Ex vivo non-viral vector-mediated neurotrophin-3 gene transfer to olfactory ensheathing glia: effects on axonal regeneration and functional recovery after implantation in rats with spinal cord injury

Objective Combine olfactory ensheathing glia （OEG） implantation with ex vivo non-viral vector-based neurotrophin- 3 （NT-3） gene therapy in attempting to enhance regeneration after thoracic spinal cord injury （SCI）. Methods Primary OEG were transfected with cationic liposome-mediated recombinant plasmid pcDNA3.1 （＋）-NT3 and subsequently implanted into adult Wistar rats directly after the thoracic spinal cord （T9） contusion by the New York University impactor. The animals in 3 different groups received 4x 1050EG transfected with pcDNA3.1 （＋）-NT3 or pcDNA3.1 （＋） plasmids, or the OEGs without any plasmid transfection, respectively; the fourth group was untreated group, in which no OEG was implanted. Results NT-3 production was seen increased both ex vivo and in vivo in pcDNA3.1 （＋）-NT3 transfected OEGs. Three months after implantation of NT-3-transfected OEGs, behavioral analysis revealed that the hindlimb function of SCI rats was improved. All spinal cords were filled with regenerated neurofilament-positive axons. Retrograde tracing revealed enhanced regenerative axonal sprouting. Conclusion Non-viral vector-mediated genetic engineering of OEG was safe and more effective in producing NT- 3 and promoting axonal outgrowth followed by enhancing SCI recovery in rats.

Surface Modification of Biomimetic PLGA-(ASP-PEG) Matrix with RGD-Containing Peptide:a New Non-Viral Vector for Gene Transfer and Tissue Engineering

RGD-containing peptide （ K16-GRGDSPC） , characterized as non-viral gene vectors, was fabricated to modify the surface of PLGA-[ASP- PEG] matrix, which offered the foundation for gene transfer with porous matrix of gene activated later. Peptide was synthesized and matrix was executed into chips A, B and chip C. Chip C was regarded as control. Chips A and B were reacted with cross-linker. Then chip A was reacted with peptide. MS and HPLC were ased to detect the .14W and purity of peptide. Sulphur, existing on the surface of biomaterials, was detected by XPS. The purity of un-reacted peptide in residual solution was detected by a spectrophotometer. HPLC shows that the peptide purity was 94%- 95% , and MS shows that the MW was 2 741. 3307. XPS reveals that the binding energy of sulphur was 164 eV and the ratio of carbon to sulphur （C/S） was 99. 746 ：0. 1014 in reacted chip A. The binding energy of sulphur in reacted chip B was 164 eV and 162 eV, C/ S was 99.574：0.4255, aM there was no sulphur in chip C. Peptide was manufactured and linked to the surface of biomimetic and 3-D matrix, which offered the possibilities for gene transfer and tissue engineering with this new kind of non-viral gene vector.

Non-viral targeted delivery system mediates transfection of thymidine kinase gene of herpes simplex virus into ovarian cancer cells:a comparison between one time and continuous mediation

Objective： To compare the transferring efficiency and killing effects of one time and continuous mediation with GE7, a non-viral targeted delivery system, in transfection of thymidine kinase gene of herpes simplex virus （HSV-tk） into ovarian cancer cells. Methods： GE7 was used to prepare recombinants with β-galactosidase （β-gal） and HSVI-tk; the recombinants were then used to transfect human ovarian cancer line CaOV3 once and continuously. β-gal staining was used to compare the efficiencies of one time and continuous mediation with GE7 system. Ganciclovior （GCV） was introduced into HSVI-tk transfected ovarian cells. Through drawing the cell growth curve and flow cytometry, the killing effects of GCV on once and continuously GE7/HSVI-tk transfected cells were observed. Results： We found that the one time and continuous exogenous gene transfer efficiencies were about 80% and 85%, respectively. When 1 μg/mL GCV was used to treat ovarian cell transfected with HSVI-tk gene, growth inhibiting rates of ovarian cells of one time and continuous transferring were 82% and 90%, respectively; their apoptosis indices were 15 and 30, respectively. Under same GCV concentration, continuous mediation of GE7/pCMV-tk transfection into ovarian cancer cells had more significant inhibitory effect than one time mediation （P 〈 0.05）. Conclusion： Compared with one time mediation, continuous mediation of transfection with GE7 gene delivery system has higher efficiency. Continuous mediation of GE7/HSVI-tk/GCV therapeutic gene system has more powerful killing effect.

