Gene therapy: a key technology for the treatment of severe diseasesProgress in the field of invasive and non-invasive diagnostic techniques now allows the detection of many diseases earlier than in former times. Furthermore, advances in biotechnology, proteomics and genomics, as well as the deeper understanding of the molecular and cellular basics of human health and disease, have allowed the development of novel therapeutic strategies. In particular, severe genetic, hereditary and acquired diseases such as AIDS, cancer, cystic fibrosis or cardiovascular diseases, which are often not accessible to efficient and causal therapies, may benefit from the new therapeutic concepts [1,2].
Gene therapy is one of these key technologies that boomed during the last 20 years, raising great hopes for being effective in therapy, diagnostics and prophylaxis. Gene therapy products are def ined by the regulatory authorities as all products that mediate their effects in vivo or ex vivo prior to administration to the recipient, by transcription and/or translation of transferred genetic material and/or by integrating into the host genome [3,4]. They are administered as nucleic acids, viruses or genetically engineered microorganisms. Therefore, the term ‘gene therapy’ accounts for the regulation or replacement of a defect or missing gene, or the expression of a new genetic sequence creating a site for therapeutic invention. The nucleic acid may encode for shRNA or antisense oligonucleotides to delete the expression of disease-related endogenous genes at the transcriptional or translational
level. This def init ion would exclude oligonucleotide products such as synthetic DNA or RNA material. Although the original concept dates back to 1944 when Avery et al. demonstrated that genes could be transferred by nucleic acids, it took a long time until the first approved clinical trial in humans in 1990, treating patients with adenosine deaminase deficiency [3,5].
The goal of the therapeutic interventions is the transfer of genetic material, particularly in somatic cells. Gene therapy on germline cells passing the gene to the next generation is still not allowed by legislation due to ethical concerns. Currently, two different approaches are used [3,6]: for the ex vivo procedure, the patient’s cells can be explanted and expanded, transfected with the genetic information and re-infused or re-transplanted into the same subject. The main advantage of this technique is that all cellular manipulations are performed outside the body without exposure to the patient. On the other hand, this approach is very time consuming and cost-intensive, and the acceptance by the patient and physician is limited due to the sophisticated surgeries. Therefore, in vivo gene transfer is preferred where the transfection takes place in the patient’s body after direct local or systemic delivery of the genetic material.
Gene therapy is still an experimental approach. Although for in vitro gene delivery various commercially available transfer reagents have been introduced, the in vivo situation is considerably more complex, especially in the case of systemic administration. After more
Recent developments and perspectives on gene therapy using synthetic vectors
Nonviral vector technology is attracting increasing importance in the biomedical community owing to unique advantages and prospects for the treatment of severe diseases by gene therapy. In this review, synthetic vectors that allow the controlled design of efficient and biocompatible carriers are highlighted. The current benefits, potentials, problems and unmet needs of synthetic gene delivery systems, as well as the strategies to overcome the obstacles are also discussed. Common design principles and structure–activity trends have been established that are important for stable and targeted transport to regions of interest in the body, efficient uptake into cells as well as controlled release of drugs inside the cells, for example, in specialized compartments. The status quo of the use of these systems in preclinical and clinical trials is also considered.
Florian Schlenk†, Stefan Grund† & Dagmar Fischer*Institute of Pharmacy, Department of Pharmaceutical Technology, Friedrich-Schiller-University Jena, Otto-Schott-Strasse 41, D-07745 Jena, Germany *Author for correspondence: Tel.: +49 3641 949941 Fax: +49 3641 949942 E-mail: [email protected]†Authors contributed equally
than 1700 clinical trials since 1989, only one gene therapy product has been approved to market by the authorities in the western countries today. In most cases, the efficiency of the nucleic acid-based drugs themselves is not the limiting factor. One of the main issues lies in the safe and efficient delivery to a given target site of action, seriously hampering their therapeutic utility so far [7].
Viral vectors, which were the first carriers described for the use in human gene therapy, demonstrated high transduction efficiencies. However, they suffered from issues such as strong immune response, especially after repeated injections, an oncogenic potential due to insertional mutagenesis and limited DNA-carrying capacity [3,8]. These aspects forced the development of vectors without viral elements per se, but with the intention to mimic the successful key features of the viruses. These so-called nonviral vectors based on lipid (lipoplexes) or polymeric (polyplexes) materials show several advantages, including lower safety risks and the ability to carry larger amounts of DNA, as well as the potential for easy modifications and the safe and cost-saving large-scale synthesis [9,10]. They can be custom-designed for specific therapeutic needs by tailoring their molecular weights, hydrophobic/hydrophilic balance, solubility and stability of the complexes as well as the binding to specific cells or tissues. The first polymer for gene transfer was introduced in 1965 by Vaheri and Pagano using diethylaminoethyl modified dextran [11]. N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl-ammonium chloride (DOTMA), the f irst cationic lipid was published by Felgner in 1987 [12]. Since that time the number and variety of structures has dramatically increased.
The present review covers the current progress in nonviral gene delivery using synthetic vectors with a special focus on cationic polymers and lipids. The challenges of the use of plasmid DNA, the barriers and issues of a safe and efficient gene delivery by nonviral vectors, as well as the current strategies to overcome these will be discussed. The concept of multifunctional nonviral vectors, the status quo of preclinical and clinical trials, as well as the needs for reproducible and controlled manufacturing and scale-up to provide sufficient amounts of complexes for broader applications, will be highlighted. The most important trends and troubleshootings for the development of improved and more sophisticated nonviral vector systems will be presented.
DNA as an active ingredient: from a technological & biological point of viewPlasmid DNA molecules used for gene therapy are highly susceptible to physical and chemical degradation, which plays a role during packaging in and storage of delivery systems, as well as biodistribution in the organism [13]. Additionally, several biological barriers have to be overcome for successful transcription and translation. The issues related to the administration of nucleic acids in vitro and in vivo can be categorized in three main aspects: limited stability with fast degradation and excretion; low cell-penetrating ability; and as the need to apply high doses in humans [8]. These topics will be discussed in the following to elucidate the need for nonviral vector systems.
The effect of plasmid DNA is strongly dependent on an intact sequence [14]. Improper physical agitation such as mechanical stress, ultrasound treatment, shear stress under vigorous shaking, or stirring (e.g., during manufacturing) can cause fragmentation of the nucleic acid chains [15]. The open circular form preserves approximately 90% of transfection efficacy of the supercoiled plasmid, while the linear form has approximately 10% of the original efficacy [16,17]. Ultrasonication of naked plasmids results in complete fragmentation after 30 s [16]. Chemical degradation by hydrolysis of the bases, oxidation, photochemical reactions or enzymatic cleavage by nucleases may induce changes in the primary structure, cleavage of nucleic acid chains, loss of supercoiled content and, consequently, reduced biological activity [13,18,19]. Although these effects are temperature dependent and can be slowed down by storage under cooling or freezing conditions, improper freeze–thaw steps themselves (e.g., during lyophilization) may cause degradation and aggregation [13].
Following systemic administration, plasmids are rapidly eliminated from the blood stream, with a half-life of less than 5 min [20,21]. Several mechanisms have been identified that contribute to the fast clearance of DNA from the circulation including rapid degradation by enzymes, opsonization and phagocytosis, or filtering by capillaries. The high clearance from the circulation may be due to metabolic degradation rather than the extensive uptake by cells and tissues [20,22]. DNA is known to undergo degradation by endo- and exo-nucleases within 5–10 min [23]. Free plasmid DNA was
Key Terms
Transfection: Transfer of nucleic acids into cells using nonviral vectors or physical methods.
Transduction: Transfer of nucleic acids into cells by viral vectors.
Lyophilization: A gentle method for dehydration after freezing of high-quality and sensitive material. The frozen water sublimates under reduced pressure from the solid to the gas phase.
Review | Schlenk, Grund & Fischer
Therapeutic Delivery (2013) 4(1)96 future science group
found to be mainly accumulated in the liver and excreted by the hepatic pathway. Plasmids were taken up by liver nonparenchymal cells such as endothelial and Kupffer cells via a specific scavenger receptor-like mechanism that recognizes a wide range of polyanions [21].
Enzymatic and physical barriers efficiently protect mammalian cells from foreign genetic material. Once the plasmid DNA has reached its target cells, uptake is hindered by the high molar mass and hydrophilic character, as well as the negative charge of the plasmid DNA leading to electrostatic repulsion with the also negatively charged cell membrane [8]. Molecules taken up by clathrin- or caveolin-dependent mechanisms, phagocytosis or macropinocytosis end up in endosomal/lysosomal compartments, where the DNA has to escape nucleases and a low pH of approximately 5 in order to avoid degradation. After its release into the cytoplasm, the DNA has to enter the nucleus for transcription followed by translation into the related protein sequence [24]. Passive diffusion of materials in the cytoplasm is slow due to its high viscosity and high protein concentration. It seems unlikely that plasmid DNA can move in the cytoplasm because it demonstrated a size-dependent mobility with DNA >2000 base pairs being unable to diffuse in the highly crosslinked actin cytoskeleton [25,26]. Furthermore, due to cytoplasmatic DNases, the half-life of free plasmids was estimated to range between 50 min and 5 h [27,28]. Vaughan et al.
calculated that, assuming an average half-life of 3 h, during a typical 24 h transfection less than 0.4% of the input DNA would remain [24]. The nuclear envelope represents the most substantial barrier for the transport of plasmids. Only during mitosis when the nuclear barrier breaks down, or by an energy- and dose-dependent process over a nuclear pore complex can DNA reach the nuclear machinery for transcription. With the exception of DNA modified by a nuclear localization sequence facilitating its nuclear entry, only limited amounts (one in 1000 plasmids) are taken up [24]. Plasmids required in the nucleus for measurable transgene expression, however, were reported to vary between 75 and 4000 plasmid copies depending on the type of vector.
