基因治疗在创伤愈合中的应用

创伤修复是一个复杂的生物学过程,各种生长因子、炎症介质等在创面愈合过程中扮演着重要角色。基因治疗是现代生物治疗的一项重要技术,在创伤修复,尤其是难愈性创面的治疗中具有广阔的应用前景。基因治疗分为病毒载体导入系统（逆转录病毒、慢病毒、腺病毒、单纯疱疹病毒和腺病毒相关病毒等）和非病毒载体导入系统的基因感染（直接注射、显微注射、基因枪、电穿孔、脂质体和脂质体复合物、阳离子多聚物等）。本文对创伤修复及基因治疗在该领域的应用进行文献综述。     

Histidinylated poly-L-lysine-based vectors for cancer-specific gene expression via enhancing the endosomal escape

In this work, we synthesized a series of poly-L-lysine (PLL)-based polymers for gene delivery, by modifying the PLL with both cationic peptide and histidine. The peptide moieties serve as cationic centers for polyplex formation, and also as substrates for protein kinase Cα (PKCα), which is specifically activated in many types of cancer cells, to achieve cancer-specific gene expression. The histidine groups serve as buffering moieties to increase the ability of the plasmid DNA (pDNA)-polymer complex (polyplex) to escape the endosome and thus to promote expression of the pDNA in the transfected cells. The facile synthesis of the polymers proceeded by modifying the PLL with side-group-protected peptide and protected histidine, followed by deprotection of the functional groups. The synthesized polymers showed significant buffering capacity over the neutral to acidic pH range and showed less cytotoxicity in vitro compared with histidine-unmodified polymers. The polyplexes successfully showed PKCα-responsive gene expression immediately after their introduction into cancer cells and the gene expression continued for at least 24?h. These PLL-based carriers thus show promise for cancer-targeted gene therapy.     

The role of PEG architecture and molecular weight in the gene transfection performance of PEGylated poly(dimethylaminoethyl methacrylate) based cationic polymers

In this study, we report the synthesis of well-defined model PEGylated poly(dimethylaminoethyl methacrylate) based cationic polymers composed of different PEG architecture with controlled PEG and nitrogen content via reversible addition-fragmentation chain transfer (RAFT) polymerization, and study the effects of PEG architecture and polymer molecular weight on gene delivery and cytotoxicity. Investigation of the physico-chemical interactions of these model cationic polymers with DNA demonstrated that all these polymers effectively complexed with DNA, and PEG topology did not significantly affect the abilities of the polymers to complex and release DNA. However the size and zeta potential of the complexes were found to be influenced by PEG architecture. The polymers with the block-like configurations formed nanosized DNA complexes. In contrast, considerably higher molecular weight was necessary for the copolymer with the statistical configuration of short PEG chains to form such a small complex. Cell line-dependent influence of PEG architecture on cellular uptake, gene expression efficiency and cell viability of the polymer-DNA complexes was observed. The diblock copolymer-DNA complexes induced higher gene expression than the brush-like block copolymer-DNA complexes, and the statistical copolymer-DNA complexes mediated much lower gene expression than the block-like copolymers-DNA complexes. Increasing the molecular weight of statistical polymer to some extent improved gene expression efficiency. The statistical copolymer was less cytotoxic as compared to the block-like copolymers. These findings provide important insights into the effect of PEGylation nature on gene expression, which will be useful for the design of PEGylated gene delivery polymers.     

Synthetic peptides as non-viral DNA vectors

The use of multiple peptide motifs to provide effective gene delivery holds great promise as an elegant, non-immunogenic approach to gene therapy. The molecular understanding of cell and viral biology provides a strong foundation on which to pursue this objective. Synthetic peptides containing multiple lysines and/or arginines (occasionally ornithines) provide natural polycations for multivalent electrostatic binding of DNA, and for DNA compaction into particles suitable for gene delivery. These cationic peptides can incorporate additional functional motifs (e.g. for translocating DNA into the nucleus) and they can be linked by disulphide bonds to produce high molecular reducible polycations with superior properties for gene therapy. Many factors influence the size, surface charge and stability of peptide/DNA particles. For in vivo use, uncharged particles resistant to disruption by salt and protein, and targeted to tissue-specific membrane molecules, will be required. Entry into the cell is via one of the endocytic pathways, depending on particle size and (in principle) the target cell surface molecule. Peptide motifs for endocytic escape are based mainly on the anionic fusogenic peptide of influenza virus haemagglutinin and on histidine-rich peptides (where the buffering properties of the imidazole group cause osmotic swelling and probably rupture of endocytic vesicles). Once in the cytosol, translocation of DNA plasmids across the nuclear pore complex into the nucleus is a crucial step, because most target cells for gene therapy are either non-dividing or slowly dividing. Nuclear translocation can be achieved by classical nuclear localising motifs, or more simply by (Lys)16 and other cationic peptides.     

