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.

阳离子固体脂质纳米粒/pDNA二元复合物的初步研究

目的制备阳离子固体脂质纳米粒/pDNA二元复合物(SLNs-pDNA),并研究该复合物的药剂学和生物学特征。方法复乳法制备空白阳离子固体脂质纳米粒,与报告基因复合,加入不同浓度Ca^(2+)调节体系的荷正电量,分别对纳米粒的相关理化性质,细胞毒性,体外释药特性及其保护pDNA抵抗核酸酶降解的能力进行考察,通过体外基因转染实验对复合物在COS-7细胞中的转染效率进行了初步评价。结果本实验中制备的纳米粒呈球形或类球形,平均粒径(51.9±0.58)nm,Zeta电位(47.4±1.1)mV,加入Ca^(2+)显著提高了纳米粒复合pDNA的能力,制得的复合物平均粒径为(91.6±5.3)nm,Zeta电位为(31.5±1.4)mV。细胞毒性实验表明,纳米粒对COS-7细胞的毒性较小,凝胶阻滞分析表明,阳离子固体脂质纳米粒能够充分结合pDNA,形成稳定的复合物。pDNA保护性实验表明,该复合物对pDNA有很好的保护作用。体外释放实验结果表明,SLNs-pDNA具有缓释能力。体外基因转染实验表明,该复合物能够转染COS-7细胞,复合物所荷基因能够在该细胞中表达。结论阳离子固体脂质纳米粒/DNA复合物是一种制备工艺简单,复合物粒径较小,细胞毒性小,对pDNA保护作用强,所载DNA能够从复合物中缓慢释放,体外转染效率较高的具有一定开发前景的非病毒纳米基因载体。

农杆菌介导5种中草药叶片瞬时表达的研究

农杆菌介导的瞬时基因表达因其操作简单、可重复性高和实验成本低而成为研究基因功能、生产活性蛋白的有效方法。该研究以非病毒型(载体Ⅰ)和病毒型二元载体(载体Ⅱ)及两种农杆菌(EHA105、LBA4404)为介导,通过叶片渗透法在5种中草药(甘草、黄芩、黄芪、大黄、板蓝根)叶片中进行报告基因GFP的瞬时表达,并对影响表达的因素进行分析,以明确中药材中适宜瞬时表达技术应用的物种材料。结果显示:(1)农杆菌在5种中草药叶片中的瞬时转化效率受到载体、菌株和植物物种类型的影响。(2)在所选的5种中草药植物中,与无转化载体的菌株相比,当农杆菌重悬液OD600为0.8、叶片侵染5~7 d时,含非病毒型二元载体的EHA105菌株在甘草叶片中荧光强度最高,大黄次之;含病毒型二元载体的LBA4404菌株在黄芩叶片中GFP荧光表达量最高;含不同载体的不同菌液侵染的甘草、黄芪叶片仅有注射孔处检测出荧光或几乎无荧光蛋白表达,即未向四周扩散表达;在板蓝根叶片中检测出弱荧光或无荧光强度。研究表明,甘草和黄芩植物叶片可作为农杆菌介导基因瞬时表达的材料。

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.

CRISPR/Cas9 systems:Delivery technologies and biomedical applications

The emergence of the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)genome-editing system has brought about a significant revolution in the realm of managing human diseases,establishing animal models,and so on.To fully harness the potential of this potent gene-editing tool,ensuring efficient and secure delivery to the target site is paramount.Consequently,developing effective delivery methods for the CRISPR/Cas9 system has become a critical area of research.In this review,we present a comprehensive outline of delivery strategies and discuss their biomedical applications in the CRISPR/Cas9 system.We also provide an indepth analysis of physical,viral vector,and non-viral vector delivery strategies,including plasmid-,mRNA-and protein-based approach.In addition,we illustrate the biomedical applications of the CRISPR/Cas9 system.This review highlights the key factors affecting the delivery process and the current challenges facing the CRISPR/Cas9 system,while also delineating future directions and prospects that could inspire innovative delivery strategies.This review aims to provide new insights and ideas for advancing CRISPR/Cas9-based delivery strategies and to facilitate breakthroughs in biomedical research and therapeutic applications.

Clinical applications of the CRISPR/Cas9 genome-editing system: Delivery options and challenges in precision medicine

CRISPR/Cas9 is an effective gene editing tool with broad applications for the pre-vention or treatment of numerous diseases.It depends on CRiSPR(clustered regularly inter-spaced short palindromic repeats)as a bacterial immune system and plays as a gene editing tool.Due to the higher specificity and efficiency of CRISPR/Cas9 compared to other editing ap-proaches,it has been broadly investigated to treat numerous hereditary and acquired ill-nesses,including cancers,hemolytic diseases,immunodeficiency disorders,cardiovascular diseases,visual maladies,neurodegenerative conditions,and a few X-linked disorders.CRISPR/Cas9 system has been used to treat cancers through a variety of approaches,with sta-ble gene editing techniques.Here,the applications and clinical trials of CRisPR/Cas9 in various illnesses are described.Due to its high precision and efficiency,CRISPR/Cas9 strategies may treat gene-related illnesses by deleting,inserting,modifying,or blocking the expression of specific genes.The most challenging barrier to the in vivo use of CRISPR/Cas9 like off-target effects will be discussed.The use of transfection vehicles for CRISPR/Cas9,including viral vectors(such as an Adeno-associated virus(AAV),and the development of non-viral vectors is also considered.

