Biological Gene Delivery Vehicles: Beyond Viral Vectors

review

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Biological Gene Delivery Vehicles: Beyond Viral Vectors Yiqi Seow1 and Matthew J Wood1 Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK

1

Gene therapy covers a broad spectrum of applications, from gene replacement and knockdown for genetic or acquired diseases such as cancer, to vaccination, each with different requirements for gene delivery. Viral vectors and synthetic liposomes have emerged as the vehicles of choice for many applications today, but both have limitations and risks, including complexity of production, limited packaging capacity, and unfavorable immunological features, which restrict gene therapy applications and hold back the potential for preventive gene therapy. While continuing to improve these vectors, it is important to investigate other options, particularly nonviral biological agents which include bacteria, bacteriophage, virus-like particles (VLPs), erythrocyte ghosts, and exosomes. Exploiting the natural properties of these biological entities for specific gene delivery applications will expand the repertoire of gene therapy vectors available for clinical use. Here, we review the prospects for nonviral biological delivery vehicles as gene therapy agents with focus on their unique evolved biological properties and respective limitations and potential applications. The potential of these nonviral biological entities to act as clinical gene therapy delivery vehicles has already been shown in clinical trials using bacteria-mediated gene transfer and with sufficient development, these entities will complement the established delivery techniques for gene therapy applications. Received 22 December 2008; accepted 5 February 2009; published online 10 March 2009. doi:10.1038/mt.2009.41

Introduction Nucleic acids and their analogs have many therapeutic applications, ranging from correction of genetic defects to gene augmentation for chronic disease including cancer to acting as adjuvants for vaccination. Nucleic acids have been exploited to deliver genes as DNA plasmids, to mediate gene knockdown via RNA interference (RNAi) mechanisms or to alter pre-mRNA splicing to ameliorate disease-causing mutations. Although most nucleic acid–based technology is currently used as therapeutics, there is potential for gene therapy in disease prevention by replacing disease-predisposing alleles with innocuous versions before the onset of disease. Moreover, as the knowledge of underlying genetic risk factors accrues, pre-emptive gene therapy will increasingly be feasible in order to reduce the burden of chronic disease. Naked therapeutic genetic molecules are generally difficult to deliver primarily due to rapid clearance,1 nucleases which limit serum half-life of unmodified small interfering RNA to 5–60 minutes2 and DNA to 10 minutes,3 the lack of organ-specific distribution, and the low efficiency of cellular uptake following systemic delivery. Although nucleic acid modifications, including incorporation of targeting ligands and the use of physical delivery systems, such as hydrodynamic injection, can overcome some of these limitations, specialized gene delivery vehicles (GDVs) that improve delivery efficiency and cell-specificity whilst protecting against immune recognition are preferred. In addition, GDVs can enhance the therapeutic value of the transgene by providing complementary effects such as codelivery of

inflammatory suppressors to reduce cytokine production triggered by plasmid DNA.4 Viral vectors and cationic liposomes are at the forefront of GDV technology with a large number already in clinical trial.5 Despite their potential, limitations remain (Table 1), with immune recognition6–8 for most viral GDVs, mutagenic integration9 for some viruses, and inflammatory toxicity and rapid clearance for liposomes10 being the most significant. For example, immune activation can require the concomitant use of immunosuppressive strategies to overcome uptake and readministration problems with current GDVs.11–13 Antibodies generated against the GDVs can also dramatically decrease transgene expression on readministration.14 Furthermore, viral vectors have packaging size constraints limiting their genetic cargo capacity. This is particularly important given that additional plasmid maintenance and replication genes are required for nonintegrating DNA vectors to maintain persistent expression within their host cells (reviewed in ref. 15). The inherent risks and limitations of current GDVs have generally limited their application to life-threatening diseases,16 in which the benefits of therapy clearly outweigh the risks, to diseases in special tissue environments, for example ­immune-privileged sites such as the eye,17 or for genetic vaccination.18 However, for genetic diseases that are chronic and debilitating but not necessarily life-threatening, a much lower risk profile and the ability to sustain corrective gene therapy for decades is required for curative intervention. An example of an unacceptable risk lies in the aforementioned immunosuppressive strategy, and is highlighted

Correspondence: Matthew J Wood, Department of Physiology, Anatomy and Genetics, Le Gros Clark Building, University of Oxford, South Parks Road, Oxford OX1 3QX, UK. E-mail: [email protected] Molecular Therapy vol. 17 no. 5, 767–777 may 2009

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Table 1  Limitations of viral vectors and cationic liposomes Vehicle

Limitations

References

Adeno-associated viral vectors

Small packaging capacity (~4.7 kb)

Reviewed in ref. 126

Neurons Exosomes Herpesviruses

Allows only DNA-based cargo Circulatory

Low probability of integration Reduced efficacy of repeat administration Retroviral/lentiviral Insertional mutagenesis vectors

127 9,128

Transcriptional silencing leads to reduced expression over time

Reviewed in ref. 129

Adenoviral vectors

Triggers strong immune response against vehicle and transgene

Reviewed in ref. 130

Cationic liposome

Immune recognition

131, reviewed in ref. 132

Generally low transduction efficiency compared to viruses Risk of inflammatory toxicity 133

by the death of a healthy patient due to opportunistic infection in a recent adeno-associated virus gene therapy trial for rheumatoid arthritis.19 With an increasing number of common diseases shown to possess a genetic component, there is potential for safe and sustained pre-emptive genetic solutions. Hence, it is imperative to develop efficient gene delivery technologies that are able to avoid immune recognition and inflammation. Despite sustained efforts to improve the safety and efficacy of liposome formulations and viral vectors, research into alternative GDVs may provide the least complex solution for novel therapeutic applications.

