Design,preparation and application of nucleic acid delivery carriers

Gene delivery vectors must deliver their cargoes into the cytosol or the nucleus, where DNA or siRNA functions in vivo. Therefore it is crucial for the rational design of the nucleic acid delivery carriers. Compared with viral vectors, non-viral vectors have overcome some fatal defections in gene therapy. Whereas the most important issue for the non-viral vectors is the low transfection efficiency, which hinders the progress of non-viral carriers. Sparked by the structures of the virus and understanding of the process of virus infection, various biomimic structures of non-viral carriers were designed and prepared to improve the transfection issues in vitro and in vivo. However, less impressive results are achieved. In this review, we will investigate the evolution of the virus-mimicking carriers of nucleic acids for gene therapy, especially in cancer therapy; explore and discuss the relationship between the structures, materials and functions of the carriers, to provide guidance for establishing safe and highly efficient non-viral carriers for gene therapy.     

Nucleocytoplasmic transport of DNA: enhancing non-viral gene transfer

Gene therapy, the correction of dysfunctional or deleted genes by supplying the lacking component, has long been awaited as a means to permanently treat or reverse many genetic disorders. To achieve this, therapeutic DNA must be delivered to the nucleus of cells using a safe and efficient delivery vector. Although viral-based vectors have been utilized extensively due to their innate ability to deliver DNA to intact cells, safety considerations, such as pathogenicity, oncogenicity and the stimulation of an immunological response in the host, remain problematical. There has, however, been much progress in the development of safe non-viral gene-delivery vectors, although they remain less efficient than the viral counterparts. The major limitations of non-viral gene transfer reside in the fact that it must be tailored to overcome the intracellular barriers to DNA delivery that viruses already master, including the cellular and nuclear membranes. In particular, nuclear transport of the therapeutic DNA is known to be the rate-limiting step in the gene-delivery process. Despite this, much progress had been made in recent years in developing novel means to overcome these barriers and efficiently deliver DNA to the nuclei of intact cells. This review focuses on the nucleocytoplasmic delivery of DNA and mechanisms to enhance to non-viral-mediated gene transfer.     

Effect of endonuclease G depletion on plasmid DNA uptake and levels of homologous recombination in hela cells

Endonuclease G (EndoG) is a mitochondrial apoptosis regulator that also has roles outside of programmed cell death. It has been implicated as a defence DNase involved in the degradation of exogenous DNA after transfection of mammalian cells and in homologous recombination of viral and endogenous DNA. In this study, we looked at the effect of EndoG depletion on plasmid DNA uptake and the levels of homologous recombination in HeLa cells. We show that the proposed defence role of EndoG against uptake of non-viral DNA vectors does not extend to the cervical carcinoma HeLa cells, as targeting of EndoG expression by RNA interference failed to increase intracellular plasmid DNA levels. However, reducing EndoG levels in HeLa cells resulted in a statistically significant reduction of homologous recombination between two plasmid DNA substrates. These findings suggest that non-viral DNA vectors are also substrates for EndoG in its role in homologous recombination.     

Delivery of Full-Length Factor VIII Using a piggyBac Transposon Vector to Correct a Mouse Model of Hemophilia A

Viral vectors have been used for hemophilia A gene therapy. However, due to its large size, full-length Factor VIII (FVIII) cDNA has not been successfully delivered using conventional viral vectors. Moreover, viral vectors may pose safety risks, e.g., adverse immunological reactions or virus-mediated cytotoxicity. Here, we took advantages of the non-viral vector gene delivery system based on piggyBac DNA transposon to transfer the full-length FVIII cDNA, for the purpose of treating hemophilia A. We tested the efficiency of this new vector system in human 293T cells and iPS cells, and confirmed the expression of the full-length FVIII in culture media using activity-sensitive coagulation assays. Hydrodynamic injection of the piggyBac vectors into hemophilia A mice temporally treated with an immunosuppressant resulted in stable production of circulating FVIII for over 300 days without development of anti-FVIII antibodies. Furthermore, tail-clip assay revealed significant improvement of blood coagulation time in the treated mice.piggyBac transposon vectors can facilitate the long-term expression of therapeutic transgenes in vitro and in vivo. This novel gene transfer strategy should provide safe and efficient delivery of FVIII.     