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.

Gene therapy: light is finally in the tunnel

After two decades of ups and downs,gene therapy has recently achieved a milestone in treating patients with Leber’s congenital amaurosis(LCA).LCA is a group of inherited blinding diseases with retinal degeneration and severe vision loss in early infancy.Mutations in several genes,including RPE65,cause the disease.Using adenoassociated virus as a vector,three independent teams of investigators have recently shown that RPE65 can be delivered to retinal pigment epithelial cells of LCA patients by subretinal injections resulting in clinical benefits without side effects.However,considering the whole field of gene therapy,there are still major obstacles to clinical applications for other diseases.These obstacles include innate and immune barriers to vector delivery,toxicity of vectors and the lack of sustained therapeutic gene expression.Therefore,new strategies are needed to overcome these hurdles for achieving safe and effective gene therapy.In this article,we shall review the major advancements over the past two decades and,using lung gene therapy as an example,discuss the current obstacles and possible solutions to provide a roadmap for future gene therapy research.

Efficient drug and gene delivery to liver fibrosis:rationale, recent advances, and perspectives

Liver fibrosis results from chronic damages together with an accumulation of extracellular matrix,and no specific medical therapy is approved for that until now.Due to liver metabolic capacity for drugs,the fragility of drugs,and the presence of insurmountable physiological obstacles in the way of targeting,the development of efficient drug delivery systems for anti-fibrotics seems vital.We have explored articles with a different perspective on liver fibrosis over the two decades,then collected and summarized the information by providing corresponding in vitro and in vivo cases.We have discussed the mechanism of hepatic fibrogenesis with different ways of fibrosis induction in animals.Furthermore,the critical chemical and herbal anti-fibrotics,biological molecules such as micro-RNAs,siRNAs,and growth factors,which can affect cell division and differentiation,are mentioned.Likewise,drug and gene delivery and therapeutic systems on in vitro and in vivo models are summarized in the data tables.This review article enlightens recent advances in emerging drugs and nanocarriers and represents perspectives on targeting strategies employed in liver fibrosis treatment.

Gene delivery in peritoneal dialysis related peritoneal fibrosis research

Objective To summarize the development of gene delivery vectors in peritoneal fibrosis research and discuss the feasibility and superiority of lentiviral vectors. Data sources The data in this article were collected from PubMed database with relevant English articles published from 1995 to 2011. Study selection Articles regarding the gene therapy in peritoneal fibrosis research using non-viral vectors, adenoviral vectors, retroviral vectors, and lentiviral vectors were selected. Data were mainly extracted from 60 articles, which are listed in the reference section of this review. Results Non-viral vector-mediated gene delivery （including naked DNA for ex vivo, oligonucleotides, ultrasound- contrast agent mediated naked gene delivery, etc.） and viral vector-mediated gene delivery （including adenovirus, helper-dependant adenovirus, and retrovirus vectors） have been successfully applied both in the mechanistic investigation and the potential prevention and treatment of peritoneal fibrosis. Conclusions Peritoneal fibrosis is a major complication of peritoneal dialysis （PD）. Recently, the wide use of the gene delivery technique made it possible to access and further research peritoneal fibrosis. The use of lentiviral vector is expected to be widely used in PD research in the future due to its advantages in gene delivery.

Peptide-based DNA delivery system

Gene therapy is defined as the ability to change individual's genetics by correcting genetic mutations or making site-specific alterations that target therapeutic treatment.The focus of gene therapy is optimization of delivery vectors.Peptide-based DNA delivery vaccine is a field in non-viral gene delivery systems and not many researchers have looked after it.DNA vaccines have many appealing production potentials,including the ability to provide cross-protection against various virus subtypes,mass manufacturing in a reasonably short period,and greater stability than other delivery systems.Non-viral approaches are considered benign than viral delivery because of factors such as lack of immunogenicity,low toxicity,and the potential for tissue specificity.DNA vaccines have several limitations but,in this review,we focus on how a novel peptide R9-K-GALA can be used in DNA vaccine and overcome almost all the pitfalls.It is also expected that this delivery system can be used to protect against Malaria.R9-K-GALA is a novel peptide,and arginine(R)is used in this peptide because GALA cannot directly bind to DNA.So,Arginine will help to bind DNA as arginine being positively charged and DNA is negatively charged.This review mainly focuses on how peptides can be used to make DNA vaccines and how DNA vaccines in the coming future can be a breakthrough for many diseases.