Biological GDVs Biological entities are well suited for gene delivery. Many forms of microorganism, exemplified by viruses, have evolved to infect cells effectively and stably while evading host immune responses. Furthermore, many such microorganisms are tolerated by the immune system, including commensal bacteria in the gut and transfusion-transmitted virus in the liver.20 Numerous cell types can also endocytose membrane-bound bodies, for example macrophage phagocytosis of apoptotic cells21 and mast cell uptake of exosomes,22 which could potentially serve as novel routes for therapeutic cargo delivery (Figure  1). The ‘ideal’ biological GDV should have the appropriate packaging size for its cargo, the ability to evade immune recognition, target cell-specificity, and achieve efficient cargo delivery. The requirements at each of these steps differ between applications and thus different GDVs are likely to be required. Although the most-developed GDVs in this class, viruses, show great promise for a wide range of disease applications, they have the aforementioned limitations and unconventional biological GDVs with unique properties might fill therapeutic niches currently poorly served by mainstream viral and liposomal GDVs, for example the use of gut bacteria to deliver transgenes to gut epithelium23 or exosomes for delivery to mast cells.22 Such a specialized delivery philosophy may allow 768

Erythrocyte ghosts Exosomes Herpesviruses AAV

Liver Erythrocyte ghosts Exosomes Transfusion transmitted virus Hepatitis C virus Hepatitis B virus Herpesviruses

Gastrointestinal Escherichia coli Bifidobacteria Lactobacilli Streptococci Bacteriophages Herpesviruses Human papilloma viruses

Figure 1  Examples of potential biological vehicles tolerated naturally in selected organs. Organisms and cell-derived particles are found naturally in certain organs as indicated, and are well-tolerated. These particles are promising delivery vehicles that can be exploited for gene therapy. AAV, adeno-associated virus.

the immune and inflammatory problems that have plagued other delivery systems to be eliminated. Here, we review the current state-of-the-art in unconventional biological GDVs, including bacteria, bacteriophage, virus-like particles (VLPs), erythrocyte ghosts, and exosomes, and how their unique properties might be exploited for specific gene therapy applications.

Bactofection Bactofection refers to the use of bacteria for transgene delivery, which are naturally or artificially engineered to be invasive but attenuated to prevent pathogenesis. Strains of bacteria currently used include Listeria monocytogenes,24–28 certain Salmonella strains,29–34 Bifidobacterium longum,35 and modified Escherichia coli.23,36,37 Cellular entry typically occurs through endocytosis followed by endosomal escape into the cytoplasm. Once in the cytoplasm, the transgene product can be expressed in two distinct ways. The first is via host cell–mediated expression of the genetic cargo released by the bacteria; the alternative is the production and secretion of the transgene product33,38 or RNA39 directly by the bacteria, efficiently acting as an expression cassette within the host cell (Figure 3). Although the former can be achieved by other gene delivery systems, the latter can only be accomplished by bacteria because as functional organisms, they possess the full complement of RNA polymerase and transfer RNAs for the production of mRNA, RNAi molecules, and proteins. When DNA is released into a cell by GDVs, the presence of immunostimulatory unmethylated CpG motifs in the exogenous plasmid DNA40 and epigenetic silencing of integrated viral DNA41 both result in reduced transgene expression over time. Bacteria-mediated transgene or RNAi expression may evade the host defence against exogenous DNA because the DNA is enclosed within the bacteria, potentially allowing for long-term expression of transgene. In support of this idea, a Listeria mutant with low expression of membrane pore–forming protein listeriolysin O is able to reach a ‘stalemate’ with the host cell innate defence mechanism without causing cell death, allowing for active transcription and slow replication in macrophages,42 suggesting that stable long-term bacterial expression systems are attainable, even though most cells have innate defenses against bacteria. www.moleculartherapy.org vol. 17 no. 5 may 2009

