Gene therapy and regulation最新文献_第8页

Improvement of RNA-DNA oligonucleotide design by using mammalian nuclear extracts 利用哺乳动物核提取物改进RNA-DNA寡核苷酸设计

Gene therapy and regulation Pub Date : 2000-08-15 DOI: 10.1163/156855800744593

O. Igoucheva, K. Yoon

{"title":"Improvement of RNA-DNA oligonucleotide design by using mammalian nuclear extracts","authors":"O. Igoucheva, K. Yoon","doi":"10.1163/156855800744593","DOIUrl":"https://doi.org/10.1163/156855800744593","url":null,"abstract":"An RNA-DNA oligonucleotide (RDO) has been shown to either correct or cause a specific point mutation in episomal or genomic target DNA in mammalian cells. The original design of the RDO consists of a double-hairpin capped duplex comprising a 25 nucleotide-long DNA stretch (DNA-containing strand) paired to a fully complementary 2'-O-methyl RNA stretch with a pentameric DNA interruption that carries a mismatch to target DNA (RNA-containing strand). In order to improve the gene conversion activity of the RDO, several oligonucleotides with structural and chemical modifications were synthesized and compared in their gene correction activity. Previously, we established an in vitro system capable of RDO-mediated gene correction of a point mutation (G → A) in the E. coli β-galactosidase gene by using mammalian nuclear extracts. Conversion frequencies among six mammalian cell types and the chicken DT40 cell line were compared by using a convenient bacterial assay that score blue or white colonies. This in vitro reaction with DT40 nuclear extract is now used to study the structure and activity relationship of the RDO. Modifications of the original RDO design including a complete sequence complementarity of the RNA-containing strand to target DNA, a replacement of the central five DNA residues with 2'-O-methyl RNA, and chemical modification of the hairpin loops result in a ten-fold increase in gene correction activity. Moreover, we show that the single-base correction in the target DNA is preferentially driven by the DNA-containing strand of the RDO by comparing two RDOs that carry a mismatch to target DNA either on the RNA- or DNA-containing strand. Thus, the highly sensitive and convenient assay utilizing E. coli β-galactosidase is not only useful to compare the gene correction frequency among different cell types but also to optimize the RDO structure.","PeriodicalId":93646,"journal":{"name":"Gene therapy and regulation","volume":"1 1","pages":"165-175"},"PeriodicalIF":0.0,"publicationDate":"2000-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1163/156855800744593","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64793730","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 9

RNA and gene repair/alteration: from inherited diseases to acquired disorders and tantalizing applications for non-disease conditions RNA和基因修复/改变:从遗传性疾病到获得性疾病和非疾病条件的诱人应用

Gene therapy and regulation Pub Date : 2000-08-15 DOI: 10.1163/156855800744566

R. Bertolotti

引用次数: 2

Alphavirus vectors: from protein production to gene therapy 甲病毒载体:从蛋白质生产到基因治疗

Gene therapy and regulation Pub Date : 2000-03-07 DOI: 10.1163/156855800744520

C. Smerdou, P. Liljeström

{"title":"Alphavirus vectors: from protein production to gene therapy","authors":"C. Smerdou, P. Liljeström","doi":"10.1163/156855800744520","DOIUrl":"https://doi.org/10.1163/156855800744520","url":null,"abstract":"Alphaviruses are enveloped viruses containing a single positive strand RNA molecule as genome. Several vectors derived from alphaviruses have been developed, which include Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan Equine Encephalitis virus (VEE). Alphavirus self-replicating RNA containing heterologous genes can be synthesized in vitro from plasmids having the recombinant alphavirus replicon sequences under the control of a prokaryotic promoter, such as SP6 or T7. High level expression of the heterologous proteins/RNA is obtained in cells transfected with this RNA. Although the system can be used for gene delivery directly as naked RNA, several packaging systems have been developed which allow the encapsidation of the alphaviral recombinant RNA into suicidal viral particles, increasing the efficiency of delivery of the recombinant genome into cells. A more recent variant of the system is based on a DNA/RNA layered alphaviral vector in which the recombinant replicon is transcribed from an RNA polymerase-II promoter, allowing direct delivery of DNA into cells. Alphavirus vectors have been used to express a great number of proteins with many different purposes, including protein production and characterization, functional studies, vaccination, and gene therapy. Both the recombinant alphavirus particles and the alphavirus nucleic acid vectors have shown to be able to induce protective immune responses in animal models. The possible application of these vectors in gene therapy faces, however, two limitations, which are the lack of specific targeting and the transient nature of the vector, due to the induction of apoptosis by the alphavirus replicon. Several strategies have been recently described to improve the targeting of alphavirus vectors and to develop noncytopathic vectors with potential use in gene therapy.","PeriodicalId":93646,"journal":{"name":"Gene therapy and regulation","volume":"1 1","pages":"33-63"},"PeriodicalIF":0.0,"publicationDate":"2000-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1163/156855800744520","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64792967","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 9

