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.     

Applications and developments of gene therapy drug delivery systems for genetic diseases

Genetic diseases seriously threaten human health and have always been one of the refractory conditions facing humanity. Currently, gene therapy drugs such as siRNA, shRNA, antisense oligonucleotide, CRISPR/Cas9 system, plasmid DNA and miRNA have shown great potential in biomedical applications. To avoid the degradation of gene therapy drugs in the body and effectively deliver them to target tissues, cells and organelles, the development of excellent drug delivery vehicles is of utmost importance. Viral vectors are the most widely used delivery vehicles for gene therapy in vivo and in vitro due to their high transfection efficiency and stable transgene expression. With the development of nanotechnology, novel nanocarriers are gradually replacing viral vectors, emerging superior performance. This review mainly illuminates the current widely used gene therapy drugs, summarizes the viral vectors and non-viral vectors that deliver gene therapy drugs, and sums up the application of gene therapy to treat genetic diseases. Additionally, the challenges and opportunities of the field are discussed from the perspective of developing an effective nano-delivery system.     

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.     

Degradable poly(amido amine)s as gene delivery carriers

Introduction: In recent years, there has been a great deal of interest in the development of vectors which are being developed based on the capacity of polymers to mediate appropriate interactions with the cellular environment, or to interface with specific cellular processes. Several such vectors have been synthesized, resulting in biomacromolecules with low cytotoxicity and higher gene delivery ability.

Areas covered: This review briefly describes the recent success of poly(amido amine)s (PAAs) as non-viral vectors, and highlights their promising future in the development of nucleic acid-based therapy. It also provides an overview on the synthesis, characterization and application of PAAs as gene carriers, which will be useful for various biological motifs. This review helps the readers to better understand the emergence of non-viral vectors for gene therapy, especially PAAs, their properties, their advantages and disadvantages and the gene therapy based on them.

Expert opinion: The future of gene-based therapy needs to identify approaches to develop new carriers, depending on the properties of the biological membranes they face, and their physicochemical properties, in order to successfully deliver the genes to the target sites. With the emergence of a variety of non-viral vectors, such as biodegradable polymers, it may not take long before non-viral vectors are observed that are not just safe and tissue-specific, but even more efficient than viral vectors.     

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.     

Polyethylenimine-based non-viral gene delivery systems.

Gene therapy has become a promising strategy for the treatment of many inheritable or acquired diseases that are currently considered incurable. Non-viral vectors have attracted great interest, as they are simple to prepare, rather stable, easy to modify and relatively safe, compared to viral vectors. Unfortunately, they also suffer from a lower transfection efficiency, requiring additional effort for their optimization. The cationic polymer polyethylenimine (PEI) has been widely used for non-viral transfection in vitro and in vivo and has an advantage over other polycations in that it combines strong DNA compaction capacity with an intrinsic endosomolytic activity. Here, we give some insight into strategies developed for PEI-based non-viral vectors to overcome intracellular obstacles, including the improvement of methods for polyplex preparation and the incorporation of endosomolytic agents or nuclear localization signals. In recent years, PEI-based non-viral vectors have been locally or systemically delivered, mostly to target gene delivery to tumor tissue, the lung or liver. This requires strategies to efficiently shield transfection polyplexes against non-specific interaction with blood components, extracellular matrix and untargeted cells and the attachment of targeting moieties, which allow for the directed gene delivery to the desired cell or tissue. In this context, materials, facilitating the design of novel PEI-based non-viral vectors are described.     

