RESUMO
Many applications for human gene therapy would be facilitated by high levels and long duration of physiologic gene expression. Adenoviral vectors are frequently used for gene transfer because of their high cellular transduction efficiency in vitro and in vivo. Expression of viral proteins and the low capacity for foreign DNA limits the clinical application of first- and second-generation adenoviral vectors. Adenoviral vectors with all viral coding sequences deleted offer the prospect of decreased host immune responses to viral proteins, decreased cellular toxicity of viral proteins and increased capacity to accommodate large regulatory DNA regions. Currently most vectors used in vivo for preclinical and clinical studies express cDNAs under the control of heterologous eukaryotic or viral promoters. Using an adenoviral vector with all viral coding sequences deleted and containing the complete human alpha1-antitrypsin (PI) locus, we observed tissue-specific transcriptional regulation in cell culture and in vivo; intravenous injection in mice resulted in high levels of very stable expression for more than ten months and decreased acute and chronic toxicity. These results indicate significant advantages of regulated gene expression using genomic DNA for gene transfer and of adenoviral gene transfer vectors devoid of all viral coding sequences.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Proteínas de Homeodomínio , Transfecção/métodos , Adenoviridae , Animais , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Genes RAG-1 , Terapia Genética/métodos , Vetores Genéticos , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Transcrição Gênica , alfa 1-Antitripsina/biossíntese , alfa 1-Antitripsina/genéticaRESUMO
Primary human cells are relatively refractory to transformation by adenoviral E1 functions. For almost two decades, human embryonic kidney (HEK)-derived 293 cells have been the only E1-complementing cell line suitable for production of E1-deleted adenoviral vectors. More recently, new vector production cell lines have been derived from human embryonic retina (HER) cells, a cell type that is difficult to obtain. We were surprised to find that readily available primary human amniocytes are efficiently transformed by adenoviral E1 functions. We selected cell lines that allow high-titer production of recombinant adenoviral vectors. The generation of replication-competent adenovirus (RCA) during production, caused by homologous recombination between vector and cellular DNA, was excluded by designing the transforming plasmid to lack sequence overlap with current adenoviral vectors. In addition, we generated an infectious plasmid that can be used for convenient generation of first-generation adenoviral vectors in Escherichia coli and that matches the E1 complementation in the new production cell lines.
Assuntos
Adenoviridae/genética , Proteínas E1 de Adenovirus/genética , Líquido Amniótico/citologia , Terapia Genética/métodos , Vetores Genéticos , Transformação Genética , Western Blotting , Linhagem Celular , Clonagem Molecular , Humanos , Modelos Genéticos , Plasmídeos/metabolismo , Recombinação Genética , Fatores de Tempo , TransfecçãoRESUMO
High-capacity adenoviral (HC-Ad) vectors contain only the noncoding termini of the viral genome, can deliver large DNA fragments of up to 36 kb into target cells, and feature reduced toxicity and prolonged transgene expression in vivo. To enhance the potential of HC-Ad vectors to transduce specific cell types, we constructed a versatile infectious new helper virus plasmid that can be used readily to introduce peptide ligands into the HI loop of the fiber knob domain of Ad5-based HC-Ad vectors. Helper viruses with a 6x-His epitope or Arg-Gly-Asp (RGD) peptide insertion retained the full infectivity of the wild-type helper virus. The RGD-modified helper virus was used for production of a capsid-modified HC-Ad vector expressing beta-galactosidase. The RGD HC-Ad vector transduced the ovarian carcinoma cell lines SK-OV-3 and OVCAR-3 with 4- to 20-fold higher efficiency, compared to unmodified vectors. Transduction of both primary vascular smooth muscle cells as well as primary human endothelial cells was increased up to 15-fold with the RGD-modified vector. Competition experiments with recombinant knob protein and different RGD peptides indicated that the RGD-mediated transduction was Coxsackie and Adenovirus receptor (CAR)-independent and involved integrin alpha(v)beta(5). The use of fiber-modified helper viruses in the last amplification step of HC-Ad vector production allows for convenient and efficient targeting of these vectors towards different cell types. Targeting strategies will increase the spectrum of applications for HC-Ad vectors and will further add to their safety.
