further analysis of the F29 TP exhibited its N-terminal domain harbors the predicted NLS. Moreover, an in vitro F29 TPprimed amplification system was applied to confirm that the attached TP accounts for the nuclear translocation step [99]. The exploitation of this amplification system that produces linear DNA molecules with TP covalently attached to their 5¢ ends revealed that the coupling of F29 TP enhances DNA delivery to the nucleus in comparison with a control linear DNA. This is in agreement with the fact that bacteriophage TP through its NLS fraction contributes to the localization of the linear DNA in the nucleus of mammalian cells. The ability to generate TP-DNA molecules provides a novel approach for the development of therapeutic GDVs. Another report from this group indicated the TP of the lately discovered phage YS61, suggested to form a new independent subfamily in the virus family Podoviridae, possesses a putative NLS [100]. Altogether, these observations might support the notion that targeting to the nucleus of eukaryotic cells seems to be an evolutionary prevalent characteristic of bacteriophage TPs from diverse families and hosts. The presence of functional eukaryotic NLSs within bacteriophage proteins has been proposed to play a possible role in the horizontal gene transfer between prokaryotes and eukaryotes, highlighting its function in inter-kingdom genetic exchange [101].

Although these findings may provide important evidence to the possible widespread existence of NLS in proteins derived from phages, many questions regarding the presence of NLS-containing proteins in phages and their evolutionary relevance remain to be answered. Obviously, gaining further insight into the role these sequences play in the evolution of phages paves the way for their application in the development of phage-based efficient platforms for the delivery of therapeutic genes in gene therapy. Besides the issue of nuclear localization, postnuclear delivery is also important for efficient expression of the transgene delivered into mammalian cells. Consistent with this, intranuclear events can influence the expression level of transgenes [102]. These intranuclear events involve transcriptional activity and posttranscriptional processes such as the nuclear export of mRNA. It has been demonstrated that the intranuclear disposition of the transgene is a major aspect of postnuclear delivery events and plays a major role in controlling foreign gene transcription [103]. To the best of our knowledge, so far, there has not been any report on investigating the role played by postnuclear delivery processes and intranuclear events in phage-mediated gene expression. Evolutionarily speaking, bacteriophages have not developed the required machinery----comparable with eukaryotic viruses----to efficiently transcribe their encoded genes and translate their mRNAs in mammalian cells. In contrast, eukaryotic viruses have evolved the specialized mechanisms for controlling the intranuclear events involved in enhancing the expression of virus-encoded genes. These evolutionary adaptations enable eukaryotic viruses to bypass postnuclear delivery barriers of gene expression, effectively replicate and efficiently express their encoded therapeutic genes in mammalian cells. Many viruses hijack the host cell transcription machinery for successful replication and gene expression. For example, influenza virus uses high mobility group protein B1 (HMGB1) (an important chromatin-associated protein) for growth and enhancing transcriptional activity of its polymerase [104]. Similar to the previously described approaches in which the tools developed by eukaryotic viruses were applied to overcome the prenuclear and nuclear delivery barriers of phage-mediated gene expression, the strategies used by eukaryotic viruses for enhancing the efficiency of intranuclear gene transcription can also be exploited for the development of phage vectors with enhanced intranuclear gene expression. In this regard, the sequences mediating the binding of eukaryotic viruses to the transcription machinery of the host cell can be utilized to promote the interaction of phage vectors with cellular machinery underlying nucleosome positioning, chromatin remodeling, transcription, mRNA splicing and mRNA nuclear export. These interactions enhance the decomposition of phage structural capsid proteins in the intranuclear environment. The unpacking of phage virions may ultimately lead to the expression of phage-encoded genes. The postnuclear events involved in the expression of the phage-carried therapeutic transgene have been largely ignored.


9


Table 1. Intracellular barriers for phage-mediated gene expression and the solutions used to bypass theses barriers.


