RESUMO
Ever since its introduction 40 years ago l-3,4-dihydroxyphenylalanine (l-DOPA) therapy has retained its role as the leading standard medication for patients with Parkinson's disease. With time, however, the shortcomings of oral l-DOPA treatment have become apparent, particularly the motor fluctuations and troublesome dyskinetic side effects. These side effects, which are caused by the excessive swings in striatal dopamine caused by intermittent oral delivery, can be avoided by delivering l-DOPA in a more continuous manner. Local gene delivery of the l-DOPA synthesizing enzymes, tyrosine hydroxylase and guanosine-tri-phosphate-cyclohydrolase-1, offers a new approach to a more refined dopaminergic therapy where l-DOPA is delivered continuously at the site where it is needed i.e. the striatum. In this study we have explored the therapeutic efficacy of adeno-associated viral vector-mediated l-DOPA delivery to the putamen in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated rhesus monkeys, the standard non-human primate model of Parkinson's disease. Viral vector delivery of the two enzymes, tyrosine hydroxylase and guanosine-5'-tri-phosphate-cyclohydrolase-1, bilaterally into the dopamine-depleted putamen, induced a significant, dose-dependent improvement of motor behaviour up to a level identical to that obtained with the optimal dose of peripheral l-DOPA. Importantly, this improvement in motor function was obtained without any adverse dyskinetic effects. These results provide proof-of-principle for continuous vector-mediated l-DOPA synthesis as a novel therapeutic strategy for Parkinson's disease. The constant, local supply of l-DOPA obtained with this approach holds promise as an efficient one-time treatment that can provide long-lasting clinical improvement and at the same time prevent the appearance of motor fluctuations and dyskinetic side effects associated with standard oral dopaminergic medication.
Assuntos
Antiparkinsonianos/administração & dosagem , GTP Cicloidrolase/administração & dosagem , Vetores Genéticos/uso terapêutico , Levodopa/biossíntese , Transtornos Parkinsonianos/terapia , Putamen/metabolismo , Tirosina 3-Mono-Oxigenase/administração & dosagem , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/efeitos adversos , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/análogos & derivados , Animais , Antiparkinsonianos/uso terapêutico , Dependovirus/genética , Avaliação Pré-Clínica de Medicamentos , Feminino , GTP Cicloidrolase/análise , GTP Cicloidrolase/genética , GTP Cicloidrolase/metabolismo , Genes Reporter , Genes Sintéticos , Vetores Genéticos/administração & dosagem , Humanos , Macaca mulatta , Masculino , Atividade Motora/efeitos dos fármacos , Transtornos Parkinsonianos/induzido quimicamente , Parte Compacta da Substância Negra/química , Parte Compacta da Substância Negra/patologia , Estudo de Prova de Conceito , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/análise , Proteínas Recombinantes/uso terapêutico , Tirosina 3-Mono-Oxigenase/análise , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
UNLABELLED: The life cycle of the human parvovirus adeno-associated virus (AAV) is orchestrated by four Rep proteins. The large Rep proteins, Rep78 and Rep68, are remarkably multifunctional and display a range of biochemical activities, including DNA binding, nicking, and unwinding. Functionally, Rep78 and Rep68 are involved in transcriptional regulation, DNA replication, and genomic integration. Structurally, the Rep proteins share an AAA(+) domain characteristic of superfamily 3 helicases, with the large Rep proteins additionally containing an N-terminal origin-binding domain (OBD) that specifically binds and nicks DNA. The combination of these domains, coupled with dynamic oligomerization properties, is the basis for the remarkable multifunctionality displayed by Rep68 and Rep78 during the AAV life cycle. In this report, we describe an oligomeric interface formed by Rep68 and demonstrate how disruption of this interface has drastic effects on both the oligomerization and functionality of the Rep proteins. Our results support a role for the four-helix bundle in the helicase domain of Rep68 as a bona fide oligomerization domain (OD). We have identified key residues in the OD that are critical for the stabilization of the Rep68-Rep68 interface; mutation of these key residues disrupts the enzymatic activities of Rep68, including DNA binding and nicking, and compromises viral DNA replication and transcriptional regulation of the viral promoters. Taken together, our data contribute to our understanding of the dynamic and substrate-responsive Rep78/68 oligomerization that is instrumental in the regulation of the DNA transitions that take place during the AAV life cycle. IMPORTANCE: The limited genome size of small viruses has driven the evolution of highly multifunctional proteins that integrate different domains and enzymatic activities within a single polypeptide. The Rep68 protein from adeno-associated virus (AAV) combines a DNA binding and endonuclease domain with a helicase-ATPase domain, which together support DNA replication, transcriptional regulation, and site-specific integration. The coordination of the enzymatic activities of Rep68 remains poorly understood; however, Rep68 oligomerization and Rep68-DNA interactions have been suggested to play a crucial role. We investigated the determinants of Rep68 oligomerization and identified a hydrophobic interface necessary for Rep68 activity during the AAV life cycle. Our results provide new insights into the molecular mechanisms underlying the regulation of the versatile Rep proteins. Efficient production of AAV-based gene therapy vectors requires optimal Rep expression levels, and studies such as the one presented here could contribute to further optimization of AAV production schemes.