Taking all of the discussed challenges for the in vitro and in vivo use of plasmid DNA into consideration, they highlight the necessity for the formulation of therapeutic DNA by vector systems. In the following section, the characteristics and, most importantly the design principles of nonviral vectors will be summarized.
Synthetic nonviral vector materialsThe limitations of the viral vectors have stimulated the search for nonviral alternatives based on polymeric or lipid macromolecules for the transfection of cells. Tables 1 & 2 give an overview of frequently used, most advanced
Table 1. Current developmental status of the most advanced polymeric gene-delivery systems.
Structure Synthetic polymer Delivered genes Developmental status
EBV: Epstein–Barr virus; EGFP: Enhanced green fluorescent protein.
Recent developments & perspectives on gene therapy using synthetic vectors | Review
www.future-science.com 97future science group
polymeric and lipidic nonviral vector materials, respectively. Based on electrostatic interactions between the cationic carrier materials and the negatively charged DNA, nanoassemblies can spontaneously form that mask the anionic charge of the DNA (complexation) as well as the condensation of the DNA to small-sized units [29]. Whereas most of the cationic materials are able to complex plasmid DNA, the condensation defined as a remarkable reduction in molecular volume followed by a molecular collapse that protects plasmids against enzymatic degradation, is rarely achieved [30–32]. From the chemical point of view, synthetic polymers and lipids applied for gene therapy are a highly versatile and diverse group of macromolecules. Therefore, the design of new vectors based on studies investigating structure–activity relationships has boomed in the recent years [33]. The majority of these molecules show some common structural features, which provide the basis for the rational design of new and improved DNA carriers [1].
For polycationic polymers, the molar mass, type, number and density of cationic charges as well as the 3D structure and flexibility of the polycations, which determine their acces-sibility to the negative DNA and cell mem-brane charges, were reported to determine the physico chemical complex characteristics, biological efficiency and safety [1,34]. Highly branched macromolecules as well as polyca-tions with high-molecular-weights, high cati-onic charge densities and a high 3D flexible architecture (globular < linear < branched) are characterized by strong electrostatic inter-actions with plasmid DNA, resulting in an
efficient protection against enzymatic degra-dation. They are able to form small and stable polyplexes with high cell-uptake and transfec-tion efficiencies [34,35]. However, their action is limited due to toxicological effects such as hemolysis, erythrocyte aggregation and cell and nuclear membrane damaging effects, as well as reduction of metabolic cell activity related to strong interactions with the negatively charged cell membranes and the extracellu-lar matrix [36]. This phenomenon was termed the ‘charge dilemma’ [1]. Branched polycations (e.g., poly(ethylenimine) [PEI]) displayed higher cytotoxic effects than linear (poly(l-lysine), cationic poly(vinylpyrrolidone) [PVP]) or globular and rigid structures (dendrimers or cationized albumin) due to increased pos-sibilities to come into contact with cellular membranes [36]. Additionally, the molar mass of the polymers strongly influences cytotoxic-ity and transfection. As an example, branched PEIs with sizes >25 kDa are highly effective in transfection but presented higher cytotoxicity when compared with branched PEIs <5 kDa. In contrast, smaller PEIs of <2 kDa were reported to prevent lower toxicity, but showed insuffi-cient transfection efficiency. It has to be taken into consideration that conflicting data regard-ing the safety of PEI exist, which is mainly due to the different molecular weights of PEIs used in different studies [34,37].
Lipids for the transfection of plasmid DNA exhibit common design principles such as a cationic head group with a single or with multiple charges (mostly polyamines or quaternized ammonium salts), which is
Table 2. Current developmental status of the most advanced lipid and liposomal gene-delivery systems.
Head group Cationic lipid Delivered gene Developmental status
Therapeutic Delivery (2013) 4(1)98 future science group
covalently linked by a spacer to a hydrophobic hydrocarbon tail consisting of fatty acids, alkyl groups or cholesterol derivatives [9,38]. While for monovalent head groups unsaturated C18-fatty acids were reported to be highly effective in vitro and in vivo, saturated C12- and C14-chain lengths were preferred for multivalent macromolecules. The cholesterol-based cholesteryl-3β-carboxyamidoethylene-dimethylamine was the first cationic lipid tested in clinical trials. The function of the head group is related to electrostatic interactions with the DNA and the cell membranes and is thought to be responsible for cytotoxic effects, as described above for polymers. Dependent on its chemical nature (ether, ester, amide or carbamate), the spacer contributes to the stability and biodegradability of the lipidic macromolecules. Whereas amides and carbamoyl groups display sufficient stability and biodegradation, ester groups were found to be too labile during storage and ether groups suffer from a low biodegradation. The length of the linker does not seem to be relevant for transfection efficiency. DOTMA was the f irst monovalent cationic lipid introduced, dioctadecylamido-glycylspermine (DOGS) the first polyvalent cationic lipid. The high electrostatic repulsion of the cationic head groups during carrier formation can be reduced by addition of neutral helper lipids such as dioleoylphosphatidylethanolamine (DOPE), which additionally forms inverse hexagonal nonbilayer structures and supports the fusion of membranes, for example, during endosomal release [9,38,39].
Today, all of these polymers and lipids can be prepared as chemically def ined substances under controlled conditions free of contaminants and immunogenic components. As established for the use of other drug delivery systems, they have to ensure the nucleic acid accumulation at the intended sites; rerouting of the genes from sites of toxicity; and increasing the plasma half-life of the labile and rapidly eliminated nucleic acids (FiguRe 1) . The biodistribution and elimination patterns of injected polyplexes or lipoplexes are dictated mainly by their overall physicochemical properties. Size and stability of the complexes, hydrophobicity of the particle surface as well as density and type of surface charge were found to be the key parameters for their plasma half-lives, extravasation through endothelial gaps as well as adsorption of opsonizing proteins onto
the surface of carrier systems and subsequent uptake by the reticuloendothelial system [33]. Gene delivery systems should allow a reduction of the administered therapeutic dose, thereby reducing toxic side effects and costs for the treatment. FiguRe 2 gives an overview of the requirements of synthetic nonviral vectors for gene delivery.
The need of multifunctional conceptsAs stated above, the transport and expression of plasmid DNA is a multistep process facing many efficient barriers for gene transfer that have to be overcome by nonviral vector systems (FiguRe 1). The main hurdles where synthetic vectors require an improvement are: extracellular delivery and targeting; enhanced intracellular release and persistence of expression; reduced cyto- and hemo-toxicity; and nonimmunogenicity [10,40]. To address all of these needs, multifunctional carrier systems consisting of the polymeric or lipidic components combined with stealth polymers, biodegradable linkers, ligands for cell-specific targeting and lysosomotropic or fusogenic agents for endosomal release were developed.
Stealth polymersTo overcome the adverse effects of opsonization during systemic application, hydrophilic, nonionic polymers can be incorporated in the complexes to prevent recognition by the
Figure 1. Summary of the main barriers for synthetic nonviral gene-delivery systems. Needs for synthetic vectors are (A) formulation of a stable application form; (B) hemocompatibility; (C) successful cell uptake; (D) endosomal release of DNA; and (E) uptake into the nucleus.
Recent developments & perspectives on gene therapy using synthetic vectors | Review
www.future-science.com 99future science group
reticuloendothelial system and rapid clearance. This so-called stealth or brush effect was discussed to be related not only to reducing protein adsorption and opsonization, but also to selective adsorption of dysopsonins or influencing the rates of adsorption of different opsonization/desopsonization proteins. PEG technology provides the ability of stealth effects also in the sense that the molecules coated with PEG have the ability to bypass different defence mechanisms depending in which system of the human body they are administered (e.g., lung). The stealth effect may lead to less frequent dosings supporting the patient’s compliance. Additionally, higher water solubility, reduced aggregation, higher biocompatibility, and reduced immunogenicity of the delivery systems can be accomplished [41].