Barrier

Phage-eliminating agent

Solution

Endosome

Low pH

Lysosome Proteasome Nuclear membrane

Proteases Degradative machinery The lack of nuclear localization sequences

It is of interest to note that elucidation of the intranuclear mechanisms responsible for regulating gene expression of bacteriophages can pave the way for improving the efficiency of phage-based GDVs. Surely, future studies will extend the current knowledge on this research area adding another piece to solve the puzzle of phage-mediated gene expression. 8.

Conclusion

On the tortuous road to search for novel GDVs, bacteriophages have attracted attention as a promising category of gene carriers with interesting characteristics. Within the recent years, a large body of evidence has shown the practicability of using bacterial viruses for entry and subsequent gene expression in mammalian cells as their atypical hosts. Herein, we devoted efforts to provide an insightful overview of the capacity of bacteriophages for gene delivery into mammalian cells and also addressed the prospects and problems of this class of gene carriers. The attractiveness of phages for delivery purposes comes from the fact that these biological entities make up a biologically significant part of the natural microflora of the mammalian body that has led to the extensive exposure of mammalian cells to phages. This evolutionarily long communication between mammalian organisms and phage particles makes these viral agents highly safe for application in the clinical context. In contrast to eukaryotic viruses, the wide tropism of which triggers major concerns, the lack of tropism of phages towards mammalian cells obviates the need to modify them for eliminating ectopic and nonselective delivery to nontarget cells and tissues. On the other hand, the enormous genetic flexibility of bacteriophages can be exploited to manipulate their surface features for molecularly targeting them to specialized cells or tissues of interest. In spite of these benefits, the question of efficiency of phages in gene delivery remains a matter of debate and needs to be the focus of further investigation. This is due to the fact that phages are faced with some obstacles for expressing their encoded genes inside mammalian cells. These intracellular hurdles can lead to the elimination of phage virions. However, information gathered within the past several years has shed light on some aspects of this multifaceted phenomenon. This information can be helpful in adapting phage particles for more efficient gene expression in human cells. In line with this, several strategies have 10

Endosomal escape factors Endosome-disrupting factors Inhibitors of lysosomal proteases Proteasomal inhibitors Incorporation of nuclear localization signals for nuclear targeting

been suggested to reach a higher rate of phage-mediated gene expression in mammalian cells. A number of these strategies are largely based on mimicking the tactics used by eukaryotic viruses including the exploitation of endosomal escape and endosome-disrupting factors as well as NLSs. Some other scenarios involve the utilization of pharmacological agents with inhibitory activity on the function of lysosomal proteases and proteasome complex (Table 1). These pharmacological agents have shown promise to be used as instrumental tools for filling some gaps in our understanding of postuptake steps of phage-encoded gene expression. The use of phages as therapeutic tools against bacterial infections can be seen as far back as the initial decades of the twentieth century. However, their entry into the field of gene delivery is a newly developed idea. This cutting-edge field is currently under huge investigation. To date, phage researchers have made great strides in the manipulation of these carriers for improving their properties in gene delivery. Engineering bacteriophages for delivery purposes have added these biological entities as a new armamentarium to the arsenal of GDVs. Nevertheless, many issues remain unexplored and there is still much to be learned. Undoubtedly, future studies will help phage-based gene therapy to move from conception to realization. 9.

Expert opinion

Phage display as a powerful molecular engineering methodology has played a crucial role in expanding the repertoire of potential applications of bacteriophages in biomedicine and biotechnology. This technology, through generating diverse surface modifications and formulating fusion phages with distinctive surface properties, has revolutionized the area of phage research being a turning point in the development of phage-based gene delivery platforms. The advent of phage display allowed taking advantage of the immense structural flexibility of phages in order to optimize their characteristics for delivery purposes. This goal can be materialized through targeting of phages towards specialized diseased cells and tissues (e.g., malignant sites in the body). Phage display-based targeting of phages into receptors on the surface of mammalian cells has made it possible to use the scenario of RME to selectively and specifically deliver phage-encoded therapeutic genes into mammalian cells. Also, combinatorial chemistry