Assuntos
Adenoviridae/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Genoma Viral , Ligação Competitiva , Western Blotting , Células Cultivadas , Clonagem Molecular , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Endotélio/citologia , Epitopos , Vírus Auxiliares/genética , Humanos , Ligantes , Modelos Genéticos , Músculo Liso/citologia , Oligopeptídeos/genética , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas de Protozoários , Receptores Virais/metabolismo , Transdução Genética , Células Tumorais Cultivadas , beta-Galactosidase/metabolismoRESUMO
The utility of first-generation adenovirus vectors for long-term gene transfer in humans is limited by preexisting antiadenoviral immunity. We demonstrate here that new-generation high-capacity adenovirus vectors (HC-Ads) can efficiently transduce the brain and mediate stable transgene expression for at least 2 months, even in the presence of a preexisting antiadenoviral immune response. First-generation vector-mediated transduction was almost completely abolished in preimmunized animals within 60 days of the vector injection. Levels of HC-Ad-mediated transduction by 3 days postinjection were not significantly affected by preimmunization, were reduced within 14 days to 56% of those levels seen in nonimmunized animals, and remained stable until day 60 postinjection. Acute brain inflammation elicited by the HC-Ad vector injection was more transient, and was reduced in intensity compared with brain inflammation elicited by the first-generation vector injection in immunized animals. Inflammation was significantly higher in all immunized animals than in nonimmunized animals. Our results show that preexisting antiadenoviral immunity does not significantly reduce initial HC-Ad-mediated infection of the brain and is not a barrier to stable HC-Ad vector-mediated transduction of the CNS. Although input HC-Ad capsid proteins injected into the brain may contain transient targets for a brain-infiltrating cellular adenovirus-specific immune response, this fails to eliminate transgene expression. Thus HC-Ads show promise for gene therapy of chronic brain disease.
Assuntos
Adenoviridae/genética , Adenoviridae/imunologia , Encéfalo/metabolismo , Vetores Genéticos/genética , Transgenes/genética , Animais , Encéfalo/imunologia , Encéfalo/patologia , Expressão Gênica , Terapia Genética/métodos , Soros Imunes/imunologia , Imunização , Imuno-Histoquímica , Inflamação/imunologia , Inflamação/patologia , Testes de Neutralização , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Transdução Genética , Transgenes/imunologiaRESUMO
Optimal gene therapy for many disorders will require efficient transfer to cells in vivo, high-level and long-term expression, and tissue-specific regulation, all in the absence of significant toxicity or inflammatory responses. While recombinant adenoviral vectors are efficient for gene transfer to hepatocytes, their usefulness is limited by short duration of expression related, at least in part, to immune responses to viral proteins and by a low capacity for foreign DNA. A number of systems have been developed for producing adenoviral vectors devoid of all viral coding sequences. Using AdSTK109, a vector lacking all viral coding sequences and carrying the complete human alpha1-antitrypsin (hAAT) genomic DNA locus, we have demonstrated sustained expression for longer than 10 months in mice. Utilizing high doses of this vector for hepatic gene transfer in mice, we find that supraphysiological levels of hAAT can be achieved without hepatotoxicity.
Assuntos
Adenoviridae/genética , Vetores Genéticos/genética , Vírus Auxiliares/genética , alfa 1-Antitripsina/metabolismo , Adenoviridae/fisiologia , Animais , Ensaio de Imunoadsorção Enzimática , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Vírus Auxiliares/fisiologia , Humanos , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Many RNA polymerase II- or III-transcribed genes are inactive when their promoter is methylated at critical CpG dinucleotides. We have applied the genomic sequencing method and a direct DNA blotting technique to analyze the extent of DNA methylation in the 5'-CpG-3' rich promoter region of the RNA polymerase I-transcribed ribosomal RNA genes (rDNA) in DNA from primary human cells, primary human tumor cells and human cell lines. In none of the analyzed primary human cells and primary human tumor cells was the DNA in the rDNA promoter region found to be detectably methylated. In contrast, in some of the cell lines this promoter is methylated in all 5'-CpG-3' dinucleotides in the majority of the approximately 200 ribosomal RNA gene copies. In actively growing cells, rDNA gene activity is a prerequisite for cell viability. The high levels of DNA methylation in the promoter region of rDNA in the human cell lines raise questions on the role of promoter methylation in these RNA polymerase I-transcribed genes. It is, however, conceivable that a subset of the about 200 rDNA copies per haploid genome have escaped methylation and account for the rRNA synthesis in these cell lines. Alternatively, complete 5'-CpG-3' promoter methylation may be compatible with promoter activity as demonstrated for certain viral genomes.