Assuntos
Replicação do DNA , DNA Viral/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , DNA Viral/química , DNA Viral/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Mutagênese Sítio-Dirigida , Mutação/genética , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas Virais/genéticaRESUMO
High-throughput integration site (IS) analysis of wild-type adeno-associated virus type 2 (wtAAV2) in human dermal fibroblasts (HDFs) and HeLa cells revealed that juxtaposition of a Rep binding site (RBS) and terminal resolution site (trs)-like motif leads to a 4-fold-increased probability of wtAAV integration. Electrophoretic mobility shift assays (EMSAs) confirmed binding of Rep to off-target RBSs. For the first time, we show Rep protein off-target nicking activity, highlighting the importance of the nicking substrate for Rep-mediated integration.
Assuntos
Motivos de Aminoácidos , Proteínas de Ligação a DNA/metabolismo , Dependovirus/fisiologia , Proteínas Virais/metabolismo , Integração Viral , Linhagem Celular , Células Epiteliais/virologia , Fibroblastos/virologia , HumanosRESUMO
Adeno-associated virus type 2 is known to inhibit replication of herpes simplex virus 1 (HSV-1). This activity has been linked to the helicase- and DNA-binding domains of the Rep68/Rep78 proteins. Here, we show that Rep68 can bind to consensus Rep-binding sites on the HSV-1 genome and that the Rep helicase activity can inhibit replication of any DNA if binding is facilitated. Therefore, we hypothesize that inhibition of HSV-1 replication involves direct binding of Rep68/Rep78 to the HSV-1 genome.
Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dependovirus/genética , Genoma Viral/genética , Herpesvirus Humano 1/genética , Proteínas Virais/metabolismo , Sítios de Ligação/genética , Western Blotting , Dependovirus/metabolismo , Herpesvirus Humano 1/metabolismo , HumanosRESUMO
UNLABELLED: Adeno-associated virus serotype 2 (AAV2) can efficiently replicate in cells that have been infected with helper viruses, such as adenovirus or herpesvirus. However, in the absence of helper virus infection, AAV2 establishes latency by integrating its genome site specifically into PPP1R12C, a gene located on chromosome 19. This integration target site falls into one of the most gene-dense regions of the human genome, thus inviting the question as to whether the virus has evolved mechanisms to control this complex transcriptional environment in order to facilitate integration, maintain an apparently innocuous latency, and/or establish conditions that are conducive to the rescue of the integrated viral genome. The viral replication (Rep) proteins control and direct every known aspect of the viral life cycle and have been shown to tightly control all AAV2 promoters. In addition, a number of heterologous promoters are repressed by the AAV2 Rep proteins. Here, we demonstrate that Rep proteins efficiently repress expression from the target site PPP1R12C promoter. We find evidence that this repression employs mechanisms similar to those described for Rep-mediated AAV2 p5 promoter regulation. Furthermore, we show that the repression of the cellular target site promoter is based on two distinct mechanisms, one relying on the presence of a functional Rep binding motif within the 5' untranslated region (UTR) of PPP1R12C, whereas the second pathway requires only an intact nucleoside triphosphate (NTP) binding site within the Rep proteins, indicating the possible reliance of this pathway on interactions of the Rep proteins with cellular proteins that mediate or regulate cellular transcription. IMPORTANCE: The observation that repression of transcription from the adeno-associated virus serotype 2 (AAV2) p5 and integration target site promoters is mediated by shared mechanisms highlights the possible coevolution of virus and host and could lead to the identification of host factors that the virus exploits to navigate its life cycle.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Dependovirus/fisiologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Regiões Promotoras Genéticas , Proteína Fosfatase 1/genética , Proteínas Virais/metabolismo , Integração Viral , Linhagem Celular , Humanos , Latência ViralRESUMO