Fusion Wheat Histone H4 Protein Increases Transfection Efficiency of Non-viral DNA Vector

The lack of efficient and non-toxic gene delivery, preferably with non-viral DNA vectors, is generally regarded as a major limitation for gene therapy. In this study, a wheat histone H4 gene was cloned from Triticum aestivum, sequenced, modified and expressed in E. coli. The wheat histone H4 gene and reconstructed H4TL gene encoded wheat histone H4 and a recombinant protein of 141 amino acids with an approximate molecular weight of 15500. Gel electrophoresis mobility shift assays demonstrated that the purified protein had high affinity for DNA. Most significantly, the complex of plasmid pEGFP/C1 with H4TL was transfected with increased efficiency into MCF-7, HO8910, LNCap, A549 and HeLa cells in vitro. These results demonstrate that the targeting of non-viral vectors to tumor-specific receptors provides a cheap, simple and highly efficient tool for gene delivery.

Non-viral gene coating modified IOL delivering PDGFR-αshRNA interferes with the fibrogenic process to prevent posterior capsular opacification

Posterior capsule opacification(PCO),the most common complication after cataract surgery,is caused by the proliferation,migration and epithelial-mesenchymal transition(EMT)of residual lens epithelial cells in the capsule bag.Although the surface modification and drug loading of intraocular lens(IOLs)have been effective in preventing PCO to some extent,the intraocular safety of anti-proliferative drug application is still a major limitation in clinical application.In this study,we used non-viral gene delivery systems in combination with layer-by-layer(LBL)self-assembly technology,and the modified IOL could effectively prevent the development of PCO by interfering with the EMT process mediated by the platelet-derived growth factor receptor-α(PDGFR-α).Herein,the gene fragments were wrapped by electrostatic conjugation using polyethyleneimine-graft-poly(ethylene glycol)to form gene complexes.Gene complexes were characterized by dynamic light scattering,transmission electron microscopy(TEM)and agarose gel electrophoresis,and evaluated for storage and serum stability.The layer assembly behavior of the IOL surface,changes in optical properties and the release behavior of the gene complexes were characterized using quartz crystal microbalance,UV-vis,contact angle and TEM.In vitro experiments showed that the IOL coating has good bio-compatibility and can achieve the corresponding transfection effect,and the released gene complexes exhibited excellent cell internalization and lysosomal escape behaviors,as well as effective inhibition of PDGFR-αexpression and its mediated EMT process.The early PCO prevention effect and bio-compatibility evaluation of the modified IOL in vivo were evaluated by implantation into animal eyes.This study provides a new strategy for the development of surface modifications of small nucleic acid drugs and non-toxic EMT interference therapies for PCO.

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.