Assuntos
DNA Ribossômico/metabolismo , Regiões Promotoras Genéticas , RNA Ribossômico/metabolismo , Sequência de Bases , Linhagem Celular , Células Cultivadas , DNA , Primers do DNA , Desoxicitidina/metabolismo , Fosfatos de Dinucleosídeos/metabolismo , Células HeLa , Humanos , Células KB , Metilação , Dados de Sequência Molecular , RNA Polimerase I/genética , Células Tumorais CultivadasRESUMO
Adenoviral vectors are promising gene transfer vehicles for different gene therapy applications. High-capacity adenoviral (HC-Ad) vectors address some of the problems that have been observed with replication-defective, E1-deleted first-generation adenoviral vectors: toxicity and immunogenicity due to viral gene expression and 7 to 8 kb capacity limit for the transport of therapeutic DNA. This review summarizes HC-Ad vector-related publications from the past 18 months that are mainly concerned with vector design/production and in vivo applications in different murine models.
Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Vetores Genéticos , Animais , Capsídeo/genética , Sistema Nervoso Central/metabolismo , DNA Recombinante/genética , Expressão Gênica , Técnicas de Transferência de Genes , Técnicas Genéticas , Terapia Genética/tendências , Genoma Viral , Humanos , Fígado/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Integração Viral/genéticaRESUMO
Human adenovirus type 12 (Ad12) induces undifferentiated sarcomas in neonate Syrian hamsters and hence presents a suitable model for studies of the molecular mechanism of viral oncogenesis. Since we submit that an understanding of the early steps in the interaction between Ad12 and hamster cells might shed light on the initiation of malignant transformation, the abortive infection of BHK21 hamster cells with Ad12 has been investigated in detail. Ad12 replication in these cells is blocked in early stages, while Ad2 can replicate to moderate titers. Early Ad12 genes are expressed in BHK21 hamster cells, but there is a total block in Ad12 DNA replication and late gene transcription. The Ad5-transformed hamster cell line BHK297-C131, with the left terminus of Ad5 DNA chromosomally integrated and constitutively expressed, allows limited levels of Ad12 DNA replication and late transcription, probably through Ad5 E1 functions, but not the translation of late Ad12 gene products. We have now investigated the capacities of binding of nuclear proteins NFI and NFIII from permissive human KB cells, nonpermissive hamster BHK21 cells, and complementing BHK297-C131 cells to the origin of replication (ori) of Ad2 or Ad12 DNA. The electrophoretic mobility shift assay has been used to assess these binding reactions. The data support the notions that NFIII of BHK21 cells has a lower affinity for the ori of Ad12 DNA than for the ori of Ad2 DNA and that the levels of NFIII in BHK21 cells are markedly reduced compared with the levels in the permissive human KB cells or the complementing BHK297-C131 hamster cells. These deficiencies are contributing factors for the abortive infection of BHK21 hamster cells with Ad12. The lack of sufficient levels of NFIII in BHK21 cells is also consistent with the decreased replication capacity of Ad2 in hamster compared with human cell lines.