Bacterial type IV secretion systems (T4SSs) are involved in processes such as bacterial conjugation and protein translocation to animal cells. In this work, we have switched the substrates of T4SSs involved in pathogenicity for DNA transfer. Plasmids containing part of the conjugative machinery of plasmid R388 were transferred by the T4SS of human facultative intracellular pathogen Bartonella henselae to both recipient bacteria and human vascular endothelial cells. About 2% of the human cells expressed a green fluorescent protein (GFP) gene from the plasmid. Plasmids of different sizes were transferred with similar efficiencies. B. henselae codes for two T4SSs: VirB/VirD4 and Trw. A ΔvirB mutant strain was transfer deficient, while a ΔtrwE mutant was only slightly impaired in DNA transfer. DNA transfer was in all cases dependent on protein TrwC of R388, the conjugative relaxase, implying that it occurs by a conjugation-like mechanism. A DNA helicase-deficient mutant of TrwC could not promote DNA transfer. In the absence of TrwB, the coupling protein of R388, DNA transfer efficiency dropped 1 log. The same low efficiency was obtained with a TrwB point mutation in the region involved in interaction with the T4SS. TrwB interacted with VirB10 in a bacterial two-hybrid assay, suggesting that it may act as the recruiter of the R388 substrate for the VirB/VirD4 T4SS. A TrwB ATPase mutant behaved as dominant negative, dropping DNA transfer efficiency to almost null levels. B. henselae bacteria recovered from infected human cells could transfer the mobilizable plasmid into recipient Escherichia coli under certain conditions, underscoring the versatility of T4SSs.
Assuntos
Angiomatose Bacilar/microbiologia , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Bartonella henselae/genética , Conjugação Genética , Células Endoteliais/microbiologia , Plasmídeos/genética , Transfecção , Angiomatose Bacilar/genética , Proteínas de Bactérias/genética , Bartonella henselae/metabolismo , Bartonella henselae/patogenicidade , Linhagem Celular , Escherichia coli/genética , Humanos , Plasmídeos/metabolismoRESUMO
TrwC is a bacterial protein involved in conjugative transfer of plasmid R388. It is transferred together with the DNA strand into the recipient bacterial cell, where it can integrate the conjugatively transferred DNA strand into its target sequence present in the recipient cell. Considering that bacterial conjugation can occur between bacteria and eukaryotic cells, this protein has great biotechnological potential as a site-specific integrase. We have searched for possible TrwC target sequences in the human genome. Recombination assays showed that TrwC efficiently catalyzes recombination between its natural target sequence and a discrete number of sequences, located in noncoding sites of the human genome, which resemble this target. We have determined the cellular localization of TrwC and derivatives in human cells by immunofluorescence and also by an indirect yeast-based assay to detect both nuclear import and export signals. The results indicate that the recombinase domain of TrwC (N600) has nuclear localization, but full-length TrwC locates in the cytoplasm, apparently due to the presence of a nuclear export signal in its C-terminal domain. The recombinase domain of TrwC can be transported to recipient cells by conjugation in the presence of the helicase domain of TrwC, but with very low efficiency. We mutagenized the trwC gene and selected for mutants with nuclear localization. We obtained one such mutant with a point A904T mutation and an extra peptide at its C terminus, which maintained its functionality in conjugation and recombination. This TrwC mutant could be useful for future TrwC-mediated site-specific integration assays in mammalian cells.
