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.
RNA-DNA寡核苷酸(RDO)已被证明可以纠正或引起哺乳动物细胞外体细胞或基因组靶DNA的特定点突变。RDO的原始设计由双发夹帽双链组成，包括25个核苷酸长的DNA片段(含DNA链)与完全互补的2'- o -甲基RNA片段配对，五聚体DNA中断携带与靶DNA(含RNA链)不匹配的DNA片段。为了提高RDO的基因转换活性，合成了几种经过结构修饰和化学修饰的寡核苷酸，并比较了它们的基因校正活性。在此之前，我们利用哺乳动物核提取物建立了rdo介导的大肠杆菌β-半乳糖苷酶基因点突变(G→a)的基因校正体外系统。采用一种方便的细菌检测方法，对6种哺乳动物细胞类型与鸡DT40细胞系之间的转化频率进行了比较。该体外反应与DT40核提取物现用于研究RDO的结构和活性关系。对原始RDO设计的修改，包括将含有RNA的链与目标DNA的完整序列互补，用2'- o -甲基RNA取代中心的五个DNA残基，以及对发夹环的化学修饰，导致基因校正活性增加了10倍。此外，通过比较RNA或DNA链上携带与目标DNA不匹配的两个RDO，我们发现靶DNA中的单碱基校正优先由RDO的含DNA链驱动。因此，利用大肠杆菌β-半乳糖苷酶进行高灵敏度、便捷的检测，不仅可用于比较不同细胞类型间基因校正频率，还可用于优化RDO结构。
{"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":null,"pages":null},"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}
{"title":"RNA and gene repair/alteration: from inherited diseases to acquired disorders and tantalizing applications for non-disease conditions","authors":"R. Bertolotti","doi":"10.1163/156855800744566","DOIUrl":"https://doi.org/10.1163/156855800744566","url":null,"abstract":"","PeriodicalId":93646,"journal":{"name":"Gene therapy and regulation","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2000-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1163/156855800744566","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64793006","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}
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.
{"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":null,"pages":null},"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}
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.
{"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":null,"pages":null},"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}
{"title":"Transient or long-term transgene expression and gene repair/inactivation","authors":"R. Bertolotti","doi":"10.1163/156855800744502","DOIUrl":"https://doi.org/10.1163/156855800744502","url":null,"abstract":"","PeriodicalId":93646,"journal":{"name":"Gene therapy and regulation","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2000-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1163/156855800744502","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64792949","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}
A wide stream of in vivo studies have now confirmed the prediction that rAAV vectors have the primary requirements for effective gene transfer. AAV vectors can efficiently transduce both dividing and non-dividing cells, they are able to mediate long-term gene expression, and are showing no signs of cytotoxicity or immune response. In addition, recent advancements in the production and purification technologies of AAV vectors have lead to the ability to generate high titer clinical grade vector. This review will focus on studies which have fueled the emergence of AAV as an attractive vector for gene delivery. Discussion will center on the ability of AAV to mediate long-term transgene expression in vivo , the recent success of AAV vectors in animal models, and current clinical trials that are testing rAAV vectors for the treatment of cystic fibrosis.
{"title":"Adeno-associated viral vectors and successful gene therapy, the gap is closing","authors":"C. Summerford, J. Bartlett, R. Samulski","doi":"10.1163/156855800744511","DOIUrl":"https://doi.org/10.1163/156855800744511","url":null,"abstract":"A wide stream of in vivo studies have now confirmed the prediction that rAAV vectors have the primary requirements for effective gene transfer. AAV vectors can efficiently transduce both dividing and non-dividing cells, they are able to mediate long-term gene expression, and are showing no signs of cytotoxicity or immune response. In addition, recent advancements in the production and purification technologies of AAV vectors have lead to the ability to generate high titer clinical grade vector. This review will focus on studies which have fueled the emergence of AAV as an attractive vector for gene delivery. Discussion will center on the ability of AAV to mediate long-term transgene expression in vivo , the recent success of AAV vectors in animal models, and current clinical trials that are testing rAAV vectors for the treatment of cystic fibrosis.","PeriodicalId":93646,"journal":{"name":"Gene therapy and regulation","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2000-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1163/156855800744511","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64792529","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}
Ken Suzuki, H. Nakashima, Y. Sawa, R. Morishita, H. Matsuda, Y. Kaneda
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.
{"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":null,"pages":null},"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}
Gene transfer to the liver by receptor-mediated endocytosis represents a promising method for therapeutic intervention of genetic disorders and acquired diseases affecting the liver. Based on the observation that asialoglycoprotein receptors are abundantly expressed on the surface of human hepatocytes in vivo, gene delivery and expression by DNA-ligand complexes targeted to liver have been developed in several different experimental models. This review will cover general aspects related to targeting to the liver via the asialoglycoprotein receptor, and the relationship between gene expression, persistence, the structure and size of DNA complexes, as well as the current strategies aimed to improve the overall efficiency of receptor-mediated gene therapy.
{"title":"Liver-selective nucleic acid targeting using the asialoglycoprotein receptor","authors":"T. Fukuma, George Y Wu, Catherine H. Wu","doi":"10.1163/156855800744548","DOIUrl":"https://doi.org/10.1163/156855800744548","url":null,"abstract":"Gene transfer to the liver by receptor-mediated endocytosis represents a promising method for therapeutic intervention of genetic disorders and acquired diseases affecting the liver. Based on the observation that asialoglycoprotein receptors are abundantly expressed on the surface of human hepatocytes in vivo, gene delivery and expression by DNA-ligand complexes targeted to liver have been developed in several different experimental models. This review will cover general aspects related to targeting to the liver via the asialoglycoprotein receptor, and the relationship between gene expression, persistence, the structure and size of DNA complexes, as well as the current strategies aimed to improve the overall efficiency of receptor-mediated gene therapy.","PeriodicalId":93646,"journal":{"name":"Gene therapy and regulation","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2000-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1163/156855800744548","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"64793219","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}
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