The role of the disulfide group in disulfide-based polymeric gene carriers

Background: An essential prerequisite for successful gene therapy is the development of safe and efficient gene delivery carriers. For this purpose, cationic polymers have been widely studied as non-viral carriers, but they generally suffer from low transfection efficiency and/or high cytotoxicity. To address these problems, disulfide-based cationic polymers have been designed as intelligent gene carriers that are capable of inducing highly efficient gene transfection with low cytotoxicity. Objective: The present review discusses the effects of the disulfide linker on the gene delivery properties of cationic polymers in relation to various gene delivery barriers. Methods: The literature regarding the gene delivery barriers encountered by polymeric gene delivery is reviewed and discussed in relation to the presence of the disulfide moiety in these gene carriers. Conclusions: The presence of disulfide linkages in cationic polymers can in many aspects favorably influence the gene delivery properties, such as increasing DNA binding ability, enabling de-shielding of ‘stealth’ (PEG) groups, fine-tuning of the buffer capacity for enhanced endosomal escape, improving carrier-unpacking and decreasing cytotoxicity. Therefore, disulfide-based cationic polymers are promising candidates for the next generation of non-viral carriers.     

Non-viral vectors in cancer gene therapy: principles and progress

This review focuses on the use of synthetic (non-viral) delivery systems for cancer gene therapy. Therapeutic strategies such as gene replacement/mutation correction, immune modulation and molecular therapy/'suicide' gene therapy type approaches potentially offer unique and novel ways of fighting cancer, some of which have already shown promise in early clinical trials. However, the specific and efficient delivery of the genetic material to remote tumors/metastases remains a challenge, which is being addressed using a variety of viral and non-viral systems. Each of these disparate systems has distinct advantages and disadvantages, which need to be taken into account when a specific therapeutic gene is being used. The review concentrates on particulate gene delivery systems, which are formed through non-covalent complexation of cationic carrier molecules (e.g. lipids or polymers) and the negatively charged plasmid DNA. Such systems tend to be comparatively less efficient than viral systems, but have the inherent advantage of flexibility and safety. The DNA-carrier complex acts as a protective package, and needs to be inert and stable while in circulation. Once the remote site has been reached the complex needs to efficiently transfect the targeted (tumor) cells. In order to improve overall transfection specificity and efficiency it is necessary to optimize intracellular trafficking of the DNA complex as well as the performance after systemic administration. Common principles and specific advantages or disadvantages of the individual synthetic gene delivery systems are discussed, and their interaction with tumor-specific and generic biological barriers are examined in order to identify potential strategies to overcome them.     

Current status of polymeric gene delivery systems

Gene therapy provides great opportunities for treating diseases from genetic disorders, infections and cancer. To achieve successful gene therapy, development of proper gene delivery systems could be one of the most important factors. Several non-viral gene transfer methods have been developed to overcome the safety problems of their viral counterpart. Polymer-based non-viral gene carriers have been used due to their merits in safety including the avoidance of potential immunogenecity and toxicity, the possibility of repeated administration, and the ease of the establishment of good manufacturing practice (GMP). A wide range of polymeric vectors have been utilized to deliver therapeutic genes in vivo. The modification of polymeric vectors has also shown successful improvements in achieving target-specific delivery and in promoting intracellular gene transfer efficiency. Various systemic and cellular barriers, including serum proteins in blood stream, cell membrane, endosomal compartment and nuclear membrane, were successfully circumvented by designing polymer carriers having a smart molecular structure. This review explores the recent development of polymeric gene carriers and presents the future directions for the application of the polymer-based gene delivery systems in gene therapy.     