Assuntos
Adenovírus Humanos/metabolismo , DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Origem de Replicação , Fatores de Transcrição/metabolismo , Adenovírus Humanos/genética , Animais , Extratos Celulares , Linhagem Celular , Linhagem Celular Transformada , Núcleo Celular/metabolismo , Cricetinae , DNA Viral/biossíntese , Fator C1 de Célula Hospedeira , Humanos , Fator 1 de Transcrição de Octâmero , Biossíntese de Proteínas , RNA Viral/metabolismoRESUMO
Hamster cells are completely nonpermissive for the replication of human adenovirus type 12 (Ad12), whereas types 2 and 5 can replicate in hamster cells. The Ad5-transformed hamster cell line BHK297-C131, which carries the left terminal 18.7% of the Ad5 genome and expresses at least the viral E1A region, can somehow complement Ad12 DNA replication and the transcription of the late Ad12 genes. Since the interaction of Ad12 with hamster cells must constitute a significant factor in the induction of Ad12 tumors in neonatal hamsters, we have continued to examine details of this abortive virus infection. The late Ad12 mRNAs in BHK297-C131 cells are polyadenylated but are synthesized in reduced amounts compared with the Ad12 products in Ad12-infected human cells, which are permissive for viral replication. The late mRNA derived from the Ad12 fiber gene has been assessed for its structural properties. By cloning cDNA transcripts from this region and determining their nucleotide sequences, the authenticity of the complete Ad12 fiber sequence and the completeness of the Ad12-typical tripartite leader have been confirmed. Moreover, in Ad12-infected BHK297-C131 cells the Ad12 virus-associated RNA, a virus-encoded translational activator with the correct nucleotide sequence, is synthesized. Nevertheless, the synthesis of detectable amounts of Ad12 virion-specific proteins, and in particular that of the main viral antigens, hexons and fibers, cannot be documented. Cellular factors needed to promote late mRNA translation might be missing, or inhibitory factors might exist in Ad12-infected BHK297-C131 cells.
Assuntos
Proteínas E1 de Adenovirus/fisiologia , Adenovírus Humanos/genética , Proteínas do Capsídeo , Regulação Viral da Expressão Gênica , Animais , Sequência de Bases , Capsídeo/biossíntese , Linhagem Celular , Cricetinae , DNA Viral/genética , Teste de Complementação Genética , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Nuclear Pequeno/genética , RNA Viral/genética , Replicação ViralRESUMO
Simian virus 40 large tumour antigen (T antigen) is shown to catalyse the formation of duplex DNA from complementary strands in specific conditions. The activity is dependent on an excess of unspecific double-stranded DNA and seems not to function by T antigen mediated destabilization of secondary structure. Rather, protein-protein contacts between T antigen molecules appear to be involved. Protein-protein interactions between T antigen molecules bound to physically separated DNA sites are also demonstrated by the formation of specific DNA loops and by cyclization of DNA molecules with 3'-extended single-stranded ends where T antigen specifically binds to the single-stranded/double-stranded junctions. The relevance of these properties for T antigen functions in DNA replication, transcription and(or) recombination is discussed.
Assuntos
Antígenos Transformantes de Poliomavirus , DNA Viral/ultraestrutura , Renaturação de Ácido Nucleico , Vírus 40 dos Símios/imunologia , Clonagem Molecular , DNA Viral/genética , Vetores Genéticos , Microscopia Eletrônica , Conformação de Ácido Nucleico , Mapeamento por Restrição , Vírus 40 dos Símios/genéticaRESUMO
Although adenoviral vectors provide prolonged gene expression in the brain by comparison to peripheral organs, expression is eliminated by a severe inflammatory infiltration (i.e., activated macrophages/microglia and T-lymphocytes) after peripheral infection with adenovirus. Here, we demonstrate that high-capacity adenoviral (HC-Ad) vectors succeed in maintaining long-term transgene expression in the brain, even in the presence of an active peripheral immunization with adenovirus that completely eliminates expression from first-generation vectors within 60 days. Importantly, even 60 days after the peripheral infection, brains injected with first-generation vectors exhibited evidence of a chronic infiltration of CD8(+) cells, macrophage/microglial activation, and up-regulation of brain MHC-I expression. No inflammation was observed in the brains injected with the HC-Ad vector. Thus, these results demonstrate that HC-Ad vectors will allow safe, stable, and long-term transgene expression in the brain, even in the presence of peripheral infection with adenovirus. This markedly improves the prospects for the use of adenoviral vectors for long-term gene therapy of neurological disorders.