The hydrolysis of cationic polycarboxybetaine esters to zwitterionic polycarboxybetaines with controlled properties

In this work, we report a new class of materials, cationic polycarboxybetaine esters, which have unique properties when they interact with proteins, DNAs, and bacteria. These cationic polymers can be converted to nontoxic and nonfouling zwitterionic polymers upon their hydrolysis. Due to their unique properties, they are very promising for a wide range of applications, such as highly effective gene delivery carriers and environmentally friendly antimicrobial coatings. Three positively charged polyacrylamides, of which the pedant groups bear carboxybetaine ester groups, were synthesized. These three polymers have different spacer groups between the quaternary ammonium and the ester groups. Their hydrolysis behaviors were studied using proton NMR under different NaOH concentrations. Their interactions with biomolecules and microorganisms before and after hydrolysis were demonstrated by protein adsorption/resistance, DNA condensation/release, and antimicrobial properties. The polymers were grafted onto a gold-coated surface covered with initiators using surface-initiated atom transfer radical polymerization (ATRP). Fibrinogen adsorption was measured by surface plasmon resonance (SPR) sensors. While the polymer-grafted surfaces have high protein adsorption, the surfaces became nonfouling after hydrolysis. Linear polymers were also synthesized and DNA/polymer complexes were evaluated. Agarose gel electrophoresis shows that DNA can be condensed into nanoparticles by the cationic polymers before hydrolysis and released from the DNA/polymer complexes upon the hydrolysis of the cationic polymers into zwitterionic polymers. The complexes formed were characterized by dynamic light scattering measurements. In addition, the interactions of linear polymers with bacteria were also evaluated. The polycarboxybetaine ester with a pentene spacer exhibits evident antimicrobial properties when they are incubated with Gram negative bacteria (Escherichia Coli). The polymer can be converted to a nontoxic polycarboxybetaine after hydrolysis. This work shows that the biological properties of polycarboxybetaine esters can be dramatically changed via controlled hydrolysis.     

Physicochemical parameters of non-viral vectors that govern transfection efficiency

Gene therapy is based on the vectorization of nucleic acids to target cells and their subsequent expression. Cationic lipids and polymers are the most widely used vectors for the delivery of DNA into cultured cells. Nowadays, numerous reagents made of these cationic molecules are commercially available and used by researchers from the academic and industrial field. By contrast their evaluations in preclinical programs have revealed that their use for in vivo applications will be more problematic than their massive use in vitro. This is mostly due to the physicochemical properties of cationic vectors/DNA complexes, which are the result of their mode of interaction. Indeed, these cationic vectors interact through electrostatic forces with negatively charged DNA. This results in the formation of highly organized positively charged supramolecular structures where DNA molecules are condensed. Association of DNA with cationic lipids under a micellar or liposomal form leads to lamellar organization with DNA molecules sandwiched between lipid bilayers. Although the lamellar phase is the common described structure, as evidenced by small-angle X-ray scattering and electron microscopy, some cationic lipid combined with a hexagonal forming lipid could also result with DNA in an inverted hexagonal structure. Despite a lot of effort, the precise mechanism of gene transfer with cationic vector is still ill-defined. Here, our objective was to overview the main relationships between the physico chemical properties of cationic lipid/DNA complexes and their transfection efficiency. An overview of a new class of vectors consisting of amphiphilic block copolymers designed for in vivo delivery is also presented and discussed.     

