A biomimetic lipid library for gene delivery through thiol-yne click chemistry

The delivery of nucleic acids such as plasmid DNA and siRNA into cells is a cornerstone of biological research and is of fundamental importance for medical therapeutics. Although most gene delivery therapeutics in clinical trials are based on viral vectors, safety issues remain a major concern. Non-viral vectors, such as cationic lipids and polymers, offer safer alternatives but their gene delivery efficiencies are usually not high enough for clinical applications. Thus, there is a high demand for more efficient and safe non-viral vectors. Here, we present a facile two-step method based on thiol-yne click chemistry for parallel synthesis of libraries of new biomimetic cationic thioether lipids. A library of novel lipids was synthesized using the developed method and more than 10% of the lipids showed highly efficient transfection in different cell types, surpassing the efficiency of several popular commercial transfection reagents. One of the new lipids showed highly efficient siRNA delivery to multiple cell types and could successfully deliver DNA plasmid to difficult-to-transfect mouse embryonic stem cells (mESC). Analysis of structure-activity relationship revealed that the length of the hydrophobic alkyl groups was a key parameter for efficient cell transfection and was more important for transfection efficiency than the nature of cationic head groups. The correlation of the size and surface charge of liposomes with transfection efficiency is described.     

基因治疗中的核酸药物及非病毒递送载体的研究进展

基因治疗是针对基因异常相关疾病的终极治疗技术,各种具有不同机制的核酸药物的出现为基因治疗带来了更多的可能性。但是,由于存在体内稳定性差、难以高效进入靶细胞等问题,核酸药物需要载体的帮助而进入目标细胞并到达特定的胞内位置,因此,开发安全高效的核酸递送系统是基因治疗的基石。与病毒载体相比,非病毒载体具有更高的安全性,但转染效率较低。随着纳米技术的发展,非病毒载体的效率得到了显著的提升,进入临床研究的数量逐渐增多。本文简要介绍基因治疗中的核酸药物及其递送载体,对非病毒核酸药物递送技术的瓶颈及进展做综合评述。     

Modulating polymer chemistry to enhance non-viral gene delivery inside hydrogels with tunable matrix stiffness

Non-viral gene delivery holds great promise for promoting tissue regeneration, and offers a potentially safer alternative than viral vectors. Great progress has been made to develop biodegradable polymeric vectors for non-viral gene delivery in 2D culture, which generally involves isolating and modifying cells in vitro, followed by subsequent transplantation in vivo. Scaffold-mediated gene delivery may eliminate the need for the multiple-step process in vitro, and allows sustained release of nucleic acids in situ. Hydrogels are widely used tissue engineering scaffolds given their tissue-like water content, injectability and tunable biochemical and biophysical properties. However, previous attempts on developing hydrogel-mediated non-viral gene delivery have generally resulted in low levels of transgene expression inside 3D hydrogels, and increasing hydrogel stiffness further decreased such transfection efficiency. Here we report the development of biodegradable polymeric vectors that led to efficient gene delivery inside poly(ethylene glycol) (PEG)-based hydrogels with tunable matrix stiffness. Photocrosslinkable gelatin was maintained constant in the hydrogel network to allow cell adhesion. We identified a lead biodegradable polymeric vector, E6, which resulted in increased polyplex stability, DNA protection and achieved sustained high levels of transgene expression inside 3D PEG-DMA hydrogels for at least 12 days. Furthermore, we demonstrated that E6-based polyplexes allowed efficient gene delivery inside hydrogels with tunable stiffness ranging from 2 to 175 kPa, with the peak transfection efficiency observed in hydrogels with intermediate stiffness (28 kPa). The reported hydrogel-mediated gene delivery platform using biodegradable polyplexes may serve as a local depot for sustained transgene expression in situ to enhance tissue engineering across broad tissue types.     