Toxicogenomics of cationic lipid-based vectors for gene therapy: impact of microarray technology

Implementation of the high-throughput microarray gene expression profiling technology towards "toxicogenomics" has advanced identification process for safer drugs in the century of 'omics' technology. Applying such technology, in fact, to identify mechanisms for cellular toxicity can provide a means to clarify safety liabilities early in the drug discovery and developments process. The underlying principle in gene therapy is primarily targeting a specific gene (e.g., for silencing). Hence, massive efforts have been devoted to validate the gene-based therapeutics, regardless of toxicogenomics potential of delivery systems. Of the gene delivery systems, viral and non-viral vectors, as two main paradigms, have so far been widely used for delivering of the genome-based therapeutics such as oligonucleotide, small interfering RNA and DNA. However, the use of viral vectors was narrowed due to the safety concerns. Non-viral vectors were utilized as safer alternatives for gene delivery in vitro and ex-vivo; though their success for in vivo gene therapy has been limited due to low efficiency and safety issues. Fundamental principle for gene therapy is to deliver gene-based therapeutics into target cells for specific gene targeting ideally with minimal cellular toxicity. Until now, few works have been conducted about geno-compatibility of delivery systems itself, including cationic lipid-based nanosystems. Inadvertent toxicogenomic impact of gene delivery systems (e.g., cationic lipids) may intrinsically affect the outcome of gene therapy, where often only a single desired genetic change is sought. Further, there exists a possibility that gene changes induced by the lipid delivery system itself could exacerbate, attenuate or even mask the desired effects of the gene-based therapeutics. This review will focus on toxicogenomics impact of the cationic lipid-based formulations for gene therapy.     

Degradable poly(amido amine)s as gene delivery carriers

INTRODUCTION: In recent years, there has been a great deal of interest in the development of vectors which are being developed based on the capacity of polymers to mediate appropriate interactions with the cellular environment, or to interface with specific cellular processes. Several such vectors have been synthesized, resulting in biomacromolecules with low cytotoxicity and higher gene delivery ability. AREAS COVERED: This review briefly describes the recent success of poly(amido amine)s (PAAs) as non-viral vectors, and highlights their promising future in the development of nucleic acid-based therapy. It also provides an overview on the synthesis, characterization and application of PAAs as gene carriers, which will be useful for various biological motifs. This review helps the readers to better understand the emergence of non-viral vectors for gene therapy, especially PAAs, their properties, their advantages and disadvantages and the gene therapy based on them. EXPERT OPINION: The future of gene-based therapy needs to identify approaches to develop new carriers, depending on the properties of the biological membranes they face, and their physicochemical properties, in order to successfully deliver the genes to the target sites. With the emergence of a variety of non-viral vectors, such as biodegradable polymers, it may not take long before non-viral vectors are observed that are not just safe and tissue-specific, but even more efficient than viral vectors.     

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.     

Awakening gene therapy with Sleeping Beauty transposons

Sleeping Beauty transposons have the potential for use as chromosome-integrating vectors for non-viral gene therapy. Recent preclinical data from mouse models for human genetic disorders have shown efficacy for the Sleeping Beauty transposon system in the treatment of hemophilia, tyrosinemia type I, junctional epidermolysis bullosa and type 1 diabetes. Methods have also been developed to deliver Sleeping Beauty transposons to the lung, liver and tumors for treatments for cystic fibrosis, cardiovascular and metabolic diseases, and cancer. Recent studies characterizing site selection for integration and insertional mutagenesis indicate that the Sleeping Beauty transposon system may be a safer alternative than viral approaches for gene therapy.     

Ultrasound enhances the transfection of plasmid DNA by non-viral vectors

Increasing attention has been paid to technology used for the delivery of genetic materials into cells for gene therapy and the generation of genetically engineered cells. So far, viral vectors have been mainly used because of their inherently high transfection efficiency of gene. However, there are some problems to be resolved for the clinical applications, such as the pathogenicity and immunogenicity of viral vectors themselves. Therefore, many research trials with non-viral vectors have been performed to enhance their efficiency to a level comparable to the viral vector. Two directions of these trials exist: material improvement of non-viral vectors and their combination with various external physical stimuli. This paper reviews the latter research trials, with special attention paid to the enhancement of gene expression by ultrasound (US). The expression level of plasmid DNA by various cationized polymers and liposomes is promoted by US irradiation in vitro as well as in vivo. This US-enhanced expression of plasmid DNA will be discussed to emphasize the technical feasibility of US in gene therapy and biotechnology.     