Today PEG is used as the ‘gold standard’ for shielding the cationic charge of complexes since it has demonstrated its safety and efficiency in a high number of marketed products. Depending on length and density of PEG chains, as well as the selected linker technology, the protein-repellent effect of PEG has been associated with
steric repulsion and/or hydration and water structuring due to the high flexibility and the large number of possible conformations of the PEG chains. However, more and more reports have now been published that deal with adverse effects of PEG in clinical trials [41,42]. Formation of antibodies, complement activation after systemic use leading to hypersensitivity, unclear pharmacokinetic events after repeated application (accelerated blood clearance phenomenon) and gastrointestinal issues after oral administration have been reported. Although only observed in a small number of patients, these effects triggered the search for alternative stealth polymers, which are comprehensively discussed in several reviews [41,42]. Poly(amino acid)s such as poly(glutamic acid), poly(hydroxyl-ethyl-l-asparagine) and poly(hydroxyethyl-l-glutamine) showed favorable properties, for example, prolongation of blood circulation of carriers, a decreased accelerated blood clearance effect and biodegradability. However, complement activation was induced by polymers with more than three amino acids. Polyglycerols and poly(hydroxypropyl methacrylate) showed
Easy to use in scale-up AdministrationSafe and easy
Adequate containersGood cost/risk/use profile
SynthesisUse of approved chemicalsEasy and cheap synthesis
Easy to sterilize
Technical needs Biological needs
BiocompatibilityLow cytotoxicity
High hemocompatibilityExcretion
No body accumulation
Modernnonviralvector
GMP
Figure 2. Requirements for modern synthetic nonviral vectors considering technical and biological aspects.
Key Term
Drug targeting: Selective accumulation and release of a drug in a target cell or tissue after systemic administration. Can be classified in active targeting (selective interaction between a ligand and cell surface receptor) or passive targeting (due to physicochemical properties of drug formulation and target cell or tissue).
Review | Schlenk, Grund & Fischer
Therapeutic Delivery (2013) 4(1)100 future science group
advantages and limitations comparable to PEG whereby poly(hydroxypropyl methacrylate) has already entered Phase III clinical trials. Poly(oxazoline)s behaved similar to PEG with regard to blood half-life, opsonization and organ distribution as well as in vitro cyto- and hemo-compatibility [43]. Many of these candidates are highly promising in the preclinical status, but still more preclinical, toxicological and clinical investigations are necessary for their evaluation as stealth polymers in human applications. The new stealth candidates have to compete with approximately 20 years of safe human use of PEG. Nevertheless, it is suggested that the promising potential of the alternative polymers will be explored during clinical investigations [44].
Biodegradable linker technologiesAs a disadvantage, however, the stealth effect limits cell uptake and endosomal release of the complexes as well as DNA release from the nanoassemblies. Dynamic complex shielding using PEG and a pH-cleavable linker that allows for endosomal deshielding has been achieved by acetal, pyridylhydrazone or dialkylmaleic acid linkers [45]. Conjugates of PEG and polycation with the pH-labile linker pyridylhydrazon demonstrated degradation at pH 5 in endosomes within 5 min, whereas at pH 7 complexes remained stable for several hours [46]. A dialkylmaleic acid linker was successfully used for PEG deshielding of poly(butyl/amino vinylether) carriers [47]. Hydroxyethyl starch as a PEG substitute was used for the controlled shielding and deshielding of PEI polyplexes, which offers the advantage of controllable biodegradation. The rate and extent of degradation could be regulated by careful selection of molar mass and degree of hydroxyethylation [48].
Incorporation of hydrolytically or enzy-matically cleavable groups such as disulfides, esters, amides, imines or diacrylates that medi-ate the degradation of nonviral vectors to low molecular weight metabolites followed by fast excretion have become popular for advanced drug-delivery systems [49,50]. An increasing number of gene delivery systems incorporat-ing disulf ide bonds have been reported in recent years since they take advantage of the relatively reducing environment of the intra-cellular space via the glutathione pathway. Several authors demonstrated that biodegrad-able high-molecular-weight polycations (poly-spermine, poly[ethylene imine], hyperbranched
dendrimers and poly[amido ethylene imine]) formed via disulfide bonds are efficient, yet much safer than commercially available high-molecular-weight vectors [8,50–52]. Whereas the proof-of-concept under in vitro conditions in cell culture was found to be very promising, a detailed understanding of disulfide reduction during the trafficking of such systems in the bloodstream is still missing. Using a redox-sensitive third-generation poly(amido amine) dendrimer, Brülisauer et al. were able to show that the properties of the disulfide bonds were strongly influenced by the polyionic nature of the gene delivery system [53]. Disulfide cleavage occurred to a large extent in the extracellular environment, as shown for different cell lines, depending on the unique reducing properties of each cell type. The authors concluded that a ‘one-size-fits-it-all’ approach of disulfide bonds has to be critically questioned and further investigations, especially with regard to stabil-ity in the blood and under in vivo conditions are necessary.
Cell-specific targeting moietiesIncorporation of targeting ligands for cell- and tissue-specific uptake of complexes into cells (drug targeting) was shown in numerous reports to successfully increase specific transfection of target cells in vitro by factors of 100- to 1000-fold. Several comprehensive reviews give an overview of successfully used targeting ligands such as sugars, peptides, vitamins, lectins, growth factors or antibodies [54–56]. The concept of active tissue- and cell-targeting in gene therapy has been investigated for more than 15 years without bringing a targeted product to the market, despite many successful preclinical in vitro reports. Differences between the in vitro test conditions under static conditions and the dynamic in vivo conditions in the bloodstream were suggested to be responsible. Blood flow may compromise the interaction between ligand and target, especially for ligands with low-binding aff inities. It is proposed that binding studies in cell-based perfusion assay systems that are already used for cell adhesion experiments may increase the prediction for in vivo applications [57]. Furthermore, in many in vitro experiments non-target cells have not been tested, thus leading to misleading information regarding specificity [8]. In tumor therapy, the high heterogeneity of human tumors even at different tumor stages may compromise a therapy equally successful in all patients. Thus,
Recent developments & perspectives on gene therapy using synthetic vectors | Review
www.future-science.com 101future science group
a prescreening of patients for a certain receptor at the surface of target cells may increase the success rate of targeted medicines, as already discussed in personalized medicine concepts. The first targeted synthetic nucleic acid delivery system, CALAA-01, went into clinical trials for solid tumors and has been recruiting patients since February 2012. It consists of a cyclodextrin-containing polymer, a steric stabilizing moiety (AD-PEG) and a targeting agent containing transferrin as a ligand for binding to the transferrin receptor that is typically upregulated in cancer cells [58].
Cell-penetrating peptides based on lysine-rich (e.g., transportan) or arginine-rich (e.g., homeodomain of Antennapedia, HIV-1 trans-activating transcriptional activator) facilitate a non-cell type-specif ic uptake of particulate systems directly into the cytoplasm without endosomal involvement [59]. Hydrophobic residues (alkyl chains, steroids and hydrophobic amino acids) covalently bound to polymers increase the interaction with the lipophilic parts of cell membranes and, consequently, the transfection rate in an unspecific manner [60–63].
Endosomal releaseA major limitation of many transfection agents is the low transfection efficiency due to poor endosomal escape. Polycations need to accomplish the protection of nucleic acids against this hostile environment as well as the release of the drug into the cytoplasm. For lipoplexes, release by membrane fusion has been described that is mediated by the interaction with anionic endosomal phospholipids and has the ability to transit from the lamellar to the hexagonal phase [38,64]. After electrostatic interaction between the cationic lipid and the anionic membrane of the endosomes, the lipidic bilayers form ion pairs leading to membrane destabilization and the release of DNA. The transition from the lamellar to the hexagonal phase can be enhanced by the geometry of the lipid molecules with a preference for cone-shaped systems, the integration of helper lipids (e.g., DOPE) and the transition temperature of the lipid formulation [65–67].
Polymers with a high degree of amine groups and low pKa values have been shown to act by buffering the acidic environment of the endosomes due to their proton acceptor function. This leads to swelling and rupture of the endosomes due to an increase in ionic
strength and subsequent water inf lux into the endosome, termed ‘proton sponge effect’ [68]. The buffering capacity of the polycations was reported to be influenced by molecular weight, degree of branching and type of alkaline groups [69,70]. Fusogenic peptides such as KALA, GALA or the N-terminus of the hemagglutinin subunit HA-2 of the influenza virus demonstrated changes in their 3D structure upon lowering the pH in the endosomes, thus triggering interactions with the endosomal membranes and formation of pores, fusion or lysis of the membrane [71]. Photosensitizers that selectively accumulate in endosomes accomplish the complex release from endosomes after photoactivation [72]. The different endosomal release mechanisms demonstrated their potential, but have been discussed as being far from sufficiently effective for clinical applications [8].
Since the trafficking of nonviral vectors is a multistep process, several strategies can be combined to multifunctional vector systems (FiguRe 3). As a major limitation the function and synergism of all these strategies, especially under clinical conditions, has still to be investigated. Several studies have shown that successful in vitro transfections observed for the use of nonviral vectors often fails to reproduce in in vivo studies.
Transfer of preclinical concepts to clinical trialsMultiple structurally different polymeric and lipidic vectors have been established over the last two decades, but none of them has received marketing approval by the regulatory authorities. Successful in vitro transfection by nonviral vectors often fails to reproduce the results under in vivo conditions. Several reasons are discussed in the literature for this phenomenon.
Little is known about the composition and arrangement of the nucleic acid and the carrier material in the complexes. In the case of polyplexes, a plasmid core surrounded by a polymer shell has been discussed as often as a model favoring the randomized distribution of both components in the electrostatic complex. The dynamic processes and changes that the complexes undergo in different preparation media, as well as in physiological media and the bloodstream, are barely understood while being important for the improvement of stability [29,73]. Cell culture experiments often performed in serum-free media may give
Review | Schlenk, Grund & Fischer
Therapeutic Delivery (2013) 4(1)102 future science group
misleading transfection results, suggesting higher complex stabilities than realistic under in vivo conditions. Complex stability and delivery of genes were described to be progressively compromised with increasing serum levels, with the effects of serum only being less dramatic when the serum content increases to values higher than 50% [29,73,74].