载体与基因转染的研究进展

基因载体是指将基因或其它核酸物质运载到细胞中的工具.其化学本质可以是蛋白质或多肽、核酸、脂类、糖类、其它有机分子或它们的复合物.基因传递系统是基因治疗的重要组成部分,也是目前基因治疗的瓶颈.现有的基因载体包括两类.即病毒载体和非病毒载体.病毒载体转染效率高,但由于其转染具有免疫原性和致突变性限制了它的应用;非病毒载体系统具有低毒、低免疫原性和相对靶向性等优点,是新兴发展起来的基因转移系统.就各种载体的最新研究进展作一综述.     

Synthetic and natural polycations for gene therapy: state of the art and new perspectives

Currently, the major drawback of gene therapy is the gene transfection rate. The two main types of vectors that are used in gene therapy are based on viral or non-viral gene delivery systems. There are several non-viral systems that can be used to transfer foreign genetic material into the human body. In order to do so, the DNA to be transferred must escape the processes that affect the disposition of macromolecules. These processes include the interaction with blood components, vascular endothelial cells and uptake by the reticuloendothelial system. Furthermore, the degradation of therapeutic DNA by serum nucleases is also a potential obstacle for functional delivery to the target cell. Cationic polymers have a great potential for DNA complexation and may be useful as non-viral vectors for gene therapy applications. The objective of this review was to address the state of the art in gene therapy using synthetic and natural polycations and the latest strategies to improve the efficiency of gene transfer into the cell.     

Electroporation gene therapy: new developments in vivo and in vitro

Electroporation-based gene therapy has become a "hot field" for non-viral gene delivery. This review summarizes the progress made in intramuscular and intratumoral electrogenetransfer, which include new applications and modifications. The progress in dendritic cell (DC) and stem cell transfection by use of electroporation has also been discussed. Rapid progress during the past two years clearly demonstrates the great potential of this technology, but there are challenges faced by both in vitro and in vivo applications, which include how to enhance the transfection efficiency for intratumoral delivery, how to extend the duration of gene expression for intramuscular injection, and how to increase the survival rate for in vitro cell transfection. Resolving these issues will shed new light on this field.     

非病毒型载体介导基因转染

基因载体是制约基因转移技术发展的关键。近年来,非病毒载体由于其安全、低毒、低免疫原性等特点而备受青睐。文章以脂质体和聚乙烯亚胺为代表,介绍了非病毒载体的性质、介导转染的机制。随着人们对细胞转染机制了解的深入以及生物材料科学的迅速发展,非病毒型载体将有望实现高效、低毒、靶向特异等特点,从而成为基因治疗中的理想载体。     

Progress in cationic lipid-mediated gene transfection: a series of bio-inspired lipids as an example

Over the last several years, various gene delivery systems have been developed for gene therapy applications. Although viral vector-based gene therapy has led to the greatest achievements in animal and human studies, synthetic non-viral vectors have also been developed as they offer several advantages over viral systems, including lower immunogenicity and greater nucleic acid packaging capacity. Nevertheless, the transfection efficiency of the current non-viral gene carriers still needs to be improved, especially as regards direct in vivo transfection. In particular, cationic lipid/nucleic acid complexes (termed lipoplexes) have been the subject of intensive investigation with a view to optimize their performance and to better understand their mechanisms of action, and consequently to design new approaches to overcome the critical barriers of cationic liposome-mediated gene delivery. A possible strategy may rely on considering the membrane constituents and properties of the vast variety of living organisms as a source of inspiration for the design of biocompatible, non-toxic and effective novel artificial liposomal systems. Thus, the present forward-looking review provides an overview of the progress already made during the last years in the field of cationic lipid-mediated gene transfection and also focuses on a series of novel bio-inspired lipids for both in vitro and in vivo gene transfection.     