非病毒型纳米载体在基因治疗中的研究现状及展望

近 10年来 ,新型非病毒载体在基因治疗中日益受到欢迎。其主要代表为纳米载体 ,具有无毒性及免疫原性的优势 ,已作为高效阳离子载体用于基因转移。体外基因转移实验表明 ,纳米载体的基因转移率高于普通脂质体及其它阳离子多聚体 ,如多聚氮丙啶及聚赖氨酸。本文对纳米载体的结构特点、性能、基因转移机制进行综述 ,并将其在体内外基因转移效率与其它非病毒载体作以比较     

Design of modular non-viral gene therapy vectors

Gene delivery has numerous potential applications both clinically and for basic science research. Non-viral vectors represent the long-term future of gene therapy and biomaterials are a critical component for the development of efficient delivery systems. Biomaterial development combined with fundamental studies of virus function and cellular processes will enable the molecular level design of modular vectors. Vectors are being developed based on cationic polymers or lipids that contain functional groups to mediate appropriate interactions with the extracellular environment or to interface with specific cellular processes. This review describes recent progress on the development of biomaterials for non-viral vectors and highlights opportunities for future development. Ultimately, efficient vectors will expand the traditional applications of gene therapy within the clinic and may enable numerous other opportunities within diagnostics, biotechnology, and basic science research.     

Target delivery of a gene into the brain using the RVG29-oligoarginine peptide

The development of non-viral delivery systems that are capable of mediating an efficient, exclusive, and non-invasive transfer of DNA across the blood-brain barrier into the brain is challenging, but essential for the clinical application of gene therapy to brain diseases. Compared with other non-viral DNA carriers (e.g., lipids or polymers), peptide-based DNA delivery systems have many advantages including the ease of synthesis, low immunogenicity, biocompatibility, and biodegradability in vivo. However, all of the existing peptide-based vehicles for DNA delivery lack selectivity toward cells or tissues, which largely limited their applications in vivo. In this study, we demonstrated that an RVG29-9rR peptide-based DNA delivery system was able to transfect Neuro 2a cells in vitro more efficiently and specifically than Lipofectamine LTX & Plus, one of the most efficient commercially available transfection reagents. More significantly, the peptide mediated efficient and brain-targeting reporter gene expression after intravenous injection into mice. Thus, the results herein suggest a new strategy for brain-targeting DNA delivery in vivo.     

Gene therapy for gastric diseases

Gene therapy for gastric cancer and gastric ulcer is a rationalized strategy since various genes correlate with these diseases. Since gene expressions in non-target tissues/cells cause side effects, a selective gene delivery system targeted to the stomach and/or cancer must be developed. The route of vector transfer (direct injection, systemic, intraperitoneal, gastric serosal surface and oral administration) is an important issue which can determine efficacy and safety. Strategies for cancer gene therapy can be categorized as suicide gene therapy, growth inhibition and apoptosis induction, immunotherapy, anti-angiogenesis, and others. Combination of the target gene with other genes and/or strategies such as chemotherapy and virotherapy is promising. Candidates for treatment of gastric ulcer are vascular endothelial growth factor, angiopoietin-1, serum response factor, and cationic host defense peptide cathelicidin. In this review, we discuss stomach- and cancer-targeted gene transfer methods and summarize gene therapy trials for gastric cancer and gastric ulcer.     

Effect of molecular weight of amine end-modified poly(β-amino ester)s on gene delivery efficiency and toxicity

Amine end-modified poly(β-amino ester)s (PBAEs) have generated interest as efficient, biodegradable polymeric carriers for plasmid DNA (pDNA). For cationic, non-degradable polymers, such as polyethylenimine (PEI), the polymer molecular weight (MW) and molecular weight distribution (MWD) significantly affect transfection activity and cytotoxicity. The effect of MW on DNA transfection activity for PBAEs has been less well studied. We applied two strategies to obtain amine end-modified PBAEs varying in MW. In one approach, we synthesized four amine end-modified PBAEs with each at 15 different monomer molar ratios, and observed that polymers of intermediate length mediated optimal DNA transfection in HeLa cells. Biophysical characterization of these feed ratio variants suggested that optimal performance was related to higher DNA complexation efficiency and smaller nanoparticle size, but not to nanoparticle charge. In a second approach, we used preparative size exclusion chromatography (SEC) to obtain well-defined, monodisperse polymer fractions. We observed that the transfection activities of size-fractionated PBAEs generally increased with MW, a trend that was weakly associated with an increase in DNA binding efficiency. Furthermore, this approach allowed for the isolation of polymer fractions with greater transfection potency than the starting material. For researchers working with gene delivery polymers synthesized by step-growth polymerization, our data highlight the potentially broad utility of preparative SEC to isolate monodisperse polymers with improved properties. Overall, these results help to elucidate the influence of polymer MWD on nucleic acid delivery and provide insight toward the rational design of next-generation materials for gene therapy.     