Biological Gene Delivery Vehicles

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DNA cargoes for gene therapy are normally bacteria-derived plasmids. The bacterial delivery system hence merges the production and packaging of cargo into a single step, both increasing the speed of production and decreasing the cost. Additionally, as naturally occurring plasmids in bacteria range from 2 to 200 kbps in size, they can accommodate the inclusion of large constructs and multiple genes for gene therapy. The potential of this system has already been demonstrated with plasmids encoding prodrug convertases25,31,35 and short hairpin RNAs.37 Well-characterized nutritional tropism and the ease of bacterial genetic screens for nutritional and tissue-specific tropism can provide favorable biodistribution profiles restricting bactofection to target tissues. For example, Pawelek et al.31 demonstrated that polyauxotrophic mutants of a highly invasive Salmonella typhimurium, defective in purine and multiple amino-acid biosynthesis, are able to invade nutrient-rich tumors in mice up to 9,000 times more effectively than nutrient-poor liver tissue, allowing for specific delivery of a prodrug convertase gene to tumors. Natural preferences for anaerobic conditions within tumors can also be found with attenuated B. longum35 and Vibrio cholerae;43 while E. coli (DH5α) and attenuated V. cholerae preferentially target the liver after systemic injection despite rapid clearance subsequently.43 In the absence of natural tropism, modification of bacteria surface proteins by conjugation to a cell-type specific ligand can also alter tissue specificity to suit the application. For example, Akin et al.26 recently demonstrated a system for conjugating nonbiological nanoparticles with N-acetylmuramidase-specific antibodies to L. monocytogenes to target delivery of a fluorescent transgene to N-acetylmuramidase-expressing cells. Other beneficial mutations can also be exploited, such as those in a strain of E. coli K12 that undergoes lysis to release the therapeutic cargo upon entry into mammalian cells because of impaired cell wall synthesis due to diaminopimelate auxotrophy.23 To date, bacterial GDVs have been used mainly in cancer gene therapy25,31–33,35 and for DNA vaccination27–29,34 but they have also been used for the treatment of genetic diseases, e.g., cystic ­fibrosis.36 Although transgene expression levels have been generally low compared to other delivery methods, work by Zelmer et al.44 suggests that, at least for Listeria-mediated gene transfer, the plasmid DNA is often effectively released into the target cell but nuclear transport and subsequent expression are inhibited by the macromolecular structure, perhaps related to the CpG content of the plasmid. This implies that improved design of the plasmid, not the bacterial GDV, may be what is required to improve the expression of the transgene product, such as with CpG-free plasmids.45 The intrinsic toxicity of bacteria, the most significant risk in using bactofection, varies between different bacterial strains. In clinical gene transfer trials of direct intratumoral33 and intravenous32 injection of attenuated Salmonella bacteria, no significant side effects were observed at low doses <7.6 × 107 colony forming units over the course of 4 days for intratumoral injections and 108 colony forming units/m2 of body surface area for single intravenous injections. Although bacteremia-related symptoms developed at higher doses, these were readily resolved with antibiotics and side effects were rapidly reversed without signs of toxic shock, indicating that administration of attenuated bacteria could be safe in humans if titres are kept below a certain threshold. However, Molecular Therapy vol. 17 no. 5 may 2009

IgM, IgA, and IgG antibodies developed against Salmonella in a substantial number of subjects following intravenous administration,32 resulting in strong immune priming that may render this GDV unsafe and ineffective due to the presence of neutralizing antibodies. For intranasal applications with invasive E. coli36 in mice, septic shock resulted from high titres and reflects the low therapeutic indices for attenuated bacteria in nonnative environments. However, deployment of bacteria into the gut, their natural host environment, appears to be benign, based on the oral administration of E. coli for gene delivery to the intestinal lining in mice.23 The immune system has also developed tolerance for many of the gut-derived bacteria, probably due to host–commensal interactions using pattern recognition receptors,46 making such gut-derived bacteria suitable for avoiding immune stimulation. It has been recently shown that Lactococcus lactis administered orally to mice can transduce intestinal epithelia to produce β-lactoglobulin effectively to stimulate a transitory Th1 immune response in order to modulate the Th2 response against milk protein allergens.47 Therefore, the use of native bacterial flora to target tissues appears to improve the therapeutic index and fulfill the criteria of an ‘ideal’ delivery vehicle within certain organs, including the gut, upper respiratory tract, and vagina.48 Although this selective approach has been successful in animal models, significantly more research is required before clinical adoption to ascertain safety in human subjects. Unlike revertant replication-competent viruses potentially generated from vector production, if the attenuated bacteria unexpectedly spread beyond the target organ, via increase in pathogenic potential or causing severe bacteremia, the resultant infection can easily be brought under control with an established arsenal of antibiotics. There is growing interest in the use of bacteria as GDVs with currently at least three bactofection Phase I clinical trials for different cancers delivering either tumor antigens for vaccination with S. typhimurium or cytotoxic genes with L. monocytogenes, and one Phase II trial for ulcerative colitis involving delivery of interleukin-10 by L. lactis.5 The inexpensive production, the diverse natural and modified tropism profiles, the large and diverse packaging capacity of bacteria, coupled with their immunological tolerance in certain target organs, and relative ease of control in the case of adverse events make bactofection an attractive alternative to consider for gene delivery to the gastrointestinal, respiratory, and urogenital tracts. For application of bactofection beyond these native tissue environments, the problem of immune activation and the development of methods to achieve immune tolerance will require further study.