Bio-functional inorganic materials: an attractive branch of gene-based nano-medicine delivery for 21st century

Treatment of a physiological disorder in the genetic level (gene therapy) and induction of a specific immunity by means of a genetic material (genetic vaccination), are considered two revolutionary approaches for clinical medicine. The implementation strategies for these basic concepts demand a vehicle for nucleic acid delivery. Viral delivery systems, although highly efficient, possess severe limitations in terms of life safety and thus non-viral synthetic systems have become increasingly desirable. Intensive efforts for the last 3 decades enabled the development of a lot of synthetic devices, most of which belong to cationic lipids, peptides and other polymers, but comparatively little attention was paid to inorganic materials. This is the first article aimed at reviewing the dramatic progress of non-viral gene delivery research focusing on the functional inorganic materials. Both biodegradable and non-biodegradable inorganic particles have been fabricated in the nano-scale with the attributes of binding DNA, internalizing across the plasma membrane and finally releasing it in the cytoplasm for final expression of a protein. Some in vivo trials also brought highly satisfactory results demonstrating their potential applications in the clinical medicine.     

Transposons for gene therapy!

Gene therapy is a promising strategy for the treatment of several inherited and acquired human diseases. Several vector platforms exist for the delivery of therapeutic nucleic acids into cells. Vectors based on viruses are very efficient at introducing gene constructs into cells, but their use has been associated with genotoxic effects of vector integration or immunological complications due to repeated administration in vivo. Non-viral vectors are easier to engineer and manufacture, but their efficient delivery into cells is a major challenge, and the lack of their chromosomal integration precludes long-term therapeutic effects. Transposable elements are non-viral gene delivery vehicles found ubiquitously in nature. Transposon-based vectors have the capacity of stable genomic integration and long-lasting expression of transgene constructs in cells. Molecular reconstruction of Sleeping Beauty, an ancient transposon in fish, represents a cornerstone in applying transposition-mediated gene delivery in vertebrate species, including humans. This review summarizes the state-of-the-art in the application of transposable elements for therapeutic gene transfer, and identifies key targets for the development of transposon-based gene vectors with enhanced efficacy and safety for human applications.     

Polymer-related off-target effects in non-viral siRNA delivery

Since off-target effects in non-viral siRNA delivery are quite common but not well understood, in this study various polymer-related effects observed in transfection studies were described and their mechanisms of toxicity were investigated. A variety of stably luciferase-expressing cell lines was compared concerning polymer-mediated effects after transfection with polyplexes of siRNA and poly(ethylene imine) (PEI) or poly(ethylene glycol)-grafted PEI (PEG-PEI). Cell viability, LDH release, gene expression profiles of apoptosis-related genes and promoter activation were investigated. Interestingly, PEG-PEI, which is generally better tolerated than PEI, was found to activate apoptosis in a cell line- and concentration-dependent manner. While both polymers showed sigmoidal dose-response of cell viability in L929 cells (IC(50)(PEI)?=?6?μg/ml, IC(50)(PEG-PEI)?=?11?μg/ml), H1299/Luc cells exhibited biphasic dose-response for PEG-PEI and stronger apoptosis at 2?μg/ml than at 20?μg/ml PEG-PEI, as shown in TUNEL assays. Gene expression profiling confirmed that H1299/Luc cells underwent apoptosis via thousand-fold activation of TNF receptor-associated factors. Additionally, it was demonstrated that NFkB-mediated CMV promoter activation in stably transfected cells can lead to increased target gene levels after transfection instead of siRNA-mediated knockdown. With these results, polymeric vectors were shown not to be inert substances. Therefore, alterations in gene expression caused by the delivery agent must be known to correctly interpret gene-silencing experiments, to understand the mechanisms of off-target effects, and most of all to further develop vectors with reduced side effects. Taking these observations into account, one established cell line was eventually identified to be suitable for RNAi experiments. As shown by these experiments, materials that have been used for many years can elicit unexpected off-target effects. Therefore, non-viral vectors must be screened for several levels of toxicity to make them promising candidates.     