Assuntos
DNA Nucleotidiltransferases/metabolismo , Proteínas de Escherichia coli/metabolismo , Marcação de Genes , Integrases/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Substituição de Aminoácidos , Núcleo Celular/química , Citoplasma/química , DNA Nucleotidiltransferases/genética , Proteínas de Escherichia coli/genética , Integrases/genética , Dados de Sequência Molecular , Mutagênese , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Patients with the lysosomal storage disease mucopolysaccharidosis IIIA (MPSIIIA) lack the lysosomal enzyme N-sulfoglucosamine sulfohydrolase (SGSH), one of the many enzymes involved in degradation of heparan sulfate. Build-up of un-degraded heparan sulfate results in severe progressive neurodegeneration for which there is currently no treatment. Experimental gene therapies based on gene addition are currently being explored. Following preclinical evaluation in MPSIIIA mice, an adeno-associated virus vector of serotype rh10 designed to deliver SGSH and sulfatase modifying factor 1 (SAF301) was trialed in four MPSIIIA patients, showing good tolerance and absence of adverse events with some improvements in neurocognitive measures. This study aimed to improve SAF301 further by removing sulfatase modifying factor 1 (SUMF1) and assessing if expression of this gene is needed to increase the SGSH enzyme activity (SAF301b). Second, the murine phosphoglycerate kinase (PGK) promotor was exchanged with a chicken beta actin/CMV composite (CAG) promotor (SAF302) to see if SGSH expression levels could be boosted further. The three different vectors were administered to MPSIIIA mice via intracranial injection, and SGSH expression levels were compared 4 weeks post treatment. Removal of SUMF1 resulted in marginal reductions in enzyme activity. However, promotor exchange significantly increased the amount of SGSH expressed in the brain, leading to superior therapeutic correction with SAF302. Biodistribution of SAF302 was further assessed using green fluorescent protein (GFP), indicating that vector spread was limited to the area around the injection tract. Further modification of the injection strategy to a single depth with higher injection volume increased vector distribution, leading to more widespread GFP distribution and sustained expression, suggesting this approach should be adopted in future trials.
Assuntos
Dependovirus/genética , Terapia Genética , Vetores Genéticos/genética , Mucopolissacaridose III/genética , Mucopolissacaridose III/fisiopatologia , Animais , Biomarcadores , Corpo Estriado/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Ativação Enzimática , Imunofluorescência , Expressão Gênica , Ordem dos Genes , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/isolamento & purificação , Hidrolases/genética , Camundongos , Mucopolissacaridose III/metabolismo , Mucopolissacaridose III/terapia , Neurônios/metabolismo , Especificidade de Órgãos/genética , Transdução Genética , Transgenes , Resultado do TratamentoRESUMO
Endonucleases of the HUH family are specialized in processing single-stranded DNA in a variety of evolutionarily highly conserved biological processes related to mobile genetic elements. They share a structurally defined catalytic domain for site-specific nicking and strand-transfer reactions, which is often linked to the activities of additional functional domains, contributing to their overall versatility. To assess if these HUH domains could be interchanged, we created a chimeric protein from two distantly related HUH endonucleases, containing the N-terminal HUH domain of the bacterial conjugative relaxase TrwC and the C-terminal DNA helicase domain of the human adeno-associated virus (AAV) replicase and site-specific integrase. The purified chimeric protein retained oligomerization properties and DNA helicase activities similar to Rep68, while its DNA binding specificity and cleaving-joining activity at oriT was similar to TrwC. Interestingly, the chimeric protein could catalyse site-specific integration in bacteria with an efficiency comparable to that of TrwC, while the HUH domain of TrwC alone was unable to catalyze this reaction, implying that the Rep68 C-terminal helicase domain is complementing the TrwC HUH domain to achieve site-specific integration into TrwC targets in bacteria. Our results illustrate how HUH domains could have acquired through evolution other domains in order to attain new roles, contributing to the functional flexibility observed in this protein superfamily.
Assuntos
DNA Nucleotidiltransferases/química , Dependovirus/enzimologia , Integrases/química , Biologia Computacional , Conjugação Genética , DNA/química , DNA Helicases/química , DNA Bacteriano/genética , DNA de Cadeia Simples , Endonucleases/química , Escherichia coli/metabolismo , Células HEK293 , Humanos , Plasmídeos , Domínios Proteicos , Proteínas Recombinantes/química , UltracentrifugaçãoRESUMO
Adeno-associated viral vectors are showing great promise as gene therapy vectors for a wide range of retinal disorders. To date, evaluation of therapeutic approaches has depended almost exclusively on the use of animal models. With recent advances in human stem cell technology, stem cell-derived retina now offers the possibility to assess efficacy in human organoids in vitro. Here we test six adeno-associated virus (AAV) serotypes [AAV2/2, AAV2/9, AAV2/8, AAV2/8T(Y733F), AAV2/5, and ShH10] to determine their efficiency in transducing mouse and human pluripotent stem cell-derived retinal pigment epithelium (RPE) and photoreceptor cells in vitro. All the serotypes tested were capable of transducing RPE and photoreceptor cells in vitro. AAV ShH10 and AAV2/5 are the most efficient vectors at transducing both mouse and human RPE, while AAV2/8 and ShH10 achieved similarly robust transduction of human embryonic stem cell-derived cone photoreceptors. Furthermore, we show that human embryonic stem cell-derived photoreceptors can be used to establish promoter specificity in human cells in vitro. The results of this study will aid capsid selection and vector design for preclinical evaluation of gene therapy approaches, such as gene editing, that require the use of human cells and tissues.