Bacteriophage A second GDV inspired by prokaryotic systems is the bacteriophage. Bacteriophages are natural viruses that exclusively infect bacteria and are ingested without serious side effect within fermented food, e.g., yoghurt.49 Bacteriophages are very resistant to nonenzymatic degradation when compared to other vehicles, allowing for longer and less costly storage. Bacteriophage lambda in particular has been shown to retain infectivity and, by extension, structural integrity for up to 6 months at 4 °C in water, which is longer than any other biological vehicle including viruses.50 Although bacteriophages are still susceptible to enzymatic 769

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degradation, their ability to withstand a wide range of pH, from 3 to 11, for up to 24 hours could make them suitable for oral delivery.50 Over the past century, the use of bacteriophage in research has placed many tools at our disposal. Development of phagemid vectors, plasmids with a bacteriophage origin of replication that are packaged into phage particles upon addition of helper phages (Figure 2), has made the genetic manipulation of bacteriophage of similar ease to plasmids used in production of viral vectors.51 The phagemid system allows for precise control over the relative composition of the wild type and modified coat proteins,47 while eliminating the genes of the potentially immunogenic bacteriophage proteins. The baceteriophage has generous packaging capacity beyond what most viral vectors like adenovirus (8 kb), adeno-associated virus (4.7 kb), or lentiviruses (8–10 kb) (ref. 52) offer. Lambda phage is capable of holding 53 kb (ref. 53) while M13 phage does not seem to possess a defined packaging limit.54 This allows for the insertion of mammalian plasmid maintenance sequences, such as the EBNA1/oriP system55 and endogenous control regions for the transgene. The coat proteins of the most commonly used bacteriophage species for gene delivery, namely M13 filamentous phage51,56–59 and lambda phage,60 can be engineered to incorporate targeting ligands without affecting structure significantly. Such ligands incorporated to date include a single-chain variable fragment antibody directed against HER2 receptor,57 growth factors, e.g., epidermal growth factor and fibroblast growth factor 2 (refs. 51,56,58), metabolites, e.g., galactose and succinic acid,61 adenovirus penton base,60 and targeting peptides, e.g., against gliomas.59 Beyond the standard ligands, the use of phage display, a high-throughput peptide library screening technique using bacteriophage particles, has the potential to identify novel peptides, which when conjugated to contextually relevant bacteriophage proteins can target unknown cell surface molecules59 and perform novel functions such as skin penetration.62 Phage nanoparticles with multiple peptides engineered for different functions can then be produced with phagemid technology to enhance gene delivery efficiency. A significant hurdle to clinical application is that unmodified bacteriophage particles delivered systemically are rapidly eliminated by the reticuloendothelial system (RES),61,63 with significant degradation occurring in the liver, spleen, and lungs.61 Although long-circulating lambda phage variants with mutations in their D and E capsid proteins have been developed to escape such fate in mice,64 the mechanism has not been elucidated and it is not yet known whether such mutants will retain function in human subjects. On the other hand, with the incorporation of targeting ligands, specificity and delivery efficiency can be dramatically improved. For example, Molenaar et al. have shown that M13 phage uptake in the parenchymal and Kupffer cells of the liver is dramatically enhanced when the phage surface lysines are chemically conjugated to lactose and this effect was shown to be mediated by specific lactose receptors.61 Oral delivery of an E. coli phage in rabbits also results in transient localization of the phage in major organs especially the spleen,65 a property that can be exploited to deliver genetic adjuvants, genes that enhance the immune response against an antigen, or antigenic genes for vaccination. Unfortunately, the generation of antibodies against 770

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phage particles, which are potent antigens, is likely to significantly reduce the efficacy of readministered phage. However, no adverse effect has been observed in healthy individuals66 or immunocompromised patients67 challenged with ~1011 bacteriophage ϕX 174 particles as a single intravenous administration. Further in vivo phage display panning experiments for weakly immunogenic phage mutants may overcome the phage’s intrinsic immunogenic nature. In another related development, MS2 bacteriophage particles assembled from bacteriophage proteins produced in a cell-free expression system have been shown to encapsulate exogenous RNA.68 Although MS2 bacteriophage particles have been generated previously in E. coli cells, the cell-free expression system allows the packaging of large amounts of exogenous therapeutic RNAs ex vivo. Wu et al. achieved similar results with antisense oligonucleotides,69 opening the door to RNAi-based and antisense therapeutics delivered through bacteriophage GDVs (Figure 3). The principal advantages of the bacteriophage system—the ease of developing and incorporating novel functionalities into the phage particle, the large packaging capacity, the ability to take non-DNA cargoes, and relative safety in humans—suggest that bacteriophage vectors can be considered as potential therapeutic agents. However, the potent antibody response limits the ability to readminister these vectors and hence, restricts their current use to the delivery of genes for vaccination purposes or to single-use applications especially those poorly served by existing strategies using viral vectors.

Virus-like particles Mammalian VLPs can also be used as GDVs. Although viral vectors are normally made replication-incompetent by separating their essential components into different plasmids, the vectors usually possess their full complement of viral proteins when packaged into a virion particle and the cargo plasmid containing the therapeutic gene is packaged within the host cell. In contrast, VLPs are made by transfecting a production cell line with a single plasmid encoding only viral structural proteins, followed by purification of the resultant particle and encapsulation of the cargo ex vivo (Figure 2). One of the advantages of this method over conventional viral vectors is that the VLPs can be produced in different cellular systems, including bacterial,68,70 plant,71 insect72–77 and yeast cells78–80 in addition to standard mammalian cell lines, potentially lowering the cost of mass production. Like viral vectors, clinical use of this system invariably involves the complication of removing residual cellular components, but because VLPs can be denatured and reformed, they can typically tolerate harsher purification conditions than viral vectors. However, the key advantage of VLPs over the viral vectors is their ability to separate loading from VLP purification as this allows for unusual cargoes, such as modified oligonucleotides, to be loaded.81 The efficacy of an unmodified papillomavirus L1 VLP in transducing cell lines with a reporter plasmid is comparable to liposomal methods,82 the predominant in vivo delivery system for small interfering RNAs. With an increasing interest in the use of modified small interfering RNAs as therapeutics, the ability to package small oligonucleotides and the high efficacy of transduction in vitro give VLPs the potential to play a significant role in RNAi delivery. www.moleculartherapy.org vol. 17 no. 5 may 2009