Polycation/DNA complexes for in vivo gene delivery 体内基因传递的多阳离子/DNA复合物

Gene therapy and regulation Pub Date : 2000-03-07 DOI: 10.1163/156855800744557

R. Kircheis, E. Wagner

{"title":"Polycation/DNA complexes for in vivo gene delivery","authors":"R. Kircheis, E. Wagner","doi":"10.1163/156855800744557","DOIUrl":"https://doi.org/10.1163/156855800744557","url":null,"abstract":"A major aim of in vivo gene transfer strategies is the efficient and specific delivery of therapeutic genes into the desired target tissues. Non-viral vectors, based on naked plasmid DNA or DNA complexes with cationic lipids or polycationic polymers, are attractive gene delivery vehicles because of their ease of manipulation, stability, low cost, safety, and their high flexibility concerning the size of the delivered transgene. A variety of non-viral vectors effective for gene transfer in cell culture have been developed. However, efficient and target specific in vivo gene delivery remains a major challenge. Compared to cell culture application, in vivo gene delivery faces a variety of additional obstacles including anatomical size constraints, and an environment of interactions with biological fluids and extracellular matrix. Furthermore, unspecific interactions with non-target cells can be a major obstacle to targeted gene delivery in vivo . Physical parameters of the transfection complexes, such as particle size, charge and stability, are critical factors determining circulation time, biodistribution and transfection efficacy in vivo . Transfection complexes have to be small enough to pass physiological barriers, inert against unspecific interactions with blood components and non-target cells, but allow specific binding to the target cells. After uptake into the target cell, escape from the endosomal compartment and nuclear uptake of the DNA are critical steps for efficient transfection. The present review focuses on the use of various polycation/DNA-based transfection systems for in vivo gene delivery.","PeriodicalId":93646,"journal":{"name":"Gene therapy and regulation","volume":"1 1","pages":"95-114"},"PeriodicalIF":0.0,"publicationDate":"2000-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1163/156855800744557","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64793314","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 30

Transient or long-term transgene expression and gene repair/inactivation 瞬时或长期转基因表达和基因修复/失活

Gene therapy and regulation Pub Date : 2000-03-07 DOI: 10.1163/156855800744502

R. Bertolotti

引用次数: 4

Adeno-associated viral vectors and successful gene therapy, the gap is closing 腺相关病毒载体和成功的基因治疗，差距正在缩小

Gene therapy and regulation Pub Date : 2000-03-07 DOI: 10.1163/156855800744511

C. Summerford, J. Bartlett, R. Samulski

引用次数: 2

Reconstituted fusion liposomes for gene transfer in vitro and in vivo 体外和体内基因转移的重组融合脂质体

Gene therapy and regulation Pub Date : 2000-03-07 DOI: 10.1163/156855800744539

Ken Suzuki, H. Nakashima, Y. Sawa, R. Morishita, H. Matsuda, Y. Kaneda

{"title":"Reconstituted fusion liposomes for gene transfer in vitro and in vivo","authors":"Ken Suzuki, H. Nakashima, Y. Sawa, R. Morishita, H. Matsuda, Y. Kaneda","doi":"10.1163/156855800744539","DOIUrl":"https://doi.org/10.1163/156855800744539","url":null,"abstract":"Using UV-inactivated whole HVJ (Hemagglutinating Virus of Japan; Sendai virus), we have developed HVJ-liposomes that are efficient in vitro and in vivo gene delivery vehicles based on cell fusion properties of the Sendai virus. However, all the proteins and the genome of HVJ remain within the HVJ-liposomes, although replication of the viral genome is severely impaired by prior UV-irradiation of HVJ particles. To construct more complete synthetic vehicles, we developed reconstituted fusion liposomes. Fusion proteins F1 and HN of HVJ were extracted by mild lysis of the viral particles and purified by ion-exchange column chromatography. Purified viral fusion proteins were inserted into liposome membranes by detergent-solubilization and dialysis to construct the reconstituted fusion particles. These particles retained fusion activity during more than 4 weeks. DNA-loaded liposomes, which were prepared by vortexing-sonication, were fused with the reconstituted fusion particles to deliver DNA to cells. Using the reconstituted vehicle, fluorescent isothiocyanate (FITC)-labeled oligonucleotides were introduced into 100% of the nuclei of target human amniotic FL cells. In addition, luciferase gene expression upon transfection of human 293 cells with reconstituted fusion liposomes was almost the same as with standard HVJ-liposomes. On the other hand, the LacZ gene was introduced into mouse skeletal muscle by the new vector, and 40 to 50% of the muscle fibers showed LacZ gene expression.","PeriodicalId":93646,"journal":{"name":"Gene therapy and regulation","volume":"1 1","pages":"65-77"},"PeriodicalIF":0.0,"publicationDate":"2000-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1163/156855800744539","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64793162","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 11

Liver-selective nucleic acid targeting using the asialoglycoprotein receptor 利用asialal糖蛋白受体的肝脏选择性核酸靶向

Gene therapy and regulation Pub Date : 2000-03-07 DOI: 10.1163/156855800744548

T. Fukuma, George Y Wu, Catherine H. Wu

引用次数: 4