Impact of the nature, size and chain topologies of carbohydrate-phosphorylcholine polymeric gene delivery systems

With the recent significant advances in the field polymer chemistry, it is now possible to produce well-defined and non-toxic cationic polymers with advanced molecular structures of desired molecular weights and compositions. Carefully engineered polymer architectures are found to impact significantly their DNA condensation and gene delivery efficacies. In a previous study, the statistical carbohydrates based copolymers were found to show high gene expression and low toxicity, however there aggregation in the presence of serum proteins was a major drawback. In this study, carbohydrate and phosphorylcholine based cationic polymers having a different architecture, compositions and varying molecular weights are produced and are termed as cationic 'block-statistical' copolymers. These cationic copolymers are evaluated for their gene delivery efficacies, interactions with serum protein, cellular uptake and nuclear localization ability. As compared to the statistical analogue, 'block-statistical' copolymers showed high gene expression, low interactions with serum proteins, as well as low toxicity in hepatocytes and human dermal fibroblasts. In addition, 2- methacryloyloxyethyl phosphorylcholine (MPC) based 'block-statistical' copolymers and their sugar incorporated analogues were prepared and were found to serve as improved gene delivery vectors than their statistical analogues.     

Laboratory formulated magnetic nanoparticles for enhancement of viral gene expression in suspension cell line

One factor critical to successful gene therapy is the development of efficient delivery systems. Although advances in gene transfer technology including viral and non-viral vectors have been made, an ideal vector system has not yet been constructed. Due to the growing concerns over the toxicity and immunogenicity of viral DNA delivery systems, DNA delivery via improve viral routes has become more desirable and advantageous. The ideal improve viral DNA delivery system should be a synthetic materials plus viral vectors. The materials should also be biocompatible, efficient, and modular so that it is tunable to various applications in both research and clinical settings. The successful steps towards this improve viral DNA delivery system is demonstrated: a magnetofection system mediated by modified cationic chitosan-coated iron oxide nanoparticles. Dense colloidal cationic iron oxide nanoparticles serve as an uptake-enhancing component by physical concentration at the cell surface in presence of external magnetic fields; enhanced viral gene expression (3-100-fold) due to the particles is seen as compared to virus vector alone with little virus dose.     

Multifunctional receptor-targeted nanocomplexes for the delivery of therapeutic nucleic acids to the Brain

Convection enhanced delivery (CED) is a method of direct injection to the brain that can achieve widespread dispersal of therapeutics, including gene therapies, from a single dose. Non-viral, nanocomplexes are of interest as vectors for gene therapy in the brain, but it is essential that administration should achieve maximal dispersal to minimise the number of injections required. We hypothesised that anionic nanocomplexes administered by CED should disperse more widely in rat brains than cationics of similar size, which bind electrostatically to cell-surface anionic moieties such as proteoglycans, limiting their spread. Anionic, receptor-targeted nanocomplexes (RTN) containing a neurotensin-targeting peptide were prepared with plasmid DNA and compared with cationic RTNs for dispersal and transfection efficiency. Both RTNs were labelled with gadolinium for localisation in the brain by MRI and in brain sections by LA-ICP-MS, as well as with rhodamine fluorophore for detection by fluorescence microscopy. MRI distribution studies confirmed that the anionic RTNs dispersed more widely than cationic RTNs, particularly in the corpus callosum. Gene expression levels from anionic formulations were similar to those of cationic RTNs. Thus, anionic RTN formulations can achieve both widespread dispersal and effective gene expression in brains after administration of a single dose by CED.     

Photochemical Transfection: A Technology for Efficient Light-Directed Gene Delivery

Most synthetic gene delivery vectors are taken up in the cell by endocytosis, and inefficient escape of the transgene from endocytic vesicles often is a major barrier for gene transfer by such vectors. To improve endosomal release we have developed a new technology, named photochemical internalization (PCI). PCI is based on photochemical reactions initiated by photosensitizing compounds localized in endocytic vesicles, inducing rupture of these vesicles upon light exposure. PCI constitutes an efficient light-inducible gene transfer method in vivo, which potentially can be developed into a site-specific method for gene delivery in in vivo gene therapy. In this paper the principle behind the PCI technology and the effect of PCI on transfection with different synthetic gene delivery vectors are reviewed. PCI treatment by the photosensitizer aluminum phthalocyanine (AlPcS_2a ) strongly improves transfection mediated by cationic polymers (e.g., poly-L-lysine and polyethylenimine), while the effect on transfection with cationic lipids is more variable. The timing of the light treatment relative to the transfection period was also important, indicating that release of the DNA from early endosomes is important for the outcome of PCI-induced transfection. The possibilities of using PCI as a technology for efficient, site-specific gene delivery in in vivo gene therapy is discussed.     