Biological Gene Delivery Vehicles

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A. Bacteria Transformation

B. Bacteriophage

C. Virus-like particles

Amplification

Production vector

Helper phage

Phagemid

Purification

Producer cells

Capsid monomers Assembly Purification

D. Erythrocyte ghost

E. Exosomes Patient-derived primary cells/ differentiated stem cells

Patient-derived erythrocytes

Lysis + purification

Exosomes reintroduced into patients MVB Cytoplasm removed

Erythrocyte ghosts

Ghosts reintroduced into patients Loading

Loading Purification of exosomes

Figure 2  Production of biological gene delivery vehicles. (A) Strains of bacteria with desirable properties are transformed with the plasmid cargo and amplified to generate GDVs. (B) The phagemid, a modified bacterial plasmid with phage sequences within, is used as the cargo and transformed into bacteria. The bacteria is then infected with a replication-defective helper phage that produces essential genes for the packaging of the phagemid vector into bacteriophage GDVs. (C) The virus surface proteins are produced in cell culture and purified as capsid monomers. The genetic cargo is then packaged into a virion as the monomers are transferred to a buffer that promotes assembly of the virion. (D) Erythrocytes are harvested from the patient and lysed to produce erythrocyte ghosts. The ghosts are then loaded, usually through osmotic pressure, with the genetic cargo before being reintroduced into the patient. (E) Patient-derived primary cells are first harvested and stimulated to produce exosomes, which are then purified, and loaded, likely by electroporation, with the genetic cargo before being reintroduced into the patient. GDV, gene delivery vehicle; MVB, multivesicular body.

This promise is tempered by the low yield of functional VLPs as disassembly and reassembly of VLPs after purification is complex and inefficient. An interesting concept proposed to address this limitation is chemical self-assembly of VLPs, based on the production of monomeric capsid precursors followed by ex vivo assembly of the virus capsid upon activation to encapsulate its desired cargo.83 This approach may increase the consistency of the VLP produced and the amount of cargo encapsulated; however, it is yet to be experimentally tested. VLPs that have been successfully shown to mediate transfer of nucleic acids can be generated from many families of viruses, including papillomaviruses,15,66,68,72,78,84–86 hepatitis B and E viruses,74,87,88 and polyoma viruses.73,77,89–93 Other virus families from which VLPs have been derived include lentivirus,76,94 rotavirus,69 parvovirus,70 and norovirus.71 The diverse tropisms of these viruses and their VLPs provide a range of natural targeting capabilities without the need for further genetic engineering. These tropisms include liver for hepatitis B VLPs,75,88 spleen for certain papilloma and polyoma VLPs,82,90 antigen presenting cells Molecular Therapy vol. 17 no. 5 may 2009

for certain papilloma VLPs,83 and glial cells for JC virus VLPs.77 Polyomavirus VP1 VLPs containing a β-galactosidase plasmid administered via the subcutaneous, intravenous, intraperitoneal, and intranasal routes also showed different β-galactosidase expression profiles in the heart, lung, kidney, spleen, liver, and brain, demonstrating how tropism of VLPs can also vary with delivery routes.90 The large number of permutations of natural VLPs and delivery methods is likely to produce diverse distribution profiles suitable for many disease applications. Similarly to viral vectors, VLPs can also be engineered to incorporate targeting ligands such as epidermal growth factor74 and antibodies,86,87 and the genetic and chemical modifications available to virus vectors can also successfully be applied to VLPs. Like bacteriophages, papillomavirus VLPs delivered orally are transported to Peyer’s patches, lamina propria, and the spleen, which also makes them suitable as oral gene vaccine vehicles.72,82 Empty VLPs were originally developed for vaccination as a replacement for attenuated viruses because they were easier to produce, hence most current gene delivery applications of VLPs 771

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A. Bacteria

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Biological Liposomes

1 Expression cassette C. Biological liposomes

2 Delivery of cargo D. Synthetic liposomes Nucleus B. Bacteriophage and VLPs E. Viral vectors DNA plasmid mRNA shRNA Protein Short oligonucleotides

Figure 3 Release of cargo intracellularly by delivery vehicles. (A)  Bacteria can deliver genetic cargoes in two distinct fashion after endocytosis and endosomal release. First, short oligonucleotides and DNA plasmids can be released directly into the host cells through the lysis of the bacteria. Alternatively, intracellular bacteria can produce and excrete therapeutic RNAs and proteins. (B–D) Bacteriophage, VLPs and both types of liposomes are capable of delivering mRNAs, short oligonucleotides and DNA plasmids. (E) Viral vectors are typically only capable of delivering DNA or RNA vectors that ultimately end up in the nucleus as DNA templates for transcription of mRNAs. shRNA, short hairpin RNA; VLP, virus-like particle.