Scientists are now starting to realize the importance of the selection of the biological cell-based test system. In a typical early stage of preclinical development, nonviral vectors are tested and optimized in 2D in vitro cell culture monolayers. Most of these cell culture experiments employ rapidly dividing cells that do not mimic the in vivo situation [10]. Cultured cells are usually transfected in a subconfluent state characterized by highly dividing cells. Since the breakdown of the nuclear membrane in dividing cells facilitates gene transfer in vitro, the
system is not comparable to the situation in vivo. Compared with 3D tissues, the cell monolayers lack an extracellular matrix surrounding the tissues that has to be passed by vectors in order to reach the target cells [10]. Penetration of nonviral vectors into deeper layers or tissue cannot be simulated with 2D monolayers. In 3D cultures, an antibody against β1-integrin seemed to change cancerous cells to noncancerous cells, a behavior that was not observable in 2D cultures. Gene activity in 2D and 3D cultures may be different [75]. For example, in the same breast-cancer system, it has been shown that antibodies against β1-integrin decrease signaling by EGF receptors; antibodies against EGF receptors similarly depress the activity of β1-integrin [76]. This reciprocal interaction does not occur in 2D cultures. Expression profiles in 3D cultures were reported to be closer to the in vivo situation than to the 2D situation, as determined by DNA
Figure 3. Synthetic vector modifications including surface functionalization and incorporation strategies. The objective is the formulation of multifunctional systems possessing DNA as active ingredient with biocompatibility, specific targeting and diagnostic labeling. PHPMA: Poly(N-[2-hydroxypropyl] methacrylamide).
Recent developments & perspectives on gene therapy using synthetic vectors | Review
www.future-science.com 103future science group
microarrays [77]. Additionally, more sophisticated and advanced imaging technologies will bring new insights into this area.
Different cell morphologies of cells in 2D and 3D cell layers are assumed to be responsible for differences between in vitro and in vivo transfection results [10,76,77]. In monolayers consisting of f lattened cells, the moving distance of complexes to the nucleus strongly depends on the site of cellular particle uptake. More specifically, complexes taken up in the supranuclear region have a short moving distance to the nucleus, while cytoplasmatically located complexes have to move a longer distance. In contrast, cells in vivo display a more spherical geometry where all points at the cell surface have nearly the same distance to the nucleus. Differences in endosomal and drug-release profiles, as well as in the time frames of each transport step, may lead to hardly comparable results. Led by cancer researchers, vector technologists are increasingly turning to 3D cell cultures such as 3D spheroids or Matrigel cultures that consist of structural proteins (laminin and collagen), growth factors and enzymes from mouse tumors [78].
An additional hurdle is the investigation of the complexes in 2D cell cultures under static cultivation conditions. Sedimentation can artificially enhance the association of complexes with cell membranes and elevate the transfection efficiency [10]. The larger the complexes or their tendency to aggregate, the greater the sedimentation and, therefore, the contact with the cell surface. In contrast, smaller particles <100 nm favor the advantage of faster diffusion and deeper penetration into tissues in vivo. The importance of dynamic perfusion systems has already been highlighted above.
Currently, the efficiency of the synthetic vectors in the clinic is too low for obtaining therapeutic levels of gene expression. Thus, in many preclinical animal experiments high complex doses were used, especially for systemic administration [8]. Correlating these doses to a 70 kg patient, several 100 mg would have to be applied, bearing the risk of toxic side effects of the cationic carrier as well as immunological effects by DNA. High doses of DNA with immunologically active motifs such as CpG can illicit an immune reaction [79,80]. Minicircles as an alternative to classical plasmids do not contain any bacterial sequences that could induce immunostimulatory effects [81]. Based on the recommendations of the European Medicines
Agency, minicircles without resistance gene against antibiotics or other markers were established to increase product safety.
Manufacturing of polyplexes & lipoplexes: from laboratory technology to a broad applicationAs presupposition for broad applications and clinical use, stable and highly purified nonviral vector formulations with defined physicochemical characteristics have to be produced under controlled conditions in order to accomplish batch-to-batch reproducibility. Up to date, the formulation of lipo- and poly-plexes has been performed preferentially in the laboratory scale (micrograms to milligrams), freshly prepared before each application in a bulk mixing process [82,83]. In these protocols, the DNA and carrier material are diluted separately in a suitable physiological medium (e.g., 5% glucose solution; 0.9% physiological saline) and then mixed by multiple pipetting steps, high-speed shakers or vortexing. During 10 to 30 min incubation at room temperature, the components spontaneously self-assemble by electrostatic interactions into nanoparticulate structures, which can be directly used for in vitro and in vivo transfection [82,84]. The characteristics of the resulting complexes can be controlled by careful selection of the mixing medium (ionic strength and pH value), the ratio of polymer to DNA (expressed as the N/P ratio, i.e., ratio of nitrogens [N] in the polymer to phosphate groups [P] of DNA), the order of adding the reagents, as well as the DNA concentration [17,85,86].
The order of reagent addition greatly influences the resulting particle size because addition of the cationic component to the plasmid solution was tenfold more efficient than adding the plasmid to the carrier [82,84]. The pH of the chosen medium determines the number of reactive charges of vectors containing primary, secondary or tertiary amines and therefore, the intensity of electrostatic interactions. As an example, PEI shows a degree of protonation of 20% at pH 7.4, compared with approximately 45% at pH 5 [87]. The higher the ionic strength and the number of multivalent ions of the dilution medium, the lower the binding affinity and the larger the complex size [88]. Polyelectrolyte complexes seem to undergo polyanion exchange, suggesting a charge-shielding effect at higher salt concentrations. Binding of DNA to PEI is
Key Term
Microfluidizer: For the purpose of producing emulsions and small-particle structures with uniform distribution, microfluidizers with interaction chambers composed of microchannels are used. The generated shear forces lead to a reduction of size and homogenization.
Review | Schlenk, Grund & Fischer
Therapeutic Delivery (2013) 4(1)104 future science group
thought to be mainly driven by entropic forces arising from the release of counter ions [89]. A small sized complex is a necessary prerequisite for the efficient transfection since endocytosis is more efficient with particles <200 nm and the cytoplasmic movement was reported to be a function of particle size [90]. The optimal N/P ratio for transfection has to be determined for each carrier system. While complexes at N/P 1 are characterized by neutral charge, low density and instability against enzymatic degradation, an excess of cationic charges (N/P >1) results in stable, condensed and small-sized nanoparticles [34,91]. However, the higher the N/P ratio, the higher is the risk of free, noncomplexed carrier material, which was reported to be primarily responsible for cytotoxicity compared with the complexes themselves [92].
Although the mixing techniques in the laboratory scale are easy to handle, major drawbacks are the restriction to relatively small volumes and the insufficient reproducibility and control of the complex characteristics often resulting in heterogeneously distributed, poorly defined complexes [83,93]. For clinical applications, pharmaceutical and technological aspects such as quality assurance, batch-to-batch reproducibility, adequate handling by physicians and practicability cannot easily be assured. It is difficult to establish adequate quality controls when the complexes are freshly prepared directly prior to administration to the patient [86,94,95]. Conclusively, several methods have been developed for lipo- and poly-plexes that should allow for the reproducible and automated manufacturing for large-scale preparation.
In the so-called micromixer technique based on a pumping method, equal volumes of DNA and lipidic or polymeric carrier material were diluted separately and loaded into two separate reservoirs such as syringes or closed f lasks. Both dilutions were mixed while passing a T- or Y-connector with constant mixing speed controlled by syringe size and plunger speed, and followed by automated filling into the application bottles. Clement et al. reported a large-scale manufacturing process for 3β[N-(N,N -́dimethylaminoethan)-carbamoyl] (DAC
30)-based liposomes [86]. The lipid dissolved in transfection medium was extruded once through an 800 nm pore size polycarbonate membrane for homogenization by a pump and was mixed with equal volumes of plasmid DNA in a Y-connector via a second peristaltic pump. The method was optimized with regard to lipid/plasmid ratio,
lipid pretreatment, type of the mixing medium, size of the mixing device, as well as mixing and lyophilization procedure. Resulting lipoplexes were directly aliquoted into bottles and lyophilized. In the case of 22 kDa poly(ethylene imine) as cationic carrier, an increase of mixing speed resulted in a decreased particle size and polydispersity [94,96]. The diameter and polydispersity index of the polyplexes increased with higher DNA concentrations [96]. All techniques can be conducted on a large scale under sterile conditions.
Several authors modified the microfluidizer technology using devices with three inlets and one outlet, based on the principle of hydrodynamic focusing [97,98]. The DNA solution in the middle inlet was squeezed or hydrodynamically focused by the other two inlets containing a 25 kDa branched PEI solution, into a narrow stream [98]. Molecules between the streams seemed to interdiffuse very rapidly, thereby reducing the mixing time to microseconds and forming small and uniform complexes. Debus et al. improved the scale-up of well-defined polyplexes using a microf luidic lab-on-a-chip assembly [95]. DNA and polymer solutions were pumped through tubings from a syringe to the mixing chip fitted on a microfluidic baseboard. The most important parameter for the formation of optimized complexes was the N/P ratio, while other parameters were only of minor importance. Additional delay loops increased the contact time and area of the components and, therefore, intensified the mixing of the fluids. An influence of different flow rates on mixing behavior and particle size could not be clearly established. In contrast to the pumping method [86,94], the polydispersity index decreased with increasing concentrations, which was discussed to be related to the use of different techniques and polymers [95].
In conclusion, all reported systems led to more homogeneously distributed and better defined complexes with higher cell viability and improved exogenous gene expression compared with the bulk mixing method in the laboratory scale. FiguRe 4 shows a comparison of the different automated scale-up techniques used for the manufacturing of complexes for gene therapy.
Special attention has to be paid to the purity of the polymers and lipids, as well as contaminants of the complexes, in order to avoid toxic effects or interactions with the transfection
Recent developments & perspectives on gene therapy using synthetic vectors | Review
www.future-science.com 105future science group
process. Polymers and lipids can be produced even under good manufacturing practices as shown by commercial products from several companies. In previous studies it has been discussed whether polycations not integrated into the complexes should be removed because they were reported to exert a higher toxicity than the complexes themselves. For example, PEI/DNA complexes were purified by size-exclusion chromatography [92]. The presence of the free components was found to be essential for the complex performance since transfection rates were reduced after the purification process. The contribution of free PEI to intracellular processes such as endosomal release was thought to be responsible for these effects. Therefore, despite the higher toxicity of free polymer compared with the complexes, its presence was found to be necessary for effective transfection.
A limitation in the clinical administration of complexes is the requirement of being freshly prepared prior to administration, due to their tendency to aggregate or fuse in the liquid environment. Beside PEGylation of the complexes, which leads to a steric stabilization of the complexes during storage, freeze-drying of lipoplexes and polyplexes can be applied. To avoid the formation of irreversible aggregates or degradation of the nucleic acids due to the freeze–thaw process, lyo- and cryo-protectants such as monosaccharides (glucose), disaccharides (sucrose and trehalose), oligosaccharides (inulin and isomaltotriose) or polysaccharides and polymers (PVP, dextrans and hyroxyethyl starch) were added to the formulations [86,94,99]. For each formulation, the ratio of cryoprotector and complex has to be determined experimentally. For plasmid/
Laboratory-scale Microfluidic systems Production-scale
Figure 4. Comparison of different manufacturing techniques used for controlled and reproducible production of gene-delivery systems based on polymers or lipids.
Key Term
Good manufacturing practices: Regulations to ensure that pharmaceutical products meet specific requirements for identity, quality, purity and strength. These regulations are announced and monitored by public agencies such as the US FDA or European Medicines Agency.
Review | Schlenk, Grund & Fischer
Therapeutic Delivery (2013) 4(1)106 future science group
linear PEI complexes, stable formulations that maintained particle size and transfection potential could be achieved after lyophilization using hydroxypropylbetadex/sucrose or lactosucrose, whereas PVP/sucrose as stabilizer reduced the transfection. Complexes consisting of DNA and PEI or transferrin-conjugated PEI retained their biological activity after lyophilization using a controlled two-step drying process and 10% sucrose as lyoprotectant [100]. The potential of such gene vector formulations as dry powder aerosols after freeze-drying was shown in several publications [101].
Clinical trialsThe ability to produce plasmids and vector materials under quality-controlled large-scale conditions, and the possibilities to reproducibly produce nonviral vector formulations, opened the door for clinical applications.
Having a closer look to the overall clinical situation, since 1989, approximately 1700 approved clinical trials with more than 10,000 patients have been performed, most of them using viral vectors [201]. The first nonviral vector was tested in melanoma patients in 1992. In 2011, 92 studies were reported worldwide with 59.6% in Phase I, but only 3.6% in Phase III. The indications mostly investigated are cancer, cardiovascular, monogenetic and infective diseases, using antigen, cytokine, tumor suppressor and suicide genes (FiguRe 5). A clear tendency to orphan drug indications is obvious. More than 75% of the studies were performed with naked DNA and viral vectors. However, none of the gene therapeutics were approved in western countries until 2012. In November 2012 the European Medicines Agency gave marketing approval to Glybera®, a gene therapeutic for the treatment of patients suffering from severe or multiple pancreatitis attacks due to lipoprotein lipase deficiency. Glybera uses an adenoassociated virus vector as delivery system to add copies of the lipoprotein lipase gene into muscle cells. One application for approval was withdrawn by the end of 2009 when a re-examination after a negative Committee for Medicinal Products for Human Use/Committee for Advanced Therapies opinion did not seem to be promising (Cerepro, ArkTherapeutics for glioblastoma therapy). Two gene therapeutics (Gendicine®, Oncorine®) that have received marketing approval by the Chinese authorities represent adenoviral systems [102]. The first
product, approved by Russian authorities, was Neovasculogen, developed by the Human Stem Cell Institute in Moscow. Neovasculogen contains a VEGF plasmid for treatment of peripheral arterial disease and its complication critical limb ischemia. This situation correlates well with the aspects discussed above and is related to the considerably more complex in vivo situation.
Tables 3 & 4 give an overview of currently active clinical trials using nonviral vectors based on polymers or lipids, respectively. Their actual status quo will be discussed in the following section. In position five, behind several viral vectors and the naked gene transfer, ranks the lipofection with 5.9% of all clinical trials. Eight clinical trials using lipids or liposomes as plasmid delivery systems are currently listed as effective on clinicaltrials.gov [202]. A selection are shown in Table 4, mostly related to the therapy of cancer and cystic fibrosis in Phase I or II. A cholesterol-containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) vector is used to transfer the fus1 gene into cancer cells with the attempt to replace this cancer-stimulating gene in lung cancer cells. The study is already completed and defined the maximum tolerated and recommended Phase II dose. A Phase I/II trial was now announced to test these systems in stage IV lung cancer in combination with erlotinib. DOTMA,
Recent developments & perspectives on gene therapy using synthetic vectors | Review
www.future-science.com 107future science group
DOTAP and 1,2-dimyristyloxypropyl-dimethyl-hydroxyethylammoniumbromide (DMRIE), the most well-developed lipids for plasmid delivery, are currently in or at the end of Phase I trials. Safety and efficacy of GL67A lipid vectors containing a normal CFTR gene are investigated in repeated nebulized administrations versus nasal applications in cystic fibrosis. The lipid consists of a chemical optimization of the promising DC-Chol structure coformulated with the neutral DOPE and small amounts of a 5 kDa PEGylated lipid structure [103,104]. A single dose safety study testing the administration to nose and lung has already been completed. The highest safe dose of intraperitoneal tgDCC-E1A that can be given in combination with paclitaxel as a treatment for patients with recurrent, platinum-resistant ovarian cancer is currently evaluated in a Phase I/II trial [105].
Only a few clinical studies have been performed using polymers as shown by their ranking in the category ‘others’ [201] . PEI is the most widely used vector in clinical trials, all of them focusing on local application. Preclinical and clinical trials up to Phase II with PEI are ongoing, targeting cancer, HIV and acute myocardic infarction. A diphtheria
toxin A-expressing plasmid complexed with jetPEI (22 kDa linear PEI) was successfully used in the bladder [106]. Two patients with recurrent superf icial transitional cell carcinoma were locally treated. EGEN-001, a novel gene-delivery system built from PEI, PEG and cholesterol, containing an IL-12-expressing plasmid for inhibition of tumor neovascularization has demonstrated potent antitumor activity in preclinical models of ovarian cancer. It also stimulates natural killer cells, IFN-g secretion and T-helper I response [107]. Phase I and II studies targeting different cancer models including colorectal peritoneal carcinomatosis, fallopian tube cancer, ovarian (epithelial) cancer or primary peritoneal cancer are being performed. Additionally, combinations of EGEN-001 with different cytostatics are also being tested.
After a successful clinical Phase I with the DermaVir patch and a topical vaccine for HIV treatment in clinical Phase II, linear 22 kDa PEI modified with mannose and dextrose was used to transfect a plasmid encoding for several HIV proteins into target antigen-expressing cells [108,109]. Additional trials are performed to reach therapeutic immunization using a DermaVir patch or to vaccinate dendritic
Table 3. Current clinical trials using polymeric vectors for gene delivery.
Product name
Synthetic vector
Disease Clinical phase Company Ref.
No product name
In vivo-jetPEI® Acute myocardiac infarctionPancreas gene therapyMultiple myeloma therapyBladder cancer gene therapyHIV immune therapy
Preclinical (started Phase I)Phase IPhase II
Polyplus Transfection SA(Illkirch, France)
[204]
No product name
jetPEI Bladder neoplasmsBladder cancer
Completed Phase I/IIPhase IIb
BioCancell Therapeutics Israel Ltd(Jerusalem, Israel)
[205]
EGEN-001 PEG-PEI-Chol Ovarian cancer (also in combination with other cytostatics)Colorectal peritoneal carcinomatosisPersistent or recurrent ovarian epithelial cancer Fallopian tube cancerPrimary peritoneal cancer
Completed Phase IPhase I/IIPhase II
EGEN, Inc.(Huntsville, AL, USA)
[206]
DermaVir PEI-mannose and dextrose
HIV-1-infected patients currently on HAARTDendritic cell vaccine in HIV-infected children, adolescents and young adultsExperimental therapeutic vaccine, in adults receiving HAARTHIV-1 infected treatment-naive patientsTherapeutic immunization withDermaVir patch
Completed Phase IPhase I/IICompleted Phase I/IIPhase IIPhase II
Genetic Immunity, Inc. (McLean, VA, USA)
[207]
Chol: Cholesterol; HAART: Highly active antiretroviral therapy; PEI: Poly(ethylenimine).