Liver-targeted hydrodynamic gene therapy: Recent advances in the technique

One of the major research focuses in the field of gene therapy is the development of clinically applicable, safe, and effective gene-delivery methods. Since the first case of human gene therapy was performed in 1990, a number of gene-delivery methods have been developed, evaluated for efficacy and safety, and modified for human application. To date, viral-vectormediated deliveries have shown effective therapeutic results. However, the risk of lethal immune response and carcinogenesis have been reported, and it is still controversial to be applied as a standard therapeutic option. On the other hand, delivery methods for nonviral vector systems have been developed, extensively studied, and utilized in in vivo gene-transfer studies. Compared to viral-vector mediated gene transfer, nonviral systems have less risk of biological reactions. However, the lower gene-transfer efficiency was a critical hurdle for applying them to human gene therapy. Among a number of nonviral vector systems, our studies focus on hydrodynamic gene delivery to utilize physical force to deliver naked DNA into the cells in the living animals. This method achieves a high gene-transfer level by DNA solution injections into the tail vein of rodents, especially in the liver. With the development of genome editing methods, in vivo gene-transfer therapy using this method is currently the focus in this research field. This review explains the method principle, efficiency, safety, and procedural modifications to achieve a high level of reproducibility in large-animal models.

Synthesis of N-methylene phosphonic chitosan(NMPCS)and its potential as gene carder

N-Methylene phosphonic chitosan （NMPCS）, an amphiphilic macromolecule with powerful chelating ability of Ca^2＋ ions, was synthesized and characterized. The physicochernical properties of NMPCS and the interactions between NMPCS and plasmid DNA were investigated by FTIR, ^13C NMR, X-ray, agarose gel electrophoresis retardation assay, atomic force microscopy （AFM） and circular dichroism （CD）. The results suggest that at charge ratio 2：1 or above, DNA could be completely entrapped and spherical complexes with mean size of 80-210 nm were formed. Taking HeLa as host cell, luciferase expression mediated by NMPCS improved about 100 times compared to the expression mediated by chitosan.

Synthesis and Evaluation of Novel Chitosan Derivatives for Gene Delivery

A series of novel water soluble chitosan derivatives as gene vectors was synthesized. The delivery systems were tested for their ability to form complexes with plasmid DNA by utilizing agarose gel electrophoresis, particle size analysis, zeta potential measurement and scanning electron microscopy. Furthermore, cytotoxicity of chitosan derivatives and transfection efficiency of polyplexes were evaluated in vitro. The experimental results showed that the novel chitosan derivatives had lower cytotoxicity, good DNA condensation, and higher transfection efficiencies compared to chitosan in both 293T and HeLa cell lines. It was indicated that these chitosan derivatives were promising candidates for non-viral gene vectors.

Effects of lipid shell microbubble on ultrasound mediated EGFP gene delivery to transplanted tumors:initial experience

Objective： To investigate the feasibility of ultrasound （US） mediated enhanced green fluorescent protein （EGFP） gene delivery in subcutaneous transplanted tumors of human cervical carcinoma （He/a） and the contribution of lipid shell microbubble （LSMB） on gene transfection. Methods： LSMB and plasmid were injected into nude mice by tail vein followed local US irradiation （P ＋ LSMB ＋ US group）. US exposure parameter was set at 2.0 W/cm2, 2 rain, duty cycle 20%. EGFP expression was evaluated by imaging for 7 days. Nude mice undergoing plasmid injection alone （P group）, plasmid injection and US exposure （P ＋ US group）, plasmid and LSMB injection （P ＋ LSMB group） were used as controls. Frozen section and histological examinations were conducted. Expression of EGFP was scored. Kinetics of protein expression post transfection and localization in vivo were evaluated. Results： Plasmid injection with LSMB plus US exposure strongly increased gene transfer efficiency. Strong EGFP expression was mainly seen in LSMB ＋ P ＋ US group. It was significantly higher than any of the following groups, P group, US ＋ P group, or LSMB ＋ P group （P 〈 0.01）./n vivo expression level of post-US 3 days was significantly higher than any other time points （P 〈 0.01）. There was not significant expression level of EGFP in other organs or tissues regardless of US exposure. No tissue damage was seen histologically. Conclusion： The combination of LSMB and US exposure could effectively transfer plasmid DNA to transplanted tumors without causing any apparently adverse effect. LSMB could be effective as a non-viral vector system in in vivo gene delivery. It would be a safe gene delivery method and provide an alternative to current clinical gene therapy.