are designed to generate immune responses against foreign or cancer antigens. For vaccination, cargoes delivered are typically gene adjuvants such as interleukin-2 (refs. 15,81,82) that activate the immune system against the VLP or genes for antigens targeted to antigen presenting cells to induce a cytotoxic T-lymphocyte (CTL) response.72,83 Because VLPs were optimized for their ability to stimulate the immune system, they are typically unsuitable for repeat administration because of the potentially high levels of serum and mucosal antibodies generated.85,88,91 The immune response against the VLP also seems to adversely affect tolerance to the transgene88 which makes this delivery strategy problematic if delivering neo-antigens for genetic loss-of-function diseases. However, these properties also make VLPs good candidates for cancer and vaccine gene therapy where the goal is to stimulate recognition of tumor and foreign antigens transiently. If the same strategies used for masking viral vectors, such as polyethylene glycolation (reviewed in ref. 95), were similarly applied to VLPs, repeat delivery may be feasible. The development of VLPs for vaccination has resulted in well-characterized, current good manufacturing practice–grade production processes which can be utilized to scale up VLPs designed for gene delivery. VLPs typically present the same pitfalls as their parent viruses, such as immunostimulation, in addition to the ineffective loading during production and lower transfection rates, but in return, enable the loading of alternate cargoes while potentially reducing carryover contamination during production in addition to the other benefits offered by viral vectors. For genetic vaccination and delivery of genetic adjuvants, VLPs are proven workhorses, but novel uses involving the delivery of small interfering RNAs and modified oligonucleotides deserve further focus and development. 772

Biological liposomes are phospholipid-based spherical particles derived from human cells. If derived from patients, these liposomes are likely to be recognized as self and hence, would be ideal ‘stealth’ GDVs, capable of evading host immune recognition and RES sequestration. Although conceptually appealing, the cells used for production of these GDVs tend to be difficult to harvest and maintain, requiring extraction of primary cells and the costly use of growth factors. Hence, biological liposomes are seldom explored as delivery options. Two populations of biological liposomes—erythrocyte ghosts and secretory exosomes—are currently being considered for gene delivery and although the technologies remain relatively immature, both types of biological liposome show promise. Erythrocyte ghosts are red blood cells that have been broken up into small spherical structures after removal of most cytoplasmic content (Figure  2). The spherical structures are then dialyzed in a hypotonic solution, sheared or electroporated to create pores for the loading of the therapeutic cargo.96–98 Erythrocyte ghosts have been investigated as drug delivery vehicles for several decades and more recently, they have been engineered to carry cargoes from small molecules97 to large plasmids,96 including modified oligonucleotides,99,100 and should presumably be able to incorporate RNA cargoes as well. The loading efficiency for plasmids appears to be relatively high if loaded by electroporation rather than with hypotonic dialysis,96 the loading efficiency of which is one of the main reasons erythrocyte ghosts are not used more extensively for drug delivery. Erythrocyte ghosts are relatively stable, retaining a small molecule cargo—fluorescein—for at least 3 weeks in saline at 4 °C,101 although experiments pertaining to the long-term stability of ghosts loaded with genetic cargo have not been performed. Moreover, Magnani et al. have developed a method that allows immediate reinfusion of erythrocyte ghosts loaded in hypotonic solution after red blood cell collection102 ­circumventing the need for storage. Several lines of evidence suggest that erythrocyte ghosts derived from the patient are well-tolerated by the immune system and are likely to be safe as GDVs. First, predeposit autologous blood transfusion, the use of a patient’s stored blood for blood transfusion during operation, is believed to eliminate the risk of hemolytic and allergic reactions mediated by the immune system when compared to allogenic blood transfusion, the use of someone else’s blood, partly because self erythrocytes are well tolerated.103 Also, Gressner et al. recently showed that >50% of labeled human erythrocyte ghosts injected intravenously in rats was lost from the blood within 20 minutes. Conversely, no loss was detected for rat ghosts, suggesting that the RES and immune system effectively recognized the rat erythrocyte ghosts as self as opposed to structurally similar human erythrocyte ghosts.104 In humans, a pilot trial of erythrocyte-mediated intravenous sustained drug delivery in cystic fibrosis patients registered no side effects or adverse events after a year of treatment and the ghosts appeared efficacious for drug delivery, consistently releasing 0.1– 0.2 nmol of dexamethasone-21-phosphate per milliliter of plasma for up to 1 month; however, the study yielded too few samples to draw definitive conclusions.105 Although the drug delivered was an anti-inflammatory corticosteroid, this trial nonetheless hints at www.moleculartherapy.org vol. 17 no. 5 may 2009

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Table 2 Advantages and limitations of biological delivery vehicles Method

Advantages

Limitations

Potential applications

Key References

Bactofection

Able to encapsulate shRNAs and DNA plasmids Natural tropism   Auxotropism   Intrinsic targeting Modification of delivery vehicle well-tolerated Bacteria-mediated production of shRNA and proteins may overcome limitations for nuclear delivery Scale-able purification processes, potentially cheaper than mammalian cells-based systems Single step production of vehicle and cargo if using plasmid Easily controlled by antibiotics

Triggers B-cell and T-cell response if administered systemically Potential toxicity Nuclear transport and subsequent expression of plasmids inhibited by host