Efficient removal of LoxP-flanked genes by electroporation of Cre-recombinase mRNA

Introduction of Cre-recombinase in target cells is currently achieved by transfection of plasmid DNA or by viral-mediated transduction. However, efficiency of non-viral DNA transfection is often low in many cell types, and the use of viral vectors for transduction implies a more complex and laborious manipulation associated with safety issues. We have developed a non-viral non-DNA technique for rapid and highly efficient excision of LoxP-flanked DNA sequences based on electroporation of in vitro transcribed mRNA encoding Cre-recombinase. A K562-DSRed[EGFP] cell line was developed in order to measure Cre-mediated recombination by flow cytometric analysis. These cells have a stable integrated DSRed reporter gene flanked by two LoxP sites, and an EGFP reporter gene, which could only be transcribed when the coding sequence for DSRed was removed. The presented data show recombination efficiencies, as measured by appearance of EGFP-fluorescence, of up to 85% in Cre-recombinase mRNA-electroporated K562-DSRed[EGFP] cells. In conclusion, mRNA electroporation of Cre-recombinase is a powerful, safe, and clinically applicable alternative to current technologies used for excision of stably integrated LoxP-flanked DNA sequences.     

Synthesis and characterization of poly (amino ester) for slow biodegradable gene delivery vector

Many therapeutic carrier materials were exploited for human gene therapy from viral to polymeric vectors. This research describes the evaluation of two biodegradable ester-bonded polymers synthesized by double-monomer polycondensation for a non-viral cationic polymer-based gene delivery system. The backbone was constructed to include inner tertiary amines and outer primary amines. Self-assembly with DNA resulted in the production of regularly nano-sized spherical polyplexes with good transfection efficiency, especially in the presence of serum. The polymers showed a relatively slow degradability for an amine-containing ester polymer, as they maintained DNA/polymer complex for 7 days in physiological buffer conditions. Finally, the low toxicity and slow degradation concluded these polymers reliable for long-term therapeutic applications.     

Gene therapy: progress and challenges.

Gene therapy is the delivery of new genetic material into a patient's somatic cells for the treatment of disease and is made possible through the development of viral and non-viral gene transfer vectors. In the first five years of gene therapy, clinical studies failed to yield efficacy data with the vectors available at that time. The lack of consistent clinical benefit prompted the United States National Institute of Health Recombinant DNA Advisory Committee to evaluate gene therapy research and conclude that substantial improvements in gene transfer vectors were needed in the areas of vector safety and control of the level and duration of gene expression, and to increase the understanding of the biological interaction of gene transfer vectors with the host. We will describe the progress in development of gene delivery technology, focusing on improvements in vector safety, analysis of vector biodistribution and GMP manufacturing of viral and non-viral gene transfer systems over the last six years since the report. Whereas 5 years ago, investigators tested every vector for every potential disease indication, the accumulated database now enables investigators to select a single vector based upon it's known performance in a wide number of animal models and human clinical studies. We will also highlight several directions investigators have taken to improve the safety and efficacy of gene therapy vectors.     

体内基因转染技术在肝再生研究中的应用

肝脏是一个特殊的器官,不仅因为它独特的解剖结构和生理特征,而且它还具有无限的再生能力。在各种动物模型中,应用病毒或非病毒载体将肝细胞生长因子等基因转入体内,能增强肝再生能力,这就是肝脏基因转染技术在肝再生研究中的应用。未来的研究目标就是消除病毒载体的毒副作用和增加非病毒载体的转染率,这也是目前肝内基因转染技术中面临的主要难题;另一个研究目标就是用受体介导基因靶向肝转染,使转入基因在肝细胞中特异高表达。这些研究成果将有助于肝再生基因机制研究,以及将来临床基因治疗提供参考。     

Transfection of Infectious RNA and DNA/RNA Layered Vectors of Semliki Forest Virus by the Cell-Penetrating Peptide Based Reagent PepFect6

Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus) based RNA and DNA vectors to study the transfection efficiency of the non-viral cell-penetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp) constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells.     