Virosome: a novel vector to enable multi-modal strategies for cancer therapy

Despite advancements in treatments, cancer remains a life-threatening disease that is resistant to therapy. Single-modal cancer therapy is often insufficient to provide complete remission. A revolution in cancer therapy may someday be provided by vector-based gene and drug delivery systems. However, it remains difficult to achieve this aim because viral and non-viral vectors have their own advantages and limitations. To overcome these limitations, virosomes have been constructed by combining viral components with non-viral vectors or by using pseudovirions without viral genome replication. Viruses, such as influenza virus, HVJ (hemagglutinating virus of Japan; Sendai virus) and hepatitis B virus, have been used in the construction of virosomes. The HVJ-derived vector is particularly promising due to its highly efficient delivery of DNA, siRNA, proteins and anti-cancer drugs. Furthermore, the HVJ envelope (HVJ-E) vector has intrinsic anti-tumor activities including the activation of multiple anti-tumor immunities and the induction of cancer-selective apoptosis. HVJ-E is currently being clinically used for the treatment of melanoma. A promising multi-modal cancer therapy will be achieved when virosomes with intrinsic anti-tumor activities are utilized as vectors for the delivery of anti-tumor drugs and genes.     

Strategies on the nuclear-targeted delivery of genes

Abstract

To improve the nuclear-targeted delivery of non-viral vectors, extensive effort has been carried out on the development of smart vectors which could overcome multiple barriers. The nuclear envelope presents a major barrier to transgene delivery. Viruses are capable of crossing the nuclear envelope to efficiently deliver their genome into the nucleus through the specialized protein components. However, non-viral vectors are preferred over viral ones because of the safety concerns associated with the latter. Non-viral delivery systems have been designed to include various types of components to enable nuclear translocation at the periphery of the nucleus. This review summarizes the progress of research regarding nuclear transport mechanisms. “Smart” non-viral vectors that have been modified by peptides and other small molecules are able to facilitate the nuclear translocation and enhance the efficacy of gene expression. The resulting technology may also enhance delivery of other macromolecules to the nucleus.     

Prospects of treating neurological diseases by gene therapy

Neurological disorders represent a major challenge for therapeutic interventions due to the sensitivity and complexity of the CNS and the requirement of the therapeutic agent(s) to have a long-term effect. Gene therapy applications have opened up new possibilities to specifically deliver not only drugs but also small 'drug factories' by non-viral or viral vectors to the target site of action. A number of studies have demonstrated the feasibility of the gene therapy approach in animal models, and the simultaneous engineering of safer and more reliable vectors has moved the technology one step closer to clinical administration. In particular, viral vectors can provide long-term expression extended over one year. Most promisingly, the discovery of RNA interference has provided a novel method of specific gene silencing, which presents the opportunity to develop new therapies for the treatment of neurological disorders.     

DNA and carbon nanotubes as medicine

The identification of disease-related genes and their complete nucleotide sequence through the human genome project provides us with a remarkable opportunity to combat a large number of diseases with designed genes as medicine. However, gene therapy relies on the efficient and nontoxic transport of therapeutic genetic medicine through the cell membranes, and this process is very inefficient. Carbon nanotubes, due to their large surface areas, unique surface properties, and needle-like shape, can deliver a large amount of therapeutic agents, including DNA and siRNAs, to the target disease sites. In addition, due to their unparalleled optical and electrical properties, carbon nanotubes can deliver DNA/siRNA not only into cells, which include difficult transfecting primary-immune cells and bacteria, they can also lead to controlled release of DNA/siRNA for targeted gene therapy. Furthermore, due to their wire shaped structure with a diameter matching with that of DNA/siRNA and their remarkable flexibility, carbon nanotubes can impact on the conformational structure and the transient conformational change of DNA/RNA, which can further enhance the therapeutic effects of DNA/siRNA. Synergistic combination of the multiple capabilities of carbon nanotubes to deliver DNA/siRNAs will lead to the development of powerful multifunctional nanomedicine to treat cancer or other difficult diseases. In this review, we summarized the current studies in using CNT as unique vehicles in the field of gene therapy.     