Gastrointestinal tract gene therapy Cancer gene therapy

23,24,33,34,37–39,47

Bacteriophage

Able to encapsulate antisense oligonucleotides, RNA and DNA Resistant to nonenzymatic degradation, including high temperatures, low pH Targeting ligands easily engineered into capsid proteins Well-characterized system of phage display to rapidly discover novel peptides for targeting/cell penetration Scale-able purification processes, potentially cheaper than mammalian cells-based systems Cell-free synthesis method available

Rapid eliminated by the reticuloendothelium system Very potent antigen CGMP grade manufacture difficult

Vaccine gene therapy Single administration gene therapy Repeated administration at immune-privileged sites

50,51,53,54,59–61,64

VLP

Able to encapsulate antisense oligonucleotides, siRNAs, long RNA and DNA Natural tropism for targeting Retargeting strategies (e.g. antibodies) for viruses applicable to VLPs as well Can be produced in different cellular systems including bacteria, yeast and plants Removal of residual cellular contaminants conceptually simpler than for viruses CGMP grade production processes well characterized Benefits from the large body of research on viral vectors

Limited packaging efficiency Heterogeneous population of particles As immunogenic as parental viruses

Vaccine gene therapy Single administration gene therapy Repeated administration at immune-privileged sites

81–83,85,88,90

Erthrocyte ghosts Able to encapsulate antisense oligonucleotides, RNA and DNA Coencapsulation of drug molecules Chemical modifications of cell surface can change properties Minimal immunogenicity

Short circulatory half-life of erthrocytes Sequestration by the spleen and liver Difficulty in genetic engineering for the production of targeting ligands

Repeated delivery targeted to the liver, spleen or blood

96,98,101,102,106

Exosomes

Largely uncharacterized Potentially expensive to develop

Repeated delivery targeted 22,108–110 to neurons and immune cells

Capable of transferring functional mRNA Chemical modification of cell surface proteins can change properties Immunosuppressive and anti-inflammatory Readily taken up by cells Minimal immunogenicity, may induce tolerance

Abbreviations: shRNA, short hairpin RNA; siRNA, small interfering RNA; VLP, virus-like particle.

the stealth properties of this treatment vehicle. The fact that erythrocytes and their derivatives are tolerated immunologically and are able to evade RES sequestration suggests that these delivery vehicles are both safe and capable of readministration, properties desirable in a GDV for systemic use. In contrast to the long halflife in rats measured with radioactive labels by Gressner et al.,104 the in vivo circulation half-life of plasmid-loaded allogenic erythrocyte ghosts in mouse is ~10 minutes as measured by quantitative PCR.96 The widely differing reports relating to half-life suggest that significant variation occurs between species and further research needs to be carried out to determine the pharmacokinetics of the Molecular Therapy vol. 17 no. 5 may 2009

ghosts and their likely behavior and effects in human subjects before future clinical application. Erythrocytes are constantly recycled by the spleen and RES, hence it is no surprise that untargeted erythrocyte ghosts are mainly localized to the spleen and liver.104,106 Nonetheless, targeting of erythrocyte ghosts can be achieved by various means. Attachment of targeting ligands, such as antibodies104,107 and metabolites98 by chemical conjugation can expand the cell-­specificity of these erythrocytes. Another interesting strategy lies in crosslinking of major transmembrane (band 3) proteins with chemical reagents after stimulation of clustering with zinc chloride.97,108,109 Clustering 773

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of these proteins on the erythrocyte surface induces autologous IgG binding and complement fixation, hence favoring phagocytosis by macrophages.109 As erythrocytes are anucleate, targeting ligands cannot be genetically engineered and expressed on these ghosts, but the use of erythrocytes generated from stem cells110 expressing targeting ligands can overcome this limitation. In spite of the limitations in targeting to other organs, erythrocyte ghosts are conceptually the best GDVs for persistent gene therapy for cells of hematopoietic lineage. However, significant hurdles, namely the relatively low loading efficacy by hypotonic dialysis and the difficulty of cell targeting, need to be addressed before therapeutic realization in the clinic. A second class of biological liposome is the exosome, a small membrane-bound microvesicle (30–90 nm) of endocytic origin that is released into the extracellular environment following fusion of multivesicular bodies with the plasma membrane (Figure 2). Exosomes have recently been demonstrated to act as natural GDVs for a large number of functional mRNAs and miRNA.22 Murine mast cell-derived exosomes were found to deliver functional mRNAs that produce murine proteins to human mast cells, and therefore exosomes could potentially be exploited to deliver other oligonucleotides, much like synthetic liposomes. Exosomes can be engineered to be immunosuppressive if the cells from which they are derived are genetically engineered to express immunosuppressive ligands, such as FasL,111 or if they are treated with immunosuppressive cytokines, such as interleukin-10 (ref.  112). This property makes them potentially suitable for treatment regimens requiring repeat administration. More excitingly, because exosomes appear to have a defined set of proteins and RNAs distinct from the parent cells, and are produced in numerous cell types, including hematopoietic, intestinal epithelial, tumor, and neuronal cells, Smalheiser113 has suggested that they may have a natural role in intercellular communication. As such, they may be readily taken up by cells without the need for targeting ligands. Exosomes also readily express ligands transfected into their parent cells, such as FasL mentioned above,111 providing a means for targeted delivery to desired cell types if required. Future studies will be needed to further characterize these natural liposomes and define their potential as GDVs for specific gene therapy applications. In future, biological liposome technology is likely to benefit from parallel advances in stem cell technologies. The production of erythrocytes from human embryonic stem cells110 should allow for consistency in the production of erythrocyte ghosts. Patientderived erythrocytes cannot be manipulated genetically as they lack a nucleus. However, with the use of stem cells, genes encoding targeting antibodies can be genetically engineered and expressed in the derived erythrocytes, augmenting the function of erythrocyte ghosts. Production of exosomes requires purification from primary cells, which may be difficult to harvest. The ability to generate multipotent or induced pluripotent stem cells, derived from a patient’s dermal fibroblasts,114 means that self-derived exosomes could potentially be harvested without invasive procedures, significantly increasing their ease of application. Overall, biological liposomes have the potential to rival conventional liposomal systems as they can deliver multiple types of genetic cargo, but with favorable immunological properties. However, the gene 774