Development of recombinant cationic polymers for gene therapy research

Cationic polymers created through recombinant DNA technology have the potential to fill a void in the area of gene delivery. The recombinant cationic polymers to be discussed here are amino acid based polymers synthesized in E. coli with the purpose to not only address the major barriers to efficient gene delivery but offer safety, biodegradability, targetability and cost-effectiveness. This review helps the readers to get a better understanding about the evolution of recombinant cationic polymers; and the potential advantages that they could offer over viral and synthetic non-viral vectors for gene delivery. It also discusses some of the major challenges that must be addressed in future studies to turn recombinant polymers into clinically effective gene delivery systems. Recent advances with the biopolymer design suggest that this emerging new class of gene delivery systems has the potential to address some of the major barriers to efficient, safe and cost-effective gene therapy.     

阳离子脂质体用做基因传递载体的研究进展

 基因治疗的难题之一在于研制安全有效的基因传递载体。常用的基因传递载体分为病毒载体和非病毒载体两类,其中,非病毒载体中的阳离子脂质体因具有低毒性与免疫原性、生物相容性好、易于制备等优点而受到广泛关注,具有良好的应用前景。近年来对阳离子脂质体载体的研究主要集中在对其传递基因机制的考察、各种影响其转基因效率的因素的探求、应用各种方法研制安全性和转染活性更佳的新型阳离子脂质体等方面。文中从转基因特点、传递机制、常用的制备材料、影响转基因效率的因素、近年来出现的新型阳离子脂质体等方面综述了此类基因传递载体的研究进展。     

Cell-penetrating peptides for the delivery of nucleic acids

INTRODUCTION: Different gene therapy approaches have gained extensive interest lately and, after many initial hurdles, several promising approaches have reached to the clinics. Successful implementation of gene therapy is heavily relying on finding efficient measures to deliver genetic material to cells. Recently, non-viral delivery of nucleic acids and their analogs has gained significant interest. Among non-viral vectors, cell-penetrating peptides (CPPs) have been extensively used for the delivery of nucleic acids both in vitro and in vivo. AREAS COVERED: In this review we will discuss recent advances of CPP-mediated delivery of nucleic acid-based cargo, concentrating on the delivery of plasmid DNA, splice-correcting ONs, and small-interfering RNAs. EXPERT OPINION: CPPs have proved their potential as carriers for nucleic acids. However, similarly to other non-viral vectors, CPPs require further development, as efficient systemic delivery is still seldom achieved. To achieve this, CPPs should be modified with entities that would allow better endosomal escape, targeting of specific tissues and cells, and shielding agents that increase the half-life of the vehicles. Finally, to understand the clinical potential of CPPs, they require more thorough investigations in clinically relevant disease models and in pre-clinical and clinical studies.     

Lipid-based emulsion system as non-viral gene carriers

The genetic materials for systemic administration meet a number of huddles before they reach the nucleus of the target cells, such as enzymatic degradation in the bloodstream, extravascularization around the target tissue, endocytosis by the target cells, and endosomal escape of the genes. Therefore, there have been tremendous needs of effective gene carriers that can deliver the genetic materials to the target site. Of numerous approaches, recent studies have demonstrated that the lipid-based emulsion systems have the high potential as non-viral gene carriers: 1 lipid emulsions are biocompatible because their major constituents are composed of the non-toxic oils and amphiphilic lipids; 2 the cationic lipid emulsions can form nano-sized complexes with negatively charged DNAs, through which the genetic materials can be protected from the enzymatic degradation in the body fluids; 3 The emulsion/DNA complexes are shown to be stable in the bloodstream since their surfaces are rarely recognized by the immune-related cells and serum proteins; and 4 the surfaces of the emulsion complexes are readily modified by varying the lipid composition. In this review, highlighted are the recent advances in the emulsion-based gene carriers.     

Cationic lipid vectors for plasmid DNA delivery

Successful gene therapy depends on efficient gene transfer vectors. Viral vectors and non-viral vectors have been investigated extensively. Cationic lipids are non-viral vectors, which resemble traditional pharmaceuticals, display little immunogenicity, and have no potential for viral infection. However, toxicity and low transfection efficiency are two barriers limiting the clinical applications of cationic lipids. Over the last decade, hundreds of cationic lipids have been synthesized to address these problems. In this brief review, we summarized recent research results concerning the structures of DNA/liposomes complexes, some important strategies used to design different classes of cationic lipids, and use of disulfide cationic lipids in plasmid DNA delivery.     

Polycation gene delivery systems: escape from endosomes to cytosol

Clinical success of gene therapy based on oligonucleotides (ODNs), ribozymes, RNA and DNA will be greatly dependent on the availability of effective delivery systems. Polycations have gained increasing attention as a non-viral gene delivery vector in the past decades. Significant progress has been made in understanding complex formation between polycations and nucleic acids, entry of the complex into the cells and subsequent entry into the nucleus. Sophisticated molecular architectures of cationic polymers have made the vectors more stable and less susceptible to binding by enzymes or proteins. Incorporation of specific ligands to polycations has resulted in more cell-specific uptake by receptor-mediated mechanisms. However, there are still other barriers limiting the transfection efficiency of polycation gene delivery systems. There is a consensus that polycation-DNA complexes (polyplexes) enter cells via the endocytotic pathway. It is not clearly understood, however, how the polyplexes escape (if they do) from endosomes, how DNA is released from the polyplexes or how the released DNA is expressed. The primary focus of this article is to review various polycation gene delivery systems, which are designed to translocate DNA from endosomes into cytosol. Many polycation gene delivery systems have tried to mimic the mechanisms that viruses use for the endosomal escape. Polycation gene delivery systems are usually coupled with synthetic amphipathic peptides mimicking viral fusogenic peptides, histidine-based gene delivery systems for pH-responsive endosomal escape, polycations with intrinsic endosomolytic activity by the proton sponge mechanism and polyanions to mimic the anionic amphiphilic peptides.     

Section Reviews: Biologicals & Immunologicals: Recent advances in non-viral gene therapy

Gene therapy is beginning to emerge as a safe and potentially effective means to treat human disease, and offers new opportunities to tackle conditions for which no satisfactory treatments have yet been developed. Current approaches rely upon either viral or non-viral vehicles to deliver the therapeutic gene, however, despite encouraging clinical data, it is realistic to expect that further advances are required if any are to be developed, manufactured and used in clinical practice. Non-viral gene therapy is attractive because, in general, it offers the potential to developed a synthetic, generic delivery vehicle, as well as safety and simplicity. However, a host of biological barriers exist, such as cell targeting and entry, intracellular trafficking, and maintenance and controlled expression of the therapeutic gene, which need to be overcome. Recently, non-viral gene therapy research has been an area of considerable innovation, with significant progress being made to address these issues. This review focuses on recent and potential advances to non-viral gene delivery systems and the strategies to ensure therapeutic genes are maintained and expressed in vivo Although further basic research is required, non-viral approaches promise to be an integral, if not major, component of future human gene therapy practice.     

Current developments in gene transfection agents

DNA can be delivered into the cell nucleus either using physical means or specific carriers that carry the genes into the cells for gene expression). Various carriers for delivering genes have been investigated which can be divided into two main groups: viral carriers where the DNA to be delivered is inserted into a virus, and cationic molecular carriers that form electrostatic interactions with DNA). Successful gene therapy depends on the efficient delivery of genetic materials into the cells nucleus and its effective expression within these cells). Although at present the in vivo expression levels of synthetic molecular gene vectors are lower than for viral vectors and gene expression is transient, these vehicles are likely to present several advantages including safety, low-immunogenicity, capacity to deliver large genes and large-scale production at low-cost). The two leading classes of synthetic gene delivery systems that have been mostly investigated are cationic lipids and cationic polymers). This review discusses recent developments in viral vectors, physical means and molecular gene carriers). The last part focuses on our recent studies in developing a new series of biodegradable polycations for in vitro and in vivo gene transfection).     

The features and shortcomings for gene delivery of current non-viral carriers

Since the viral vector for gene therapy has serious problems, including oncogenesity and other adverse effects, non-viral carriers have attracted a great deal of attention. Non-viral carriers are expected to achieve gene therapy without serious side effects. However, the most critical issue of gene delivery by non-viral carriers is the low-expression efficiencies of the desired gene. In order to apply non-viral carriers for gene therapy in practical clinical usage, further understanding of the cellular barriers against gene delivery is a prerequisite. Moreover, additional intelligent concepts for gene delivery are also needed. We will summarize the features and shortcomings of currently developed non-viral delivery systems. Especially, we will address the current progress of cationic lipids (lipoplex) and cationic polymers (polyplex) in terms of transfection efficiency. Furthermore, our group has developed a system that responds to the particular intracellular signals of target disease cells. We have named this gene delivery system a drug delivery system based on responses cellular signal (D-RECS). We will introduce this new concept of intelligent non-viral delivery system that our group recently developed.     

Barriers to Non-Viral Vector-Mediated Gene Delivery in the Nervous System

Efficient methods for cell line transfection are well described, but, for primary neurons, a high-yield method different from those relying on viral vectors is lacking. Viral transfection has several drawbacks, such as the complexity of vector preparation, safety concerns, and the generation of immune and inflammatory responses when used in vivo. However, one of the main problems for the use of non-viral gene vectors for neuronal transfection is their low efficiency when compared with viral vectors. Transgene expression, or siRNA delivery mediated by non-viral vectors, is the result of multiple processes related to cellular membrane crossing, intracellular traffic, and/or nuclear delivery of the genetic material cargo. This review will deal with the barriers that different nanoparticles (cationic lipids, polyethyleneimine, dendrimers and carbon nanotubes) must overcome to efficiently deliver their cargo to central nervous system cells, including internalization into the neurons, interaction with intracellular organelles such as lysosomes, and transport across the nuclear membrane of the neuron in the case of DNA transfection. Furthermore, when used in vivo, the nanoparticles should efficiently cross the blood-brain barrier to reach the target cells in the brain.     

Cell-specific delivery of genes with glycosylated carriers.

Cationic liposomes and polymers have been accepted as effective non-viral vectors for gene delivery with low immunogenicity unlike viral vectors. However, the lack of organ or cell specificity sometimes hampers their application and the development of a cell-specific targeting technology for them attracts great interest in gene therapy. In this review, the potential of cell-specific delivery of genes with glycosylated liposomes or polymers is discussed. Galactosylated liposomes and poly(amino acids) are selectively taken up by the asialoglycoprotein receptor-positive liver parenchymal cells in vitro and in vivo after intravenous injection. DNA-galactosylated cationic liposome complexes show higher DNA uptake and gene expression in the liver parenchymal cells in vitro than DNA complexes with bare cationic liposomes. In the in vitro gene transfer experiment, galactosylated liposome complexes are more efficient than DNA-galactosylated poly(amino acids) complexes but they have some difficulties in their biodistribution control. On the other hand, introduction of mannose residues to carriers resulted in specific delivery of genes to non-parenchymal liver cells. These results suggest advantages of these glycosylated carriers in cell-specific targeted delivery of genes.     

Cell-penetrating peptides for the delivery of nucleic acids

Introduction: Different gene therapy approaches have gained extensive interest lately and, after many initial hurdles, several promising approaches have reached to the clinics. Successful implementation of gene therapy is heavily relying on finding efficient measures to deliver genetic material to cells. Recently, non-viral delivery of nucleic acids and their analogs has gained significant interest. Among non-viral vectors, cell-penetrating peptides (CPPs) have been extensively used for the delivery of nucleic acids both in vitro and in vivo.

Areas covered: In this review we will discuss recent advances of CPP-mediated delivery of nucleic acid-based cargo, concentrating on the delivery of plasmid DNA, splice-correcting ONs, and small-interfering RNAs.