Delivery of non-viral gene carriers from sphere-templated fibrin scaffolds for sustained transgene expression

Delivery of safe and controlled levels of biomimetic cues to govern host response and reorganization is a fundamental component in the design of tissue engineering scaffolds. Non-viral gene delivery is an approach that exploits the cell machinery to produce proteins while avoiding genomic DNA incorporation. We describe a method to integrate polymeric non-viral gene carriers (polyplexes) within a novel three-dimensional, sphere-templated fibrin scaffold suitable for soft tissue engineering applications. After seeding the scaffolds with NIH-3T3 fibroblasts, different transgene expression profiles were achieved based on the spatial distribution of polyplexes within the scaffold. Scaffolds with polyplexes coated onto the surface of inter-connected pores showed peak transfection at day 5 and linear expression through 15 days. Scaffolds with polyplexes embedded within the fibrils of the biopolymer showed peak expression at 7-9 days and showed linear expression for 21-29 days, depending on the polymer:DNA ratio. Surface-coated polyplexes achieved one order of magnitude greater expression than polyplexes embedded within the scaffold. The integrated material formulations developed in this work provide a useful technology for tissue engineering applications by demonstrating the ability to provide long-term biomimetic cues through non-viral gene delivery.     

Overexpression of Bcl-2 as a proxy redox stimulus to enhance activity of non-viral redox-responsive delivery vectors

Redox-sensitive non-viral delivery systems exploit intracellular reducing environment to improve the efficacy of the delivery of nucleic acids by selectively releasing the cargo in the subcellular space. Bcl-2 overexpression is frequently observed in human cancers and is closely associated with increased resistance to chemotherapy and radiotherapy. One of the biochemical alterations accompanying Bcl-2 overexpression is the increase in cellular glutathione (GSH) levels. In this study, we hypothesize that such increase of GSH concentration will selectively enhance the transfection activity of redox-sensitive delivery systems in cells overexpressing Bcl-2. Transfection studies were conducted in MCF-7 mammary carcinoma cells and MCF-7 clones overexpressing Bcl-2. It was confirmed that Bcl-2 overexpression resulted in the expected increase in GSH concentration. Redox-sensitive complexes containing plasmid DNA, mRNA, antisense oligodeoxynucleotides, and siRNA exhibited selectively increased activity in cells overexpressing Bcl-2 compared to non-redox complexes. The effect of Bcl-2 overexpression on the selective enhancement of transfection was highly dependent on the type of the delivered nucleic acid, and was most pronounced for mRNA. This study shows that Bcl-2 overexpression can serve as a proxy redox stimulus to enhance the activity of all major classes of potential nucleic acid therapeutics, when delivered using redox-sensitive vectors.     

Ex Vivo Gene Delivery to Hepatocytes: Techniques, Challenges, and Underlying Mechanisms

Gene delivery to primary hepatocytes is an important tool for a number of applications including the study of liver cell biology and pathology, drug screening, and gene therapy. Robust transfection of primary hepatocytes, however, is significantly more difficult to achieve than in cell lines or readily dividing primary cells. In this report, we investigated in vitro gene delivery to both primary rat hepatocytes and Huh7.5.1 cells (a hepatoma cell line) using a number of viral and non-viral methods, including Lipofectamine 2000, FuGene HD, Nucleofection, Magnetofection, and lentiviruses. Our results showed that Lipofectamine 2000 is the most efficient reagent for green fluorescent protein (GFP) gene delivery to primary rat hepatocytes (33.3 ± 1.8% transfection efficiency) with minimal adverse effect on several hepatic functions, such as urea and albumin secretion. The lentiviral vectors used in this study exhibited undetectable gene delivery to primary rat hepatocytes but significant delivery to Huh7.5.1 cells (>80% transfection efficiency). In addition, we demonstrated lentiviral-based and spatially defined delivery of the GFP gene to Huh7.5.1 cells for use in biological microelectromechanical systems.     