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delivery efficacies of biological liposomes have not yet been well investigated by comparison against known liposomal delivery methods. For delivery to blood-based targets, erythrocyte ghosts and exosomes both appear to be suitable as they are well tolerated in the blood stream and appear to be used naturally by cells of hematopoietic lineage, such as mast cells and dendritic cells, for communication. However, for organ delivery via local or systemic administration, the development of surface modifications to these GDVs to increase epithelial permeability and permit tissue specificity will be necessary to make their use attractive.

Other Strategies Besides bacteriophages, nonmammalian pathogens currently under  investigation include baculoviruses which have insects as ­natural host species. The extensively characterized Autographa californica nuclear polyhedrosis virus is usually the baculovirus of choice. Autographa californica nuclear polyhedrosis virus is able to infect several mammalian cell types, but is incapable of replication or integration in these cells.115,116 However, transfection efficiencies in vivo have been poor despite impressive results in cell culture,117 due to the activation of the classical complement system.118 Although strategies to overcome the complement pathway have been attempted,119 the most effective route of delivery may be to sites without complement components, such as the brain.120Autographa californica nuclear polyhedrosis virus also appears to induce an innate antiviral and antitumor immune response in vivo,121–123 which is detrimental to repeated administration, but beneficial for vaccination strategies. Unfortunately, these baculoviruses do not offer the ease-of-production of bacteriophages or VLPs, nor are they more effective at gene delivery and as such are unlikely to be more advantageous than other delivery systems. Theoretically, yeast particles could also be utilized for gene delivery, although to date this possibility remains to be investigated. Recombinant nonpathogenic yeast particles expressing tumor and HIV antigens have been shown to strongly activate dendritic cells and produce a cytotoxic T-lymphocyte response when introduced subcutaneously,124 demonstrating a high specificity toward dendritic cells. If engineered to release mammalian plasmids encoding genes such as interleukin-12 or oligonucleotides that potently activate dendritic cells, the efficacy of vaccines could be significantly enhanced. Conversely, the strong targeting ability of recombinant yeast could be used to promote antigenic tolerance via the delivery of antigenic sequences to semimature dendritic cells125 in order to improve outcomes in organ transplantation and in autoimmune disease. Given the relatively low costs of yeast production, this may be an avenue of research worth pursuing.

Conclusions The biological GDVs discussed above, although less well-established than viral vectors and liposomal delivery agents, present tantalizing opportunities for niche therapeutic applications given further research and development (see Table  2). Besides their intrinsic advantages, biological GDVs can also benefit tremendously from development of tangential technologies. For example, VLP production can benefit from the research and industrial experience of vaccine production, while bacterial production and delivery could build on the experience of the food industry with www.moleculartherapy.org vol. 17 no. 5 may 2009

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probiotics. Given the expertise already developed for nondelivery applications, the barrier between bench and bedside applications is probably not as high as perceived; hence the development of biological GDVs probably deserves greater attention from the gene therapy community. It is clear that there is no one-size-fits-all solution to gene delivery, which is why in spite of various developments in liposome formulation and viral vector optimization, new compounds and viruses are constantly being proposed. What is needed is an arsenal of GDVs that can be utilized for specific diseases, routes, or tissues and it is likely that biological GDVs will emerge as viable options for repeat gene delivery for long-term treatment in the case of chronic disease. What this review has aimed to achieve is to summarize the progress in unconventional biological GDVs and focus on how best to exploit their properties and associated tools. The common limitations of current systems appear to be tractable if GDVs are chosen for their natural propensities for certain niche targets rather than adopting the current onesize-fits-all approach. Once the risks and limitations have been minimized with such delivery strategies, gene therapy can then be harnessed as a therapeutic for a wide variety of applications, including pre-emptive gene therapy and therapy for diseases with weaker genetic linkages, e.g., psychiatric illness. Hence, it is vital that further research be undertaken into the development of novel biological GDVs in order to diversify the delivery field and enable new gene therapy applications unimaginable today. Acknowledgments We thank Graham McClorey and Marc Weinberg for comments and critical reading of the manuscript. Y.S. acknowledges funding support from the Agency for Science, Technology and Research (Singapore) and M.J.A.W. from the UK MRC and Biotechnology and Biological Sciences Research Council (BBSRC), The Wellcome Trust, the UK Parkinson’s Disease Society, the Muscular Dystrophy Campaign and Action Duchenne.

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