Use of PEI-coated Magnetic Iron Oxide Nanoparticles as Gene Vectors

Summary: To evaluate the feasibility of using polyethyleneimine (PEI) coated magnetic iron oxide nanoparticles (polyMAG-1000) as gene vectors. The surface characteristics of the nanoparticles were observed with scanning electron microscopy. The ability of the nanoparticles to combine with and protect DNA was investigated at different PH values after polyMAG-1000 and DNA were combined in different ratios. The nanoparticles were tested as gene vectors with in vitro transfection models. Under the scanning electron microscope the nanoparticles were about 100 nm in diameter. The nanoparticles could bind and condense DNA under acid, neutral and alkaline conditions, and they could transfer genes into cells and express green fluorescent proteins (GFP). The transfection efficiency was highest (51 %) when the ratio of nanoparticles to DNA was 1:1 (v:w). In that ratio, the difference in transfection efficiency was marked depending on whether a magnetic field was present or not: about 10 % when it was absent but 51 % when it was present. The magnetic iron oxide nanoparticles coated with PEI may potentially be used as gene vectors.

A RGD-Containing Oligopeptide (K)_(16)GRGBSPC: A Novel Vector for Integrin-Mediated Targeted Gene Belivery

A 23 amino acid, bifunctional integrin-targeted synthetic oligopeptide was evaluated for ex vivo gene delivery to rabbit bone marrow stromal cells （BMSCs）. Synthesis of the peptide （K）16GRGDSPC was performed on a solid-phase batch peptide synthesizer. BMSCs were transfected with plasmid DNA coding for luciferase by （K）j6GRGDSPC and the transfection efficiency was assayed. The influences of chloroquine and polyethyleneimine on the transfection efficiency were also examined. The target specificity of （K）16GRGDSPC to mediate exogenous gene into BMSCs was analyzed using cell attachment test and gene delivery inhibition test. The results showed that the transfection efficiency of the oligopeptide vector was lower than that of Lipofectamine. But in the presence of endosomal buffer chloroquine or endosomal disrupting agent polyethyleneimine, the transfection efficiency of the vector was greatly enhanced. In addition, RGD-containing peptides inhibited BMSCs＇ attachment to the 96-well plates pretreated with fibronectin or vitronecfin and significantly decreased the transfection efficiency of the oligopeptide vector. These studies demonstrated that oligopeptide （K）16GRGDSPC was an ideal novel targeted non-viral gene delivery vector, which was easy to be synthesized, high efficient and low cytotoxicity. The vector could effectively deliver exogenous gene into rat BMSCs.

Vectors for gene therapy:A place for DNA transposon

Gene therapy offers important perspectives in current and future medicine but suffers from imperfect vectors for the delivery of the therapeutic gene. Most preclinical and clinical trials have been based on the use of viral vectors, which have evident advantages but also some serious disadvantages. In the past decade the use of DNA transposon-based systems for gene delivery has emerged as a non-viral alternative. DNA transposon vector engineering remains largely in a preclinical phase but some interesting results have been obtained. This mini-review aims to provide the current state of the art on DNA transposon vectors used in a gene therapy perspective.

When mRNA meets gene editing

The critical challenge of gene therapy lies in delivering gene editing agents.Compared with DNA,while RNA is less stable and more accessible to degrade,it comes with the benefit of lower off-target effects since permanent insertion is not involved.This review focuses on mRNA-based delivery of gene editing agents,highlighting novel mRNA delivery systems.To provide context,a comparison is made between three main gene editing agents:programmable nucleases,base editors,and prime editors.The potential of Cas\pi and transposons is also discussed in this review.Additionally,a summary of four main barriers to mRNAbased in vivo delivery is provided.Furthermore,this review detailedly introduced different delivery systems,both viral(lentivirus)and non-viral vectors(genome editing via oviductal nucleic acids delivery,lipid nanoparticles,polymer-based nanoparticles,viruslike-particles,extracellular vesicles,and migrasome).Each delivery strategy is assessed by comparing its advantages and disadvantages to offer a comprehensive and objective overview of the delivery system.Moreover,we emphasized the vital role of the protein corona as a critical regulator for nanodelivery.Ultimately,we concluded the challenges of mRNA-based gene editing strategies(RNA stability,targeting,potential immunogenicity,cytotoxicity,heterogeneity,and rational design).The purpose of this review is to guide further research and provide a comprehensive analysis of mRNA-based in vivo delivery of gene editing agents in this promising field.