Fast and Efficient Transfection of Mouse Embryonic Stem Cells Using Non-Viral Reagents

Reliable and efficient DNA and RNA transfection methods are required when studying the role of individual genes in mouse pluripotent stem cells. However, these cells usually grow in tight clusters and are therefore more difficult to transfect than many other cell lines. We have found that transfection is especially challenging when mouse embryonic stem (mES) cells are cultured in the newly described 2i medium, which is based on two chemical inhibitors of differentiation pathways. In the present study we have performed a side-by-side comparison of commercially available, non-viral transfection reagents with regard to their ability to deliver plasmid DNA and siRNA into adherent and/or trypsinized mES cells cultured in 2i medium, assessing transfection rates, plasmid gene expression, siRNA mediated knockdown of Oct4 and viability. Finally, we present a fast and efficient method for transfection of trypsinized mES cells using the liposomal-based Lipofectamine 2000. With only a five-minute long transfection time we obtained at least 85 % transfected cells with 80 % maintained viability. Moreover, this protocol saves up to a day of experimental time since the cells are in suspension at the time of transfection, which allows for immediately re-plating into the appropriate format. This fast, simplified and highly efficient transfection method will be valuable for both basic research and high-throughput applications.     

基因强化骨组织工程中的病毒载体

背景：不同的基因输送策略也被应用到骨组织工程中以修复破坏的骨组织,作为最有效率的基因转运载体,病毒载体在骨组织工程中的应用方兴未艾。 目的：系统回顾和讨论目前基因强化骨组织工程中常用的病毒载体相关应用。 方法：利用PubMed数据库对2002年1月至2015年1月的相关文献进行了检索,检索的文章主要聚焦在病毒载体基因转导方法和其在骨组织工程中的应用。对腺病毒、反转录病毒、腺相关病毒和嵌合病毒在骨组织工程的相关应用及不足进行了讨论。总共24篇相关文献被纳入此篇综述。 结果与结论：总结了近年来病毒载体联合基因治疗促进骨组织再生的研究工作。讨论了包括装载目的基因的病毒载体联合种子细胞例如间充质干细胞植入支架材料修复骨缺损。研究表明,基因强化的骨组织工程比传统组织工程具有更多的优点;病毒载体介导的基因转染效率比普通载体更高;病毒载体介导的基因强化骨组织工程用于人体的安全性仍需要漫长的临床观察研究。病毒载体系统仍然是最有效的将外源基因转入种子细胞的手段之一。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Maximizing gene delivery efficiencies of cationic helical polypeptides via balanced membrane penetration and cellular targeting

The application of non-viral gene delivery vectors is often accompanied with the poor correlation between transfection efficiency and the safety profiles of vectors. Vectors with high transfection efficiencies often suffer from high toxicities, making it unlikely to improve their efficiencies by increasing the DNA dosage. In the current study, we developed a ternary complex system which consisted of a highly membrane-active cationic helical polypeptide (PVBLG-8), a low-toxic, membrane-inactive cationic helical polypeptide (PVBLG-7) capable of mediating mannose receptor targeting, and DNA. The PVBLG-7 moiety notably enhanced the cellular uptake and transfection efficiency of PVBLG-8 in a variety of mannose receptor-expressing cell types (HeLa, COS-7, and Raw 264.7), while it did not compromise the membrane permeability of PVBLG-8 or bring additional cytotoxicities. Because of the simplicity and adjustability of the self-assembly approach, optimal formulations of the ternary complexes with a proper balance between membrane activity and targeting capability were easily identified in each specific cell type. The optimal ternary complexes displayed desired cell tolerability and markedly outperformed the PVBLG-8/DNA binary complexes as well as commercial reagent Lipofectamine™ 2000 in terms of transfection efficiency. This study therefore provides an effective and facile strategy to overcome the efficiency-toxicity poor correlation of non-viral vectors, which contributes insights into the design strategy of effective and safe non-viral gene delivery vectors.     