Assuntos
Infecções por Adenoviridae/virologia , Encefalite/virologia , Terapia Genética , Vetores Genéticos , Infecções por Adenoviridae/fisiopatologia , Animais , Doença Crônica , Encefalite/fisiopatologia , Vetores Genéticos/administração & dosagem , Vetores Genéticos/efeitos adversos , Injeções Intraventriculares , Camundongos , Fatores de TempoRESUMO
Adenoviral (Ad) vector-mediated gene delivery of normal, full-length dystrophin to skeletal muscle provides a promising strategy for the treatment of Duchenne muscular dystrophy (DMD), an X-linked recessive, dystrophin-deficient muscle disease. Studies in animal models suggest that successful DMD gene therapy by Ad vector-mediated gene transfer would be precluded by cellular and humoral immune responses induced by vector capsid and transgene proteins. To address the immunity induced by Ad vector-mediated dystrophin gene delivery to dystrophic muscle, we developed high-capacity adenoviral (HC-Ad) vectors expressing mouse dystrophin driven by the muscle creatine kinase promoter (AdmDys) and mCTLA4Ig (AdmCTLA4Ig) individually, or together from one vector (AdmCTLA4Ig/mDys). We found stable expression of dystrophin protein in the tibialis anterior muscles of mdx mice, coinjected with AdmCTLA4Ig and AdmDys, or injected alone with AdmCTLA4Ig/mDys, whereas the expression of dystrophin protein in the control group coinjected with AdmDys and an empty vector decreased by at least 50% between 2 and 8 weeks after administration. Additionally, we observed reductions in Ad vector-induced Th1 and Th2 cytokines, Ad vector-specific cytotoxic T lymphocyte activation and neutralizing anti-Ad antibodies in both experimental groups that received a mCTLA4Ig-expressing vector as compared to the control group. This study demonstrates that the coexpression of mCTLA4Ig and dystrophin in skeletal muscle provided by HC-Ad vector-mediated gene transfer can provide stable expression of dystrophin in immunocompetent, adult mdx mouse muscle and applies a potentially powerful strategy to overcome adaptive immunity induced by Ad vector-mediated dystrophin gene delivery toward the ultimate goal of treatment for DMD.
Assuntos
Adenoviridae/genética , Distrofina/genética , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Imunoconjugados/genética , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/terapia , Transdução Genética/métodos , Abatacepte , Adenoviridae/imunologia , Animais , Anticorpos Antivirais/imunologia , Citocinas/imunologia , Distrofina/metabolismo , Expressão Gênica , Engenharia Genética , Vetores Genéticos/imunologia , Imunoconjugados/sangue , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/sangue , Distrofia Muscular de Duchenne/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
To regulate expression of a transferred gene in response to an exogenous compound, we have combined a high capacity adenoviral vector devoid of all viral coding sequences with a regulatory system that can be used to express a target gene in vivo in a selected site and at a desired time. This system uses a chimeric transactivator, GLp65, which consists of a mutated progesterone receptor-ligand binding domain fused to the GAL4 DNA binding domain and part of the activation domain of the human p65 protein, a component of the NF-kappaB complex. In the presence of the antiprogestin mifepristone, this chimeric regulator binds to a target gene containing the 17-mer GAL4 binding site, resulting in an efficient ligand-inducible transactivation of the target gene. We inserted the regulator GLp65 and a regulable human growth hormone target gene containing the 17-mer GAL4 binding site into the same adenoviral vector. To obtain tissue-specific expression of the target gene, we coupled the regulator to a liver-specific promoter. Infection of HepG2 cells and experimental mice with the adenovirus resulted in consistently high induction levels of human growth hormone in the presence of mifepristone whereas the transgene expression was undetectable in the absence of the ligand. Taken together, our regulable adenoviral vector represents an important tool for transgene regulation that can be used for potentially diverse applications, ranging from tissue-specific gene expression in transgenic animals to human gene therapy.