载鱼精蛋白-pDNA复合物固体脂质纳米粒的初步研究

目的制备非病毒基因载体载鱼精蛋白-pDNA复合物的固体脂质纳米粒(PD-SLN),PD-SLN由多聚阳离子缩合DNA内核与一个脂质外壳组成,从而构成多功能信封式纳米载体(multifunctional envelope-type nano device,MEND)结构,研究其特征,对DNA的保护作用以及DNA的体外释放性质。方法分别采用溶剂扩散法和复乳法制备纳米粒;用透射电镜观察形态;用Zeta电位测定仪测定粒径、多分散指数和Zeta电位;用荧光分光光度法测定基因包封率;分别用琼脂糖凝胶电泳观察复合物及PD-SLN保护pDNA抵抗剧烈外力和核酸酶的降解情况;采用双室扩散法对PD-SLN进行体外释放研究。结果用2种方法制备的SLN呈球形和类球形,平均粒径分别为(231±13.7)和(627±22.9)nm,Zeta电位分别为(-17.8±3.2)和(-25.2±2.7)mV,包封率分别为(41.5±3.62)%和(56.5±5.28)%。pDNA保护性试验表明,PD-SLN对pDNA有保护作用。体外释放实验结果表明PD-SLN缓释能力强。结论PD-SLN是一种制备工艺简单,体外缓释能力好,对pDNA保护性强,具有一定应用前景的非病毒基因载体。

用于肿瘤治疗的小分子干扰RNA非病毒载体研究进展

近年来,小分子干扰RNA(siRNA)作为RNA干扰(RNAi)技术的效应分子,已被广泛用于恶性肿瘤的基因治疗领域。欲获得理想的治疗效果,其关键因素是寻找一种安全、高效、稳定、可控的基因载体。非病毒载体具有低毒、低免疫原性、制备简单、目的基因容量大、外源基因随机整合率低且携带基因大小类型不受限制等突出优势,已经成为目前siRNA载体的研究热点。在以往学者的研究基础上,从药剂学的角度,笔者对这些载体在siRNA传递系统中的研究现况做回顾性总结。

Non-viral and viral delivery systems for CRISPR-Cas9 technology in the biomedical field

The clustered regularly interspaced short palindromic repeats(CRISPR)-associated protein 9(CRISPR-Cas9) system provides a novel genome editing technology that can precisely target a genomic site to disrupt or repair a specific gene. Some CRISPR-Cas9 systems from different bacteria or artificial variants have been discovered or constructed by biologists, and Cas9 nucleases and single guide RNAs(sgRNA) are the major components of the CRISPR-Cas9 system. These Cas9 systems have been extensively applied for identifying therapeutic targets, identifying gene functions, generating animal models, and developing gene therapies.Moreover, CRISPR-Cas9 systems have been used to partially or completely alleviate disease symptoms by mutating or correcting related genes. However, the efficient transfer of CRISPR-Cas9 system into cells and target organs remains a challenge that affects the robust and precise genome editing activity. The current review focuses on delivery systems for Cas9 mRNA, Cas9 protein, or vectors encoding the Cas9 gene and corresponding sgRNA. Non-viral delivery of Cas9 appears to help Cas9 maintain its on-target effect and reduce off-target effects, and viral vectors for sgRNA and donor template can improve the efficacy of genome editing and homology-directed repair. Safe, efficient, and producible delivery systems will promote the application of CRISPR-Cas9 technology in human gene therapy.