Expert opinion: CPPs have proved their potential as carriers for nucleic acids. However, similarly to other non-viral vectors, CPPs require further development, as efficient systemic delivery is still seldom achieved. To achieve this, CPPs should be modified with entities that would allow better endosomal escape, targeting of specific tissues and cells, and shielding agents that increase the half-life of the vehicles. Finally, to understand the clinical potential of CPPs, they require more thorough investigations in clinically relevant disease models and in pre-clinical and clinical studies.     

Developing non-viral DNA delivery systems for cancer and infectious disease

Efforts to deliver therapeutic genes are frequently rebuffed by the body's adaptive immune response against viral delivery vectors. Attempts to circumvent this problem using non-viral delivery systems have encountered problems with transient expression and inflammatory responses induced by reaction of the innate immune system reacting against bacterial DNA. However, within the past decade, these barriers to non-viral DNA delivery have been recognized as potential allies in the development of novel vaccines for cancer and infectious disease. This review summarizes preclinical and current clinical studies testing the formulation, delivery route and adjuvant options in the development of novel DNA-based vaccines.     

Polymeric vectors for ocular gene delivery

Gene therapy holds promise for the treatment of many inherited and acquired diseases of the eye. Successful ocular gene therapy interventions depend on efficient gene transfer to targeted cells with minimal toxicity. A major challenge is to overcome both intracellular and extracellular barriers associated with ocular gene delivery. Numerous viral and nonviral vectors were explored to improve transfection efficiency. Among nonviral delivery systems, polymeric vectors have gained significant attention in recent years owing to their nontoxic and non-immunogenic nature. Polyplexes or nanoparticles can be prepared by interaction of cationic polymers with DNA, which facilitate cellular uptake, endolysosomal escape and nuclear entry through active mechanisms. Chemical modification of these polymers allows for the generation of flexible delivery vectors with desirable properties. In this article several synthetic and natural polymeric systems utilized for ocular gene delivery are discussed.     

Evaluation of Multicomponent Non-viral Vectors for Liver Directed Gene Delivery

Multicomponent, non-viral gene delivery vehicles are designed to have as a minimum, a DNA binding component, and a cell recognition component for specific delivery to target cells. The DNA binding component cannot only bind, but also protect DNA from serum degradation, and tends to condense DNA to sizes that can be taken up by receptor-mediated processes of target cells. Generally, cationic peptides, single chained, e.g. poly- l -lysine or branched polymers or synthetic peptides with DNA binding properties are used for DNA binding components. Ligands for binding to receptors on cell surfaces can be covalently linked to the DNA binding component. Multicomponent, non-viral vectors have been successfully used to deliver genes into cells in vitro and in vivo. Improvements have been made to the non-viral carriers resulting in increased solubility of DNA/carrier complexes and longer survival in serum. Improvements have also been made by incorporating fusogenic/lysosomolytic components that enable DNA/carrier complexes to escape intracellular degradation and enhance the levels and duration of expression of genes in vitro and in vivo.     

Evaluation of multicomponent non-viral vectors for liver directed gene delivery

Multicomponent, non-viral gene delivery vehicles are designed to have as a minimum, a DNA binding component, and a cell recognition component for specific delivery to target cells. The DNA binding component cannot only bind, but also protect DNA from serum degradation, and tends to condense DNA to sizes that can be taken up by receptor-mediated processes of target cells. Generally, cationic peptides, single chained, e.g. poly-L-lysine or branched polymers or synthetic peptides with DNA binding properties are used for DNA binding components. Ligands for binding to receptors on cell surfaces can be covalently linked to the DNA binding component. Multicomponent, non-viral vectors have been successfully used to deliver genes into cells in vitro and in vivo. Improvements have been made to the non-viral carriers resulting in increased solubility of DNA/carrier complexes and longer survival in serum. Improvements have also been made by incorporating fusogenic/lysosomolytic components that enable DNA/carrier complexes to escape intracellular degradation and enhance the levels and duration of expression of genes in vitro and in vivo.