Impact of lysine-affinity chromatography on supercoiled plasmid DNA purification

Gene therapy and DNA vaccination cover a variety of applications using viral and non-viral vectors as vehicles of choice for treatment of genetic or acquired diseases. Recently, most therapeutic applications have been performed with non-viral biological agents preparations highly enriched in supercoiled plasmid molecules and it has been concluded that this isoform is more efficient at gene transfection than open circular isoform. This work describes for the first time a new strategy that uses lysine-chromatography to efficiently eliminate Escherichia coli impurities as well as other ineffective plasmid isoforms present in a complex clarified lysate to purify and obtain pharmaceutical-grade supercoiled plasmid DNA. The quality control tests indicated that the levels of impurities in the final plasmid product were below the generally accepted specifications. Furthermore, the delivery of the purified product to eukaryotic cells, the cell uptake and transfection efficiency were also analyzed. The results showed that the transfection efficiency reached with the application of the supercoiled plasmid conformation, purified with lysine-agarose, was higher than the values achieved for other plasmid topologies. Therefore, this study presents a new enabling technology to obtain the completely purified non-viral vector, able to act with good efficiency as gene therapy delivery vehicle in several diseases like cancer.     

Selective DNA Delivery to Tumor Cells Using an Oligoarginine-LTVSPWY Peptide

DNA therapy for cancer requires efficient, selective and safe DNA delivery systems. Compared with other non-viral methods such as lipid or polymer-based DNA delivery vectors, peptide-based DNA delivery systems are biocompatible and biodegradable, which leads to lower immunogenicity and lower toxicity. Moreover, peptide vectors are easier to produce and their compositions easier to control because solid-phase peptide synthesis has been extensively developed. However, peptide-based systems for DNA delivery toward special tumor cells or tissues are still lacking. In this study, we constructed a non-viral 9rR-LTVSPWY peptide-based DNA delivery system and showed that it is able to efficiently and selectively transfect DNA into targeted tumor cells. This work presents a novel strategy for tumor cell-specific DNA delivery and a reference for designing more efficient DNA delivery systems targeted towards various types of cancer.     

Recent patents in cationic lipid carriers for delivery of nucleic acids

Gene therapy is a medical technique intended for treatment of disorders caused by defective, missing, or overexpressing genes. Efficient delivery vectors are necessary in order to transport genetic material to the target cells. Such vectors include viral and non-viral carriers. Viral vectors transfect cells efficiently, however risks associated with their use have limited their clinical applications. Nonviral delivery systems are safer, easier to prepare, more versatile and cost effective. However, their transfection efficiency still falls behind that of the viral vectors. Considerable research into nonviral gene delivery has been conducted in the last two decades on synthetic soft materials such as cationic lipids, polymers, surfactants, and dendrimers as prospective nucleotide carriers for gene delivery. So far, cationic lipids are the most widely used constituents of nonviral gene carriers, with multiple strategies employed to improve their in vitro and in vivo transfection. Efforts in synthesizing new cationic lipids were not fully successful in closing the gap between the efficiency of the viral vectors and that of binary cationic lipid/DNA complexes. Current efforts for improving lipofection efficiency are focused on the development of multicomponent carriers including cationic lipids as key constituents. This review summarizes the recent patents on new cationic lipids as well as on multicomponent formulations enhancing their efficiency as nucleotide carriers.     

Chitosan-Graft-Polyethylenimine/DNA Nanoparticles as Novel Non-Viral Gene Delivery Vectors Targeting Osteoarthritis

The development of safe and efficient gene carriers is the key to the clinical success of gene therapy. The present study was designed to develop and evaluate the chitosan-graft-polyethylenimine (CP)/DNA nanoparticles as novel non-viral gene vectors for gene therapy of osteoarthritis. The CP/DNA nanoparticles were produced through a complex coacervation of the cationic polymers with pEGFP after grafting chitosan (CS) with a low molecular weight (Mw) PEI (Mw = 1.8 kDa). Particle size and zeta potential were related to the weight ratio of CP:DNA, where decreases in nanoparticle size and increases in surface charge were observed as CP content increased. The buffering capacity of CP was significantly greater than that of CS. The transfection efficiency of CP/DNA nanoparticles was similar with that of the Lipofectamine™ 2000, and significantly higher than that of CS/DNA and PEI (25 kDa)/DNA nanoparticles. The transfection efficiency of the CP/DNA nanoparticles was dependent on the weight ratio of CP:DNA (w/w). The average cell viability after the treatment with CP/DNA nanoparticles was over 90% in both chondrocytes and synoviocytes, which was much higher than that of PEI (25 kDa)/DNA nanoparticles. The CP copolymers efficiently carried the pDNA inside chondrocytes and synoviocytes, and the pDNA was detected entering into nucleus. These results suggest that CP/DNA nanoparticles with improved transfection efficiency and low cytotoxicity might be a safe and efficient non-viral vector for gene delivery to both chondrocytes and synoviocytes.     