Molecular therapeutics of HBV

The hepatitis B virus (HBV) infection is a public health problem worldwide, particularly in East Asia. The current therapy of HBV infection is mostly based on chemical agents and cytokines that have been shown to provide limited efficacy and are also toxic to the human body. Gene therapy is a new therapeutic strategy against HBV infection, involving the transmission of gene drugs into liver cells by specific delivery systems and methods. Although this new anti-HBV infection technique is under active investigation, various promising anti-HBV viral gene drugs have been developed for gene therapy, including antisense RNA and DNA, hammerhead ribozymes, dominant negative HBV core mutants, single chain antibody, co-nuclease fusion protein, and antigen. In order to optimize their antiviral effects and/or enhance anti-HBV immunity, various novel gene delivery systems have also been developed to (specifically) deliver such DNA constructs into liver cells; some of them are viral vectors, such as adenoviral vectors, retroviral vectors and poxviral vectors, and even hepatitis B viral for its hepatocellular specificity. Others are non-viral vectors, in which naked DNA and liposomes are frequently used for DNA vaccine or nucleotide analogs for inhibiting HBV DNA polymerase. This review addresses various aspects of gene therapy for HBV infection, including gene drugs, delivery methods, animal model, and liver transplantation with combination therapy. It also discusses the problems that remain to be solved.     

A genetically synthetic protein-based cationic polymer for siRNA delivery

In recent years, a large number of researchers have paid much attention on small interfering RNA (siRNA) after the advent of RNA interference technology, which has been harnessed as an efficient way of sequence-specific gene silencing in gene therapy, enables elucidation of gene functions, and the identification of new drug targets. Despite tremendous progress has been made in novel delivery systems and vectors via formulation of polyplexes and conjugations, such as cationic polymers (LPEI, BPEI), cationic liposome (DOTAP), peptides (CPP), unmet needs still exist. Many cationic agents used for condensing siRNA often exhibits severe cytotoxicity, which limits clinical applications, and is obliged to be handled. Thus great interest in searching for novel and sophisticated polymeric vectors has been spurred. Herein we proposed a genetically synthetic protein-based polymer, which is also referred to as elastin-like polypeptides (ELPs) excerpted from human tropoelastin highly repetitive sequence, Val-Pro-Gly-Xaa-Gly, where the "guest residue" Xaa is any amino acid except Pro. Thus, if we alternate the "guest residue" Xaa to Lys or Arg, to a significant extent, it can emerge as a powerful cationic polymer for siRNA delivery carrier, and hopefully it will be put into practice in the near future.     

Viral gene therapy strategies: from basic science to clinical application

A major impediment to the successful application of gene therapy for the treatment of a range of diseases is not a paucity of therapeutic genes, but the lack of an efficient non-toxic gene delivery system. Having evolved to deliver their genes to target cells, viruses are currently the most effective means of gene delivery and can be manipulated to express therapeutic genes or to replicate specifically in certain cells. Gene therapy is being developed for a range of diseases including inherited monogenic disorders and cardiovascular disease, but it is in the treatment of cancer that this approach has been most evident, resulting in the recent licensing of a gene therapy for the routine treatment of head and neck cancer in China. A variety of virus vectors have been employed to deliver genes to cells to provide either transient (eg adenovirus, vaccinia virus) or permanent (eg retrovirus, adeno-associated virus) transgene expression and each approach has its own advantages and disadvantages. Paramount is the safety of these virus vectors and a greater understanding of the virus-host interaction is key to optimizing the use of these vectors for routine clinical use. Recent developments in the modification of the virus coat allow more targeted approaches and herald the advent of systemic delivery of therapeutic viruses. In the context of cancer, the ability of attenuated viruses to replicate specifically in tumour cells has already yielded some impressive results in clinical trials and bodes well for the future of this approach, particularly when combined with more traditional anti-cancer therapies.