has turned phage display into a high-throughput screening strategy via development of phage display libraries. These libraries harbor a rich population of molecules that can be screened for finding homing ligands to various cells and tissues in vitro and in vivo. The emergence of phage display libraries that provide access to a vast untapped pool of targeting peptide and antibody ligands has raised the prospect of using phages for gene delivery objectives. The surface of different GDVs can be decorated with these cell- or tissuehoming ligands, thereby directing these carriers to desired sites of interest. Irrespective of phage targeting for entry into mammalian cells, many processes implicated in postinternalization phage-mediated gene expression remain to be elucidated. The findings of the recent years have led us to the conclusion that investigating the fate of phage particles in mammalian cells is of great relevance to the development of efficient platforms for gene therapy. It seems, to surmount the multiple intracellular barriers for gene expression, the optimal strategy is to use the combination approach. In this approach, several simultaneous modifications are introduced into a single phage virion. Each modification aims to improve a discrete step of gene expression. These changes ultimately form a phage platform with the capacity of endosomal, lysosomal and proteasomal escape, cytoplasmic entry, nuclear localization and intranuclear gene expression. Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.

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Acknowledgment We gratefully acknowledge our colleagues and friends in 4408 laboratory (Department of Genetics, Tarbiat Modares University) for their helpful discussions.

Declaration of interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

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Affiliation

Babak Bakhshinejad1 & Majid Sadeghizadeh†2 † Author for correspondence 1 PhD Candidate in Molecular Genetics, Tarbiat Modares University, Department of Genetics, Faculty of Biological Sciences, Tehran, Iran 2 Professor in Molecular Genetics, Tarbiat Modares University, Department of Genetics, Faculty of Biological Sciences, Tehran, Iran Tel: +98 21 82884409; Fax: +98 21 82884484; E-mail: [email protected]

Lignin nanotubes as vehicles for gene delivery into human cells.

Cells as delivery vehicles for cancer therapeutics.

Chitosan nanoparticles as a potential nonviral gene delivery for HPV-16 E7 into mammalian cells.

Bacteriophages as vehicles for antibiotic resistance genes in the environment.

Cyclen-based lipidic oligomers as potential gene delivery vehicles.

Stem cells as cellular vehicles for gene therapy against glioblastoma.

Aptamers as drug delivery vehicles.

Polyphosphazenes: Multifunctional, Biodegradable Vehicles for Drug and Gene Delivery.

Novel multifunctional pH-sensitive nanoparticles loaded into microbubbles as drug delivery vehicles for enhanced tumor targeting.

DNA-carbon dots function as fluorescent vehicles for drug delivery.

Molecular imprinted polymers as drug delivery vehicles.

Cationic cell-penetrating peptides as vehicles for siRNA delivery.

Exosomes as drug delivery vehicles for Parkinson's disease therapy.

Gene delivery into plant cells for recombinant protein production.

Synthesis and characterization of layered double hydroxides and their potential as nonviral gene delivery vehicles.

PNIPAAm-MAA nanoparticles as delivery vehicles for curcumin against MCF-7 breast cancer cells.

Preparation and testing of quaternized chitosan nanoparticles as gene delivery vehicles.

HGFR-targeted Polymers as Gene Delivery Vehicles for the Theranostic Sodium Iodide Symporter (NIS) Gene.

Teratocarcinoma cells as vehicles for introducing specific mutant mitochondrial genes into mice.

Transduction of a bacterial gene into mammalian cells.

Gene transfer into mammalian somatic cells in vivo.

Design of novel cell penetrating peptides for the delivery of trehalose into mammalian cells.

Polymer-based vehicles for therapeutic peptide delivery.

Methods for introducing DNA into mammalian cells.