Review | Schlenk, Grund & Fischer
Therapeutic Delivery (2013) 4(1)108 future science group
cells for HIV-infected children, adolescents and young adults. Currently, a pilot study for the treatment of locally advanced pancreatic adenocarcinoma with intratumoral injection of jetPEI/DNA complexes with antitumoral effects and chemosensitizing activity for gemcitabine is recruiting patients. A clinical trial has been announced using a combination of plasmid DNA, siRNA and jetPEI in relapsed or refractory patients for the treatment of multiple myeloma.
These first clinical results give high reason for optimism, although final outcomes are awaited.
Future perspectiveMany lessons have been learned since the beginning of gene therapy during the 1990s. We are now skilled enough to synthesize cationic carrier materials with defined sequences and structures, link several functional moieties to form multifunctional complexes based on a huge collection of basis modules or combinations of these, and characterize the final products using a remarkable set of advanced, highly sensitive, high-resolution analytical methods. Manufacturing of vector materials and complexes, especially under good manufacturing practice conditions, has been established for use in clinical trials. Nevertheless, the number of lipids and polymers that reached clinical trials is still low. Careful selection of target cells and relevant pharmacological disease models and imaging systems to characterize composition
of the complexes as well as their changes under physiological conditions may answer some of the currently open questions. A better understanding of the differences between the preclinical test models and the in vivo situation may help to increase the success rate. In the field of regulatory requirements, new criteria regarding manufacturing, characterization and clinical safety have to be defined, which may influence the design of clinical trial protocols. New healthcare policies, technological advances, approaches in personalized medicine and increasing knowledge of pathophysiological and cell-based processes have to be taken into consideration as factors that can shape the future development of gene therapy by nonviral vectors. Although the regulatory authorities have not yet given approval for marketing any nonviral human gene therapy product to be sold, the number of gene-related research and development projects is still growsing at a fast rate and has raised great hopes for the future.
Financial & competing interests disclosureThis work was greatly supported by the Bundesministerium für Bildung und Forschung (Nanomed, 03X0104D). The authors have no other relevant affiliations or financial involvement with any organization or entity with a finan-cial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
Table 4. Selection of clinical trials using lipids or liposomal vectors for plasmid transfer.
DOTAP:Chol-fus1 Lung cancer Completed Phase I MD Anderson Cancer Center/NCI
DOTAP:Chol-fus1 Lung cancer Phase I/II MD Anderson Cancer Center/Convergen LifeSciences, Inc.
DOTMA/Chol Head and neck cancer Completed Phase II H Lee Moffitt Cancer Center and Research Institute/NCI
DOTAP:Chol Pancreatic cancer Phase I MD Anderson Cancer Center/NCI
DMRIE/DOPE Cystic fibrosis Completed Phase I University of Alabama at Birmingham/NIDDK
tgDCC-E1A Recurrent breast and head and neck cancer
Phase I Wayne State University-Karmanos Cancer Institute
tgDCC-E1A Ovarian cancer Phase I/II MD Anderson Cancer Center/NIH
GL67A Cystic fibrosis Phase I/II Imperial College London/Royal Brompton & Harefield NHS Foundation Trust University of Oxford University of Edinburgh Cystic Fibrosis Trust University of Pennsylvania
GL67A Cystic fibrosis Phase II Imperial College London/Royal Brompton & Harefield NHS Foundation TrustUniversity of Edinburgh University of Oxford
Chol: Cholesterol; NCI: National Cancer Institute; NIDDK: National Institute of Diabetes and Digestive and Kidney Diseases; NIH: National Institutes of Health. Data taken from [201]
Recent developments & perspectives on gene therapy using synthetic vectors | Review
www.future-science.com 109future science group
ReferencesPapers of special note have been highlighted as:n of interestnn of considerable interest
1 Grund S, Bauer M, Fischer D. Polymers in drug delivery – state of the art and future trends. Adv. Eng. Mat. 13(3), B61–B87 (2011).
2 Hunter AC, Moghimi SM. Therapeutic synthetic polymers: a game of Russian roulette? Drug Discov. Today 7(19), 998–1001 (2002).
3 Sivalingam J, Kon OL. Recent advances and improvements in the biosafety of gene therapy. In: Gene Therapy – Developments and Future Perspectives. Kang C (Ed.). Intech, NY, USA, 145–188 (2011).
4 U.S. Department of Health and Human Services, FDA, Center for Biologics Evaluation and Research. Gene therapy clinical trials – observing subjects for delayed adverse events. (2006).
5 Avery OT, Macleod CM, Mccarty M. Studies on the chemical nature of the substance inducing transformation of pneumococcal types: induction of transformation by a desoxyribonucleic acid fraction isolated from pneumococcus type III. J. Exp. Med. 79(2), 137–158 (1944).
6 Yla-Herttuala S, Wirth T. Therapeutic delivery using gene-delivery methods. Ther. Deliv. 2(4), 423–426 (2011).
7 Kohn DB, Candotti F. Gene therapy fulfilling its promise. N. Engl. J. Med. 360(5), 518–521 (2009).
8 Boeckle S, Wagner E. Optimizing targeted gene delivery: chemical modification of viral vectors and synthesis of artificial virus vector systems. AAPS J. 8(4), E731–E742 (2006).
9 Zhu L, Mahato RI. Lipid and polymeric carrier-mediated nucleic acid delivery. Expert Opin. Drug Deliv. 7(10), 1209–1226 (2010).
n Overview of lipid and polymeric nucleic acid delivery systems.
10 Nguyen J, Szoka FC. Nucleic acid delivery. the missing pieces of the puzzle? Acc. Chem. Res. 45(7), 1153–1162 (2012).
nn Ideas about mechanisms in nonviral nucleic acid delivery and how vectors overcome barriers.
11 Pagano JS, Vaheri A. Enhancement of infectivity of poliovirus RNA with diethylaminoethyl-dextran (DEAE-D). Arch.Virol. 17(3), 456–464 (1965).
12 Felgner PL, Gadek TR, Holm M et al. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl Acad. Sci. USA 84(21), 7413–7417 (1987).
13 Lu ZR. Stability of proteins and nucleic acids. Biomaterials for Delivery and Targeting of Proteins and Nucleic Acids. Mahato RI (Ed.) CRC Press, Fl, USA, 352–374 (2005).
14 Lindahl T. Instability and decay of the primary structure of DNA. Nature 362(6422), 709–715 (1993).
15 Fischer D, Dautzenberg H, Kunath K, Kissel T. Poly(diallyldimethylammonium chlorides) and their N-methyl-N-vinylacetamide copolymer-based DNA-polyplexes: role of molecular weight and charge density in complex formation, stability, and in vitro activity. Int. J. Pharm. 280(1–2), 253–269 (2004).
16 Adami RC, Collard WT, Gupta SA, Kwok KY, Bonadio J, Rice KG. Stability of peptide-condensed plasmid DNA formulations. J. Pharm. Sci. 87(6), 678–683 (1998).
17 Anchordoquy TJ, Koe GS. Physical stability of nonviral plasmid-based therapeutics. J. Pharm. Sci. 89(3), 289–296 (2000).
18 Armitage B. Photocleavage of nucleic acids. Chem. Rev. 98(3), 1171–1200 (1998).
19 Pogocki D, Schoneich C. Chemical stability of nucleic acid-derived drugs. J. Pharm. Sci. 89(4), 443–456 (2000).
20 Oh YK, Kim JP, Yoon H, Kim JM, Yang JS, Kim CK. Prolonged organ retention and safety of plasmid DNA administered in polyethylenimine complexes. Gene Ther. 8(20), 1587–1592 (2001).
21 Takakura Y, Nishikawa M, Yamashita F, Hashida M. Development of gene drug delivery systems based on pharmacokinetic studies. Eur. J. Pharm. Sci. 13(1), 71–76 (2001).
Executive summary
DNA as an active ingredient: synthetic nonviral vector materials
n DNA used for gene therapy is highly susceptible to chemical and physical degradation, shows a low cell penetrating ability, and needs to be applied in high doses in humans.
n Nonviral vector technology is attracting increasing attention in the biomedical community owing to unique advantages and prospects for the treatment of severe diseases by gene therapy.
The need of multifunctional concepts
n Structure–activity relationships are important tools for the rational design and application of lipids and polymers as synthetic carriers for gene delivery.
n Limited stability, toxicity issues, and inefficient cell uptake and intracellular processing of complexes may be overcome by targeting strategies, stealth effect, biodegradable linker technologies and strategies to enhance the endosomal release.
Transfer of preclinical concepts to clinical trials
n Successful in vitro transfection by nonviral vectors often fails to reproduce in in vivo studies.
n Biological in vitro models that simulate and predict in vivo conditions more precisely need to be included in the development of nonviral vectors.
n New imaging technologies to characterize the inner structure of complexes as well as the changes under physiological conditions especially during blood transport and intracellular processing are necessary.
Manufacturing of polyplexes & lipoplexes: from laboratory technology to a broad application
n Assembly and manufacturing of synthetic complexes as well as stabilization by freeze drying on a commercial scale can be realized under sterile conditions by several techniques.
Clinical trials
n Although only a limited number of gene therapeutics worldwide received approval for the market, the number of preclinical studies steadily grows at a fast rate and has raised great hopes for the future of gene therapy.
Review | Schlenk, Grund & Fischer
Therapeutic Delivery (2013) 4(1)110 future science group
22 Ward CM, Read ML, Seymour LW. Systemic circulation of poly(L-lysine)/DNA vectors is influenced by polycation molecular weight and type of DNA: differential circulation in mice and rats and the implications for human gene therapy. Blood 97(8), 2221–2229 (2001).
23 Fischer D. In vivo fate of polymeric gene carriers. Biomaterials for Delivery and Targeting of Proteins and Nucleic Acids. Mahato RI (Ed.) CRC Press, Fl, USA, 295–314 (2005).
24 Vaughan EE, Degiulio JV, Dean DA. Intracellular trafficking of plasmids for gene therapy: mechanisms of cytoplasmic movement and nuclear import. Curr. Gene Ther. 6(6), 671–681 (2006).
nn Comprehensive and detailed overview of the fate of plasmids after cell uptake.
25 Lukacs GL, Haggie P, Seksek O, Lechardeur D, Freedman N, Verkman AS. Size-dependent DNA mobility in cytoplasm and nucleus. J. Biol. Chem. 275(3), 1625–1629 (2000).
26 Dauty E, Verkman AS. Actin cytoskeleton as the principal determinant of size-dependent DNA mobility in cytoplasm: a new barrier for non-viral gene delivery. J. Biol. Chem. 280(9), 7823–7828 (2005).
27 Pollard H, Toumaniantz G, Amos JL et al. Ca2+-sensitive cytosolic nucleases prevent efficient delivery to the nucleus of injected plasmids. J. Gene Med. 3(2), 153–164 (2001).
28 James MB, Giorgio TD. Nuclear-associated plasmid, but not cell-associated plasmid, is correlated with transgene expression in cultured mammalian cells. Mol. Ther. 1(4), 339–346 (2000).
29 Xu L, Anchordoquy T. Drug delivery trends in clinical trials and translational medicine: challenges and opportunities in the delivery of nucleic acid-based therapeutics. J. Pharm. Sci. 100(1), 38–52 (2011).
30 Golan R, Pietrasanta LI, Hsieh W, Hansma HG. DNA toroids: stages in condensation. Biochemistry 38(42), 14069–14076 (1999).
31 Hansma HG, Golan R, Hsieh W, Lollo CP, Mullen-Ley P, Kwoh D. DNA condensation for gene therapy as monitored by atomic force microscopy. Nucleic Acids Res. 26(10), 2481–2487 (1998).
32 Patel MM, Anchordoquy TJ. Contribution of hydrophobicity to thermodynamics of ligand-DNA binding and DNA collapse. Biophys. J. 88(3), 2089–2103 (2005).
33 Sunshine JC, Bishop CJ, Green JJ. Advances in polymeric and inorganic vectors for nonviral nucleic acid delivery. Ther. Deliv. 2(4), 493–521 (2011).
34 Neu M, Fischer D, Kissel T. Recent advances in rational gene transfer vector design based on poly(ethylene imine) and its derivatives. J. Gene Med. 7(8), 992–1009 (2005).
35 Tang R, Palumbo RN, Nagarajan L, Krogstad E, Wang C. Well-defined block copolymers for gene delivery to dendritic cells: probing the effect of polycation chain-length. J. Control. Release 142(2), 229–237 (2010).
36 Fischer D, Li Y, Ahlemeyer B, Krieglstein J, Kissel T. In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis. Biomaterials 24(7), 1121–1131 (2003).
37 Godbey WT, Wu KK, Mikos AG. Size matters: Molecular weight affects the efficiency of poly(ethylenimine) as a gene delivery vehicle J. Biomed. Mater. Res. 45(3), 268–275 (1999)
38 Guo X, Huang L. Recent advances in nonviral vectors for gene delivery. Acc. Chem. Res. 45(7), 971–979 (2012).
39 Balazs DA, Godbey W. Liposomes for use in gene delivery. J. Drug Del. 2011, 326497 (2011).
40 Behr J-P. Synthetic gene transfer vectors II: back to the future. Acc. Chem. Res. 45(7), 980–984 (2012).
41 Knop K, Hoogenboom R, Fischer D, Schubert US. Poly(ethylene glycol) in drug delivery: pros and cons as well as potential alternatives. Angew. Chem. Int. Ed. Engl. 49(36), 6288–6308 (2010).
42 Barz M, Luxenhofer R, Zentel R, Vicent MJ. Overcoming the PEG-addiction: well-defined alternatives to PEG, from structure-property relationships to better defined therapeutics. Polymer Chem. 2(9), 1900–1918 (2011).
nn Summary of recent efforts, aspects and developments about PEG and PEG alternatives.
43 Bauer M, Lautenschlaeger C, Kempe K, Tauhardt L, Schubert US, Fischer D. Poly(2-ethyl-2-oxazoline) as alternative for the stealth polymer poly(ethylene glycol): comparison of in vitro cytotoxicity and hemocompatibility. Macromol. Biosci. 12(7), 986–998 (2012).
44 Pasut G, Veronese FM. State of the art in PEGylation: the great versatility achieved after forty years of research. J. Control. Release 161(2), 461–472 (2012)
45 Wagner E. Polymers for siRNA Delivery: inspired by viruses to be targeted, dynamic, and precise. Acc. Chem. Res. 45(7), 1005–1013 (2012).
46 Walker GF, Fella C, Pelisek J et al. Toward synthetic viruses: endosomal pH-triggered deshielding of targeted polyplexes greatly enhances gene transfer in vitro and in vivo. Mol. Ther. 11(3), 418–425 (2005).
47 Rozema DB, Lewis DL, Wakefield DH et al. Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes. Proc. Natl Acad. Sci. USA 104(32), 12982–12987 (2007).
48 Noga M, Edinger D, Rodl W, Wagner E, Winter G, Besheer A. Controlled shielding and deshielding of gene delivery polyplexes using hydroxyethyl starch (HES) and alpha-amylase. J. Control. Release 159(1), 92–103 (2012).
49 Wu C, Belenda C, Leroux JC, Gauthier MA. Interplay of chemical microenvironment and redox environment on thiol-disulfide exchange kinetics. Chemistry 17(36), 10064–10070 (2011).
50 Bauhuber S, Hozsa C, Breunig M, Gopferich A. Delivery of nucleic acids via disulfide-based carrier systems. Adv. Mater. 21(32–33), 3286–3306 (2009).
51 Zhang G, Liu J, Yang Q, Zhuo R, Jiang X. Disulfide-containing brushed polyethylenimine derivative synthesized by click chemistry for nonviral gene delivery. Bioconjug. Chem. 23(6), 1290–1299 (2012).
52 Peng Q, Zhong Z, Zhuo R. Disulfide cross-linked polyethylenimines (PEI) prepared via thiolation of low molecular meight PEI as highly efficient gene vectors. Bioconjug. Chem. 19(2), 499–506 (2008).
53 Brülisauer L, Leroux JC. Disulfide reduction considerations for cationic gene delivery systems. Presented at: CRS Annual Meeting Abstracts. Québec City, Canada, 15–18 July 2012.
54 Ogris M, Wagner E. To be targeted: is the magic bullet concept a viable option for synthetic nucleic acid therapeutics? Hum. Gene Ther. 22(7), 799–807 (2011).
nn Covers recent concepts for targeted DNA and RNA delivery to organs.
55 Basile L, Pignatello R, Passirani C. Active targeting strategies for anticancer drug nanocarriers. Curr. Drug Deliv. 9(3), 255–268 (2012).
56 Breunig M, Bauer S, Goepferich A. Polymers and nanoparticles: intelligent tools for intracellular targeting? Eur. J. Pharm. Sci. 68(1), 112–128 (2008).
57 Butler LM, Mcgettrick HM, Nash GB. Static and dynamic assays of cell adhesion relevant to the vasculature. Methods Mol. Biol. 467, 211–228 (2009).
58 Davis ME. The first targeted delivery of siRNA in humans via a self-assembling, cyclodextrin polymer-based nanoparticle: from concept to clinic. Mol. Pharm. 6(3), 659–668 (2009).
Recent developments & perspectives on gene therapy using synthetic vectors | Review
www.future-science.com 111future science group
59 Nakase I, Akita H, Kogure K et al. Efficient intracellular delivery of nucleic acid pharmaceuticals using cell-penetrating peptides. Acc. Chem. Res. 45(7), 1132–1139 (2012).
60 Wang DA, Narang AS, Kotb M et al. Novel branched poly(ethylenimine)-cholesterol water-soluble lipopolymers for gene delivery. Biomacromolecules 3(6), 1197–1207 (2002).
61 Jones SP, Gabrielson NP, Pack DW, Smith DK. Synergistic effects in gene delivery – a structure–activity approach to the optimisation of hybrid dendritic-lipidic transfection agents. Chem. Commun. 39, 4700–4702 (2008).
62 Han S, Mahato RI, Kim SW. Water-soluble lipopolymer for gene delivery. Bioconjug. Chem. 12(3), 337–345 (2001).
63 Santos JL, Oliveira H, Pandita D et al. Functionalization of poly(amidoamine) dendrimers with hydrophobic chains for improved gene delivery in mesenchymal stem cells. J. Control. Release 144(1), 55–64 (2010).
64 Xu Y, Szoka FC Jr. Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. Biochemistry 35(18), 5616–5623 (1996).
65 Cullis PR, Hope MJ, Tilcock CP. Lipid polymorphism and the roles of lipids in membranes. Chem. Phys. Lipids 40(2–4), 127–144 (1986).
66 Zelphati O, Szoka FC, Jr. Mechanism of oligonucleotide release from cationic liposomes. Proc. Natl Acad. Sci. USA 93(21), 11493–11498 (1996).
67 Hafez IM, Maurer N, Cullis PR. On the mechanism whereby cationic lipids promote intracellular delivery of polynucleic acids. Gene Ther. 8(15), 1188–1196 (2001).
68 Behr JP. The proton sponge: a trick to enter cells the viruses did not exploit. CHIMIA 51(1–2), 34–36 (1997).
69 Yang S, May S. Release of cationic polymer-DNA complexes from the endosome: a theoretical investigation of the proton sponge hypothesis. J. Chem. Phys. 129(18), 185105 (2008).
70 Fischer D, Von Harpe A, Kunath K, Petersen H, Li Y, Kissel T. Copolymers of ethylene imine and N-(2-hydroxyethyl)-ethylene imine as tools to study effects of polymer structure on physicochemical and biological properties of DNA complexes. Bioconjug. Chem. 13(5), 1124–1133 (2002).
71 Martin ME, Rice KG. Peptide-guided gene delivery. AAPS J. 9(1), E18–E29 (2007).
72 Berg K, Folini M, Prasmickaite L et al. Photochemical internalization: a new tool for
drug delivery. Curr. Pharm. Biotechnol. 8(6), 362–372 (2007).
73 Zhang Y, Anchordoquy TJ. The role of lipid charge density in the serum stability of cationic lipid/DNA complexes. Biochim. Biophys. Acta 1663(1–2), 143–157 (2004).
74 Templeton NS. Cationic liposomes as in vivo delivery vehicles. Curr. Med. Chem. 10(14), 1279–1287 (2003).
75 Wang F, Weaver VM, Petersen OW et al. Reciprocal interactions between beta1-integrin and epidermal growth factor receptor in three-dimensional basement membrane breast cultures: a different perspective in epithelial biology. Proc. Natl. Acad. Sci. USA 95(25), 14821–14826 (1998).
76 Dinh AT, Pangarkar C, Theofanous T, Mitragotri S. Understanding intracellular transport processes pertinent to synthetic gene delivery via stochastic simulations and sensitivity analyses. Biophys. J. 92(3), 831–846 (2007).
77 Zhang H, Lee MY, Hogg MG, Dordick JS, Sharfstein ST. Gene delivery in three-dimensional cell cultures by superparamagnetic nanoparticles. ACS Nano 4(8), 4733–4743 (2010).
78 Abbott A. Cell culture: biology’s new dimension. Nature 424(6951), 870–872 (2003).
79 Hodges BL, Taylor KM, JosepH MF, Bourgeois SA, Scheule RK. Long-term transgene expression from plasmid DNA gene therapy vectors is negatively affected by CpG dinucleotides. Mol. Ther. 10(2), 269–278 (2004).
80 Chen ZY, He CY, Meuse L, Kay MA. Silencing of episomal transgene expression by plasmid bacterial DNA elements in vivo. Gene Ther. 11(10), 856–864 (2004).
81 Schleef M. DNA-Pharmaceuticals. Wiley-Vch, Bielefeld, Germany (2005).
82 Boussif O, Lezoualc’h F, Zanta MA et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. USA 92(16), 7297–7301 (1995).
n The beginning of poly(ethylenimine) use in DNA transfer.
83 Beck-Broichsitter M, Ruppert C, Schmehl T et al. Biophysical investigation of pulmonary surfactant surface properties upon contact with polymeric nanoparticles in vitro. Nanomedicine 7(3), 341–350 (2011).
84 Gebhart CL, Kabanov AV. Evaluation of polyplexes as gene transfer agents. J. Control. Release 73(2–3), 401–416 (2001).
85 Schaffert D, Wagner E. Gene therapy progress and prospects: synthetic polymer-based systems. Gene Ther. 15(16), 1131–1138 (2008).
86 Clement J, Kiefer K, Kimpfler A, Garidel P, Peschka-Süss R. Large-scale production of lipoplexes with long shelf-life. Eur. J. Pharm. Biopharm. 59(1), 35–43 (2005).
87 Suh J, Paik HJ, Hwang BK. Ionization of poly(ethylenimine) and poly(allylamine) at various pHs. Bioorg. Chem. 22(3), 318–327 (1994).
88 Kabanov VA, Kabanov AV. Interpolyelectrolyte and block ionomer complexes for gene delivery: physico-chemical aspects. Adv. Drug Deliv. Rev. 30(1–3), 49–60 (1998).
89 Bronich T, Kabanov AV, Marky LA. A thermodynamic characterization of the interaction of a cationic copolymer with DNA. J. Phys. Chem. B 105(25), 6042–6050 (2001).
90 Mahato R, Furgeson D, Maheshwari A, et al. Polymeric gene delivery for cancer treatment. In: Biomaterials and Drug Delivery Towards New Millenium. Park KD, Kwon IC, Yui N, Jeong SY, Park K (Eds.). Han Rim Wonn Publishing, 249–280 (2000).
91 Clamme JP, Azoulay J, Mely Y. Monitoring of the formation and dissociation of polyethylenimine/DNA complexes by two photon fluorescence correlation spectroscopy. Biophys. J. 84(3), 1960–1968 (2003).
92 Boeckle S, Von Gersdorff K, Van Der Piepen S, Culmsee C, Wagner E, Ogris M. Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer. J. Gene Med. 6(10), 1102–1111 (2004).
93 Zelphati O, Nguyen C, Ferrari M, Felgner J, Tsai Y, Felgner PL. Stable and monodisperse lipoplex formulations for gene delivery. Gene Ther. 5(9), 1272–1282 (1998).
94 Kasper JC, Schaffert D, Ogris M, Wagner E, Friess W. Development of a lyophilized plasmid/LPEI polyplex formulation with long-term stability – a step closer from promising technology to application. J. Control. Release 151(3), 246–255 (2011).
95 Debus H, Beck-Broichsitter M, Kissel T. Optimized preparation of pDNA/poly(ethylene imine) polyplexes using a microfluidic system. Lab. Chip 12(14), 2498–2506 (2012).
96 Kasper JC, Schaffert D, Ogris M, Wagner E, Friess W. The establishment of an up-scaled micro-mixer method allows the standardized and reproducible preparation of well-defined plasmid/LPEI polyplexes. Eur. J. Pharm. Biopharm. 77(1), 182–185 (2011).
Review | Schlenk, Grund & Fischer
Therapeutic Delivery (2013) 4(1)112 future science group
97 Knight JB, Vishwanath A, Brody JP, Austin RH. Hydrodynamic focusing on a silicon chip: mixing nanoliters in microseconds. Phys. Rev. Lett. 80(17), 3863–3866 (1998).
98 Koh CG, Kang X, Xie Y et al. Delivery of polyethylenimine/DNA complexes assembled in a microfluidics device. Mol. Pharm. 6(5), 1333–1342 (2009).
99 Talsma H, Cherng J, Lehrmann H et al. Stabilization of gene delivery systems by freeze-drying. Int. J. Pharm. 157(2), 233–238 (1997).
100 Huang YC, Connell M, Park Y, Mooney DJ, Rice KG. Fabrication and in vitro testing of polymeric delivery system for condensed DNA. J. Biomed. Mater. Res. A 67(4), 1384–1392 (2003).
101 Pfeifer C, Hasenpusch G, Uezguen S et al. Dry powder aerosols of polyethylenimine (PEI)-based gene vectors mediate efficient gene delivery to the lung. J. Control. Release 154(1), 69–76 (2011).
102 Ma G, Shimada H, Hiroshima K, Tada Y, Suzuki N, Tagawa, M. Gene medicine for cancer treatment: commercially available medicine and accumulated clinical data in China. Drug Des. Devel. Ther. 2(1), 115–122 (2008).
103 Eastman SJ, Siegel C, Tousignant J, Smith AE, Cheng SH, Scheule RK. Biophysical characterization of cationic lipid: DNA
106 Ohana P. Regulatory sequences of the H19 gene in DNA based therapy of bladder cancer. Gene Ther. Mol. Biol. (8), 181–192 (2004).
107 Kendrick J. A Phase I trial of intraperitoneal EGEN-001, a novel IL-12 gene therapeutic, administered alone or in combination with chemotherapy in patients with recurrent ovarian cancer. J. Clin. Oncol. 26, 5572 (2008).