Design, mechanism, delivery and therapeutics of canonical and Dicer-substrate siRNA

Upon the discovery of RNA interference(RNAi),canonical small interfering RNA(si RNA) has been recognized to trigger sequence-specific gene silencing. Despite the benefits of si RNAs as potential new drugs,there are obstacles still to be overcome,including off-target effects and immune stimulation. More recently,Dicer substrate si RNA(Dsi RNA) has been introduced as an alternative to si RNA. Similarly,it also is proving to be potent and target-specific,while rendering less immune stimulation. Dsi RNA is 25–30 nucleotides in length,and is further cleaved and processed by the Dicer enzyme. As with si RNA,it is crucial to design and develop a stable,safe,and efficient system for the delivery of Dsi RNA into the cytoplasm of targeted cells. Several polymeric nanoparticle systems have been well established to load Dsi RNA for in vitro and in vivo delivery,thereby overcoming a major hurdle in the therapeutic uses of Dsi RNA. The present review focuses on a comparison of si RNA and Dsi RNA on the basis of their design,mechanism,in vitro and in vivo delivery,and therapeutics.

Delivery strategies for CRISPR/Cas genome editing tool for retinal dystrophies:challenges and opportunities

CRISPR/Cas,an adaptive immune system in bacteria,has been adopted as an efficient and precise tool for site-specific gene editing with potential therapeutic opportunities.It has been explored for a variety of applications,including gene modulation,epigenome editing,diagnosis,mRNA editing,etc.It has found applications in retinal dystrophic conditions including progressive cone and cone-rod dystrophies,congenital stationary night blindness,X-linked juvenile retinoschisis,retinitis pigmentosa,age-related macular degeneration,leber’s congenital amaurosis,etc.Most of the therapies for retinal dystrophic conditions work by regressing symptoms instead of reversing the genemutations.CRISPR/Cas9 through indel could impart beneficial effects in the reversal of gene mutations in dystrophic conditions.Recent research has also consolidated on the approaches of using CRISPR systems for retinal dystrophies but their delivery to the posterior part of the eye is a major concern due to high molecular weight,negative charge,and in vivo stability of CRISPR components.Recently,non-viral vectors have gained interest due to their potential in tissue-specific nucleic acid(miRNA/siRNA/CRISPR)delivery.This review highlights the opportunities of retinal dystrophies management using CRISPR/Cas nanomedicine.

Synthesis, Biophysical Characterization and <i>in Vitro</i>Transfection Activity of Novel Bivalent Amine Cationic Lipids in the Absence of Dioleoylphosphatidylethanolamine (DOPE)

In this paper, a novel series of bis [(aminoethyl)]-amine cationic lipid derivatives have been synthesized and identified to purity by NMR and Elemental analysis. B16-F0 cells were transfected with cationic lipid/pEGFP-N1 and cationic lipid/&#223-gal lipoplexes complexed at +/&#45 charge ratios of 1:1, 2:1, and 4:1. Dimyristoyl derivative showed highest activity at charge ratio 2:1 and both dimyristoyl and dioleoyl derivatives showed similar &#223-gal activity at charge ratios 4:1. In 40 mM tris buffer pH 7.2 the dioleoyl derivative was able to fully complex with and retard pDNA at charge ratios above 2:1. None of the other lipid derivatives, dilauroyl, dimyristoyl, dipalmitoyl and distearoyl were able to fully neutralize the plasmid DNA at charge ratios similar to those used in the transfection experiment. The gel-to-liquid phase transition temperatures for dimyristoyl, dipalmitoyl and distearoyl were determined by a fluorescence anisotropy method to be 27.5&degC, 32.5&degC and 39&degC, respectively. A gel-to-liquid crystalline phase transition temperature below 37&degC, appears to be the crucial property that cationic lipids have to possess in order to mediate high levels of in vitro transfection activity in the absence of other helper lipids.