Membrane fusion inducers, chloroquine and spermidine increase lipoplex-mediated gene transfection

Gene transfection into mammalian cells can be achieved with viral and non-viral vectors. Non-viral vectors, such as cationic lipids that form lipoplexes with DNA, are safer and more stable than viral vectors, but their transfection efficiencies are lower. Here we describe that the simultaneous treatment with a membrane fusion inducer (chlorpromazine or procainamide) plus the lysosomotropic agent chloroquine increases lipoplex-mediated gene transfection in human (HEK293 and C-33 A) and rat (PC12) cell lines (up to 9.2-fold), as well as in situ in BALB/c mice spleens and livers (up to 6-fold); and that the polyamine spermidine increases lipoplex-mediated gene transfection and expression in cell cultures. The use of these four drugs provides a novel, safe and relatively inexpensive way to considerably increase lipoplex-mediated gene transfection efficiency.     

Non-viral vectors in cystic fibrosis gene therapy: progress and challenges

Although the viability of cystic fibrosis (CF) gene transfer to airway epithelium has been demonstrated in vitro and in animal models, so far none of the clinical investigations using adenovirus, adeno-associated virus, lentivirus, cationic lipids or polymers has shown a persistent correction of the ion transport defects that occur in CF. Despite disappointing results, these studies have shown that non-viral vectors could represent a viable alternative for gene therapy in CF airway epithelium. The transfer efficiency of non-viral vectors is currently low, however, and thus these systems are not clinically relevant as yet. Before clinical application, several limitations encountered by non-viral delivery systems must be addressed. Recent progress has been made towards overcoming these limitations and towards making non-viral gene therapy a more realistic option for CF.     

综述——纳米材料与药物输递：基因治疗药物输递系统的研究现状及发展趋势

基因治疗是一种有效的治疗方法,可用于治疗多种严重威胁人类健康的疾病．然而,裸露的基因治疗药物存在易被核酶降解、细胞内吞效果差和细胞靶向能力差等缺点．因此,需要寻求合适的载体,将基因治疗药物有效地输递到靶细胞,实现高效的基因治疗．本文主要综述了近年来基因治疗药物输递系统的研究进展,分别总结和阐述了病毒载体,脂质体、聚合物和树状大分子等非病毒载体,以及具有示踪功能的输递系统的特点及研究和发展现状．     

Fluoride enhances transfection activity of carbonate apatite by increasing cytoplasmic stability of plasmid DNA

Intracellular delivery of a functional gene or a nucleic acid sequence to specifically knockdown a harmful gene is a potential approach to precisely treat a critical human disease. The intensive efforts in the last few decades led to the development of a number of viral and non-viral synthetic vectors. However, an ideal delivery tool in terms of the safety and efficacy has yet to be established. Recently, we have developed pH-sensing inorganic nanocrystals of carbonate apatite for efficient and cell-targeted delivery of gene and gene-silencing RNA. Here we show that addition of very low level of fluoride to the particle-forming medium facilitates a robust increase in transgene expression following post-incubation of the particles with HeLa cells. Confocal microscopic observation and Southern blotting prove the cytoplasmic existence of plasmid DNA delivered by likely formed fluoridated carbonate apatite particles while degradation of plasmid DNA presumably by cytoplasmic nucleases was noticed following delivery with apatite particles alone. The beneficial role of fluoride in enhancing carbonate apatite-mediated gene expression might be due to the buffering potential of generated fluoridated apatite in endosomal acidic environment, thereby increasing the half-life of delivered plasmid DNA.