Oligopeptide-terminated poly(β-amino ester)s for highly efficient gene delivery and intracellular localization

The main limitation of gene therapy towards clinics is the lack of robust, safe and efficient gene delivery vectors. This paper describes new polycations for gene delivery based on poly(β-amino ester)s (pBAE) containing terminal oligopeptides. The authors developed oligopeptide-modified pBAE–pDNA nanoparticles that achieve better cellular viability and higher transfection efficacy than other end-modified pBAE and commercial transfection agents. Gene expression in highly permissive cell lines was remarkably high, but transfection efficiency in less-permissive cell lines was highly dependent on oligopeptide composition and nanoparticle formulation. Moreover, the use of selected oligopeptides in the pBAE formulation led to preferential intracellular localization of the particles. Particle analysis of highly efficient pBAE formulations revealed different particle sizes and charge features, which indicates chemical pseudotyping of the particle surface, related to the oligopeptide chemical nature. In conclusion, chemical modification at the termini of pBAE with amine-rich oligopeptides is a powerful strategy for developing delivery systems for future gene therapy applications.     

Non-viral bone morphogenetic protein 2 transfection of rat dental pulp stem cells using calcium phosphate nanoparticles as carriers

Calcium phosphate nanoparticles have shown potential as non-viral vectors for gene delivery. The aim of this study was to induce bone morphogenetic protein (Bmp)2 transfection in rat dental pulp stem cells using calcium phosphate nanoparticles as a gene vector and then to evaluate the efficiency and bioactivity of the transfection. We also intended to investigate the behavior of transfected cells when seeded on 3-dimensional titanium fiber mesh scaffolds. Nanoparticles of calcium phosphate encapsulating plasmid deoxyribonucleic acid (DNA) (plasmid enhanced green fluorescent protein-BMP2) were prepared. Then, STRO-1-selected rat dental pulp stem cells were transfected using these nanoparticles. Transfection and bioactivity of the secreted BMP2 were examined. Thereafter, the transfected cells were cultured on a fibrous titanium mesh. The cultures were investigated using scanning electron microscipy and evaluated for cell proliferation, alkaline phosphatase activity and calcium content. Finally, real-time polymerase chain reaction was performed for odontogenesis-related gene expression. The results showed that the size of the DNA-loaded particles was approximately 100 nm in diameter. Nanoparticles could protect the DNA encapsulated inside from external DNase and release the loaded DNA in a low-acid environment (pH 3.0). In vitro, nanoparticle transfection was shown to be effective and to accelerate or promote the odontogenic differentiation of rat dental pulp stem cells when cultured in the 3-dimensional scaffolds. Based on our results, plasmid DNA-loaded calcium phosphate nanoparticles appear to be an effective non-viral vector for gene delivery and functioned well for odontogenic differentiation through Bmp2 transfection.     

A ligand-mediated nanovector for targeted gene delivery and transfection in cancer cells

As conventional cancer therapies struggle with toxicity issues and irregular remedial efficacy, the preparation of novel gene therapy vectors could offer clinicians the tools for addressing the genetic errors of diseased tissue. The transfer of gene therapy to the clinic has proven difficult due to safety, target specificity, and transfection efficiency concerns. Polyethylenimine (PEI) nanoparticles have been identified as promising gene carriers that induce gene transfection with high efficiency. However, the inherent toxicity of the material and non-selective delivery are the major concerns in applying these particles clinically. Here, a non-viral nanovector has been developed by PEGylation of DNA-complexing PEI in nanoparticles functionalized with an Alexa Fluor 647 near infrared fluorophore, and the chlorotoxin (CTX) peptide which binds specifically to many forms of cancer. With this nanovector, the potential toxicity to healthy cells is minimized by both the reduction of the toxicity of PEI with the biocompatible copolymer and the targeted delivery of the nanovector to cancer cells, as evaluated by viability studies. The nanovector demonstrated high levels of targeting specificity and gene transfection efficiency with both C6 glioma and DAOY medulloblastoma tumor cells. Significantly, with the CTX as the targeting ligand, the nanovector may serve as a widely applicable gene delivery system for a broad array of cancer types.