Folate-linked lipid-based nanoparticle for targeted gene delivery

Cancer gene therapy has been intensively developed using non-viral vectors, among which cationic liposomes and nanoparticles are the most thoroughly investigated. For targeted delivery to tumors, vitamin folic acid has been utilized for folate receptor (FR)-mediated drug delivery, since FR is frequently overexpressed on many types of human tumors. Liposomes conjugated to folate ligand have been used as carriers of chemotherapeutic agents and DNA to receptor-bearing tumor cells in vitro. As an alternative treatment for prostate cancer, suicide gene therapy by local injection using an adenoviral vector has been reported, but not that using non-viral vectors. The folate-linked, lipid-based nanoparticles which we developed could deliver genes extensively to FR-negative LNCaP and PC-3 cells, as well as FR-positive KB and Hela cells. In this review, we outline folate-linked liposomes and nanoparticles, and show the effectiveness of folate-linked, lipid-based nanoparticles as a vector for DNA transfection and for suicide gene therapy, to treat human nasopharyngeal and prostate tumors.     

Controlled delivery of antisense oligonucleotides: a brief review of current strategies

Antisense therapy has been investigated extensively over the past two decades, either experimentally for gene functional research or clinically as therapeutic agents owing to the conceptual simplicity, ease of design and low cost. The concept of this therapeutic approach is promising because short antisense oligonucleotides (ASOs) can be delivered into target cells for specific hybridisation with target mRNA, resulting in the inhibition of the expression of pathogenic genes. However, the efficient delivery of the ASO molecules into target cells remains challenging; this bottleneck together with several other technical hurdles need to be overcome before this approach becomes effective and widely adopted. A variety of vectors such as lipids, polymers, peptides and nanoparticles have been explored. This review outlines the recent advances of the non-viral ASO delivery strategies. Several recent scientific studies, including authors' contributions, have been selected to highlight the technical aspects of ASO delivery.     

Applications of Hemagglutinating Virus of Japan in therapeutic delivery systems

BACKGROUND: Efficient and minimally invasive vector systems appear to be the most appropriate for both gene therapy and drug delivery. Numerous viral and non-viral vectors have been developed. Each vector has its own advantages and limitations. OBJECTIVE: New vectors have been required for overcoming the limitations of both viral vectors and non-viral vectors. The idea is to compensate the limitations of one vector system with the advantages of another. This can enable efficient drug delivery and gene expression, while reducing the cytotoxicity of the various vector components. METHODS: The Hemagglutinating Virus of Japan (HVJ; Sendai virus) envelope vector was developed using fusion-competent inactivated HVJ particle. Briefly, the viral genome was destroyed by UV-irradiation, and the inactivated viral particles were mixed with plasmid DNA, proteins or siRNA in the presence of mild detergent. After centrifugation, those molecules were incorporated into the viral envelope. CONCLUSION: The HVJ-E vector can efficiently deliver therapeutic molecules such as genes, siRNA, decoy oligonucleotides, proteins, and anti-cancer drugs to various tissues in vivo. It is also available for high throughput screening of therapeutic genes. A number of anti-cancer effects of HVJ-E have been identified, specifically activation of both T cell immunity and non-T cell immunity against cancers. Furthermore, a tissue-targeting HVJ-E vector has been constructed using a unique approach for virus engineering and by conjugation with biocompatible polymers. Therefore, the HVJ-E vector is expected to enable effective cancer therapy through the delivery of molecular therapy and through its immunotherapeutic effects.     

In Vivo Application of Non-viral Vectors to the Liver

The liver plays a central role in many inherited and acquired genetic disorders, and thus is a potential target for nucleic acid therapies. Despite the great strides made in basic molecular biology over the last two decades successful gene therapy remains elusive. Most recently, there has been considerable effort to develop non-viral gene therapy approaches, in part, to overcome the potential complications associated with viral delivery systems. This review outlines the different non-viral approaches available to the liver, and includes a detailed review of recent advances in delivery vectors for use in techniques such as gene augmentation, with particular emphasis on useful applications of antisense and ribozyme technology.