Assuntos
Dependovirus/fisiologia , Vetores Genéticos/genética , Células Fotorreceptoras/citologia , Células Fotorreceptoras/metabolismo , Células-Tronco Pluripotentes/citologia , Epitélio Pigmentado da Retina/citologia , Tropismo Viral , Animais , Diferenciação Celular , Células Cultivadas , Dependovirus/classificação , Imunofluorescência , Expressão Gênica , Técnicas de Transferência de Genes , Genes Reporter , Humanos , Camundongos , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas , Transdução Genética , TransgenesRESUMO
Quantitative measurement of proteins binding to DNA is a requisite to fully characterize the structural determinants of complex formation necessary to understand the DNA transactions that regulate cellular processes. Here we describe a detailed protocol to measure binding affinity of the adeno-associated virus (AAV) Rep68 protein for the integration site AAVS1 using fluorescent anisotropy. This protocol can be used to measure the binding constants of any DNA binding protein provided the substrate DNA is fluorescently labeled.
RESUMO
Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires the presence of a helper virus, such as adenovirus or herpes virus, to efficiently replicate its genome. AAV DNA is replicated by a rolling-hairpin mechanism (Ward, 2006), and during replication several DNA intermediates can be detected. This detailed protocol describes how to analyze the AAV DNA intermediates formed during AAV replication using a modified Hirt extract (Hirt, 1967) procedure and Southern blotting (Southern, 1975).
RESUMO
The widely used pSU8 family of cloning vectors is based on a p15A replicon and a chloramphenicol acetyltransferase (cat) gene conferring chloramphenicol resistance. We frequently observed an increase in the size of plasmids derived from these vectors. Analysis of the bigger molecular species shows that they have an IS10 copy inserted at a specific site between the promoter and the cat open reading frame. Promoter activity from both ends of IS10 has been reported, suggesting that the insertion events could lead to higher CAT production. Insertions were observed in certain constructions containing inserts that could lead to plasmid instability. To test the possibility that IS10 insertions were selected as a response to chloramphenicol selection, we have grown these constructs in the presence of different amounts of antibiotic and we observed that insertions arise promptly under higher chloramphenicol selective pressure. IS10 is present in many E. coli laboratory strains, so the possibility of insertion in constructions involving cat-containing vectors should be taken into account. Using lower chloramphenicol concentrations could solve this problem.
Assuntos
Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol/farmacologia , Elementos de DNA Transponíveis/genética , Escherichia coli/genética , Vetores Genéticos/genética , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Antibacterianos/farmacologia , Cromossomos Bacterianos , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Recombinação GenéticaRESUMO
Site-specific recombinases (SSRs) have been crucial in the development of mammalian transgenesis. For gene therapy purposes, this approach remains challenging, because, for example, SSR delivery is largely unresolved and SSR DNA substrates must pre-exist in target cells. In this review, we discuss the potential of His-hydrophobic-His (HUH) recombinases to overcome some of the limitations of conventional SSRs. Members of the HUH protein family cleave single-stranded (ss)DNA, but can mediate site-specific integration with the aid of the host replication machinery. Adeno-associated virus (AAV) Rep remains the only known example to support site-specific integration in human cells, and AAV is an excellent gene delivery vector that can be targeted to specific cells and organelles. Bacterial protein TrwC catalyzes integration into human sequences and can be delivered to human cells covalently linked to DNA, offering attractive new features for targeted genome modification.
Assuntos
DNA Nucleotidiltransferases/química , Técnicas de Transferência de Genes , Genoma Humano , Histidina/química , Recombinases/química , Animais , DNA Nucleotidiltransferases/metabolismo , Histidina/genética , Histidina/metabolismo , Humanos , Recombinases/metabolismo , Especificidade por SubstratoRESUMO
BACKGROUND: Bacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering. METHODOLOGY/PRINCIPAL FINDINGS: We have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome. CONCLUSIONS/SIGNIFICANCE: The results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells.