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BACKGROUND: Mucopolysaccharidosis IIIC (MPSIIIC) is one of four Sanfilippo diseases sharing clinical symptoms of severe cognitive decline and shortened lifespan. The missing enzyme, heparan sulfate acetyl-CoA: α-glucosaminide-N-acetyltransferase (HGSNAT), is bound to the lysosomal membrane, therefore cannot cross the blood-brain barrier or diffuse between cells. We previously demonstrated disease correction in MPSIIIC mice using an Adeno-Associated Vector (AAV) delivering HGSNAT via intraparenchymal brain injections using an AAV2 derived AAV-truetype (AAV-TT) serotype with improved distribution over AAV9. METHODS: Here, intraparenchymal AAV was delivered in sheep using catheters or Hamilton syringes, placed using Brainlab cranial navigation for convection enhanced delivery, to reduce proximal vector expression and improve spread. RESULTS: Hamilton syringes gave improved AAV-GFP distribution, despite lower vector doses and titres. AAV-TT-GFP displayed moderately better transduction compared to AAV9-GFP but both serotypes almost exclusively transduced neurons. Functional HGSNAT enzyme was detected in 24-37% of a 140g gyrencephalic sheep brain using AAV9-HGSNAT with three injections in one hemisphere. CONCLUSIONS: Despite variabilities in volume and titre, catheter design may be critical for efficient brain delivery. These data help inform a clinical trial for MPSIIIC.
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Mucopolisacaridosis III , Animales , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Encéfalo , Dependovirus/genética , Modelos Animales de Enfermedad , Vectores Genéticos , Heparitina Sulfato/metabolismo , Mucopolisacaridosis/genética , Mucopolisacaridosis/terapia , Mucopolisacaridosis III/genética , Mucopolisacaridosis III/metabolismo , Mucopolisacaridosis III/terapia , Ovinos , Terapia GenéticaRESUMEN
Adeno-associated viruses (AAV) are utilized as gene transfer vectors in the treatment of monogenic disorders. A variant, rationally engineered based on natural AAV2 isolates, designated AAV-True Type (AAV-TT), is highly neurotropic compared to wild type AAV2 in vivo, and vectors based on it, are currently being evaluated for central nervous system applications. AAV-TT differs from AAV2 by 14 amino acids, including R585S and R588T, two residues previously shown to be essential for heparan sulfate binding of AAV2. The capsid structures of AAV-TT and AAV2 visualized by cryo-electron microscopy at 3.4 and 3.0 Å resolution, respectively, highlighted structural perturbations at specific amino acid differences. Differential scanning fluorimetry (DSF) performed at different pH conditions demonstrated that the melting temperature (Tm) of AAV2 was consistently â¼5 °C lower than AAV-TT, but both showed maximal stability at pH 5.5, corresponding to the pH in the late endosome, proposed as required for VP1u externalization to facilitate endosomal escape. Reintroduction of arginines at positions 585 and 588 in AAV-TT caused a reduction in Tm, demonstrating that the lack of basic amino acids at these positions are associated with capsid stability. These results provide structural and thermal annotation of AAV2/AAV-TT residue differences, that account for divergent cell binding, transduction, antigenic reactivity, and transduction of permissive tissues between the two viruses. Specifically, these data indicate that AAV-TT may not utilize a glycan receptor mediated pathway to enter cells and may have lower antigenic properties as compared to AAV2.
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Proteínas de la Cápside/genética , Cápside/metabolismo , Dependovirus/genética , Vectores Genéticos/genética , Mutagénesis Sitio-Dirigida , Animales , Sitios de Unión/genética , Cápside/química , Cápside/ultraestructura , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Línea Celular Tumoral , Microscopía por Crioelectrón , Dependovirus/química , Dependovirus/metabolismo , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HeLa , Humanos , Ratones , Modelos Moleculares , Conformación Proteica , Células Sf9 , Spodoptera , Virión/genética , Virión/metabolismo , Virión/ultraestructuraRESUMEN
Huntington's disease (HD) is an inherited neurodegenerative disorder of which skeletal muscle atrophy is a common feature, and multiple lines of evidence support a muscle-based pathophysiology in HD mouse models. Inhibition of myostatin signaling increases muscle mass, and therapeutic approaches based on this are in clinical development. We have used a soluble ActRIIB decoy receptor (ACVR2B/Fc) to test the effects of myostatin/activin A inhibition in the R6/2 mouse model of HD. Weekly administration from 5 to 11 weeks of age prevented body weight loss, skeletal muscle atrophy, muscle weakness, contractile abnormalities, the loss of functional motor units in EDL muscles and delayed end-stage disease. Inhibition of myostatin/activin A signaling activated transcriptional profiles to increase muscle mass in wild type and R6/2 mice but did little to modulate the extensive Huntington's disease-associated transcriptional dysregulation, consistent with treatment having little impact on HTT aggregation levels. Modalities that inhibit myostatin signaling are currently in clinical trials for a variety of indications, the outcomes of which will present the opportunity to assess the potential benefits of targeting this pathway in HD patients.
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Enfermedad de Huntington/patología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/fisiopatología , Miostatina/antagonistas & inhibidores , Receptores de Activinas Tipo II/farmacología , Animales , Peso Corporal/efectos de los fármacos , Fuerza de la Mano/fisiología , Proteína Huntingtina/química , Enfermedad de Huntington/complicaciones , Enfermedad de Huntington/fisiopatología , Masculino , Ratones , Músculo Esquelético/patología , Atrofia Muscular/complicaciones , Atrofia Muscular/prevención & control , Agregado de Proteínas/efectos de los fármacosRESUMEN
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).
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[This corrects the article DOI: 10.1371/journal.pone.0145425.].
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Huntington's disease (HD) is a neurodegenerative disorder for which there are no disease-modifying treatments. SIRT1 is a NAD+-dependent protein deacetylase that is implicated in maintaining neuronal health during development, differentiation and ageing. Previous studies suggested that the modulation of SIRT1 activity is neuroprotective in HD mouse models, however, the mechanisms controlling SIRT1 activity are unknown. We have identified a striatum-specific phosphorylation-dependent regulatory mechanism of SIRT1 induction under normal physiological conditions, which is impaired in HD. We demonstrate that SIRT1 activity is down-regulated in the brains of two complementary HD mouse models, which correlated with altered SIRT1 phosphorylation levels. This SIRT1 impairment could not be rescued by the ablation of DBC1, a negative regulator of SIRT1, but was linked to changes in the sub-cellular distribution of AMPK-α1, a positive regulator of SIRT1 function. This work provides insights into the regulation of SIRT1 activity with the potential for the development of novel therapeutic strategies.
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Encéfalo/metabolismo , Enfermedad de Huntington/patología , Sirtuina 1/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Proteínas de Ciclo Celular , Cerebelo/metabolismo , Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , Femenino , Enfermedad de Huntington/metabolismo , Inmunohistoquímica , Inmunoprecipitación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fenotipo , Fosforilación , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Sirtuina 1/deficiencia , Sirtuina 1/genéticaRESUMEN
Huntington disease (HD; OMIM 143100), a progressive neurodegenerative disorder, is caused by an expanded trinucleotide CAG (polyQ) motif in the HTT gene. Cardiovascular symptoms, often present in early stage HD patients, are, in general, ascribed to dysautonomia. However, cardio-specific expression of polyQ peptides caused pathological response in murine models, suggesting the presence of a nervous system-independent heart phenotype in HD patients. A positive correlation between the CAG repeat size and severity of symptoms observed in HD patients has also been observed in in vitro HD cellular models. Here, we test the suitability of human embryonic stem cell (hESC) lines carrying HD-specific mutation as in vitro models for understanding molecular mechanisms of cardiac pathology seen in HD patients. We have differentiated three HD-hESC lines into cardiomyocytes and investigated CAG stability up to 60 days after starting differentiation. To assess CAG stability in other tissues, the lines were also subjected to in vivo differentiation into teratomas for 10 weeks. Neither directed differentiation into cardiomyocytes in vitro nor in vivo differentiation into teratomas, rich in immature neuronal tissue, led to an increase in the number of CAG repeats. Although the CAG stability might be cell line-dependent, induced pluripotent stem cells generated from patients with larger numbers of CAG repeats could have an advantage as a research tool for understanding cardiac symptoms of HD patients.
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Diferenciación Celular/genética , Células Madre Embrionarias Humanas/metabolismo , Enfermedad de Huntington/patología , Mutación/genética , Miocitos Cardíacos/citología , Expansión de Repetición de Trinucleótido/genética , Línea Celular , Humanos , Células Madre Pluripotentes/citologíaRESUMEN
UNLABELLED: Adeno-associated virus (AAV) is a helper-dependent parvovirus that requires coinfection with adenovirus (AdV) or herpes simplex virus 1 (HSV-1) to replicate. In the absence of the helper virus, AAV can persist in an episomal or integrated form. Previous studies have analyzed the DNA damage response (DDR) induced upon AAV replication to understand how it controls AAV replication. In particular, it was shown that the Mre11-Rad50-Nbs1 (MRN) complex, a major player of the DDR induced by double-stranded DNA breaks and stalled replication forks, could negatively regulate AdV and AAV replication during coinfection. In contrast, MRN favors HSV-1 replication and is recruited to AAV replication compartments that are induced in the presence of HSV-1. In this study, we examined the role of MRN during AAV replication induced by HSV-1. Our results indicated that knockdown of MRN significantly reduced AAV DNA replication after coinfection with wild-type (wt) HSV-1 or HSV-1 with the polymerase deleted. This effect was specific to wt AAV, since it did not occur with recombinant AAV vectors. Positive regulation of AAV replication by MRN was dependent on its DNA tethering activity but did not require its nuclease activities. Importantly, knockdown of MRN also negatively regulated AAV integration within the human AAVS1 site, both in the presence and in the absence of HSV-1. Altogether, this work identifies a new function of MRN during integration of the AAV genome and demonstrates that this DNA repair complex positively regulates AAV replication in the presence of HSV-1. IMPORTANCE: Viral DNA genomes trigger a DNA damage response (DDR), which can be either detrimental or beneficial for virus replication. Adeno-associated virus (AAV) is a defective parvovirus that requires the help of an unrelated virus such as adenovirus (AdV) or herpes simplex virus 1 (HSV-1) for productive replication. Previous studies have demonstrated that the cellular Mre11-Rad50-Nbs1 (MRN) complex, a sensor and regulator of the DDR, negatively regulates AAV replication during coinfection with AdV, which counteracts this effect by inactivating the complex. Here, we demonstrate that MRN positively regulates AAV replication during coinfection with HSV-1. Importantly, our study also indicates that MRN also favors integration of AAV genomes within the human AAVS1 site. Altogether, this work indicates that MRN differentially regulates AAV replication depending on the helper virus which is present and identifies a new function of this DNA repair complex during AAV integration.
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Proteínas de Ciclo Celular/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Dependovirus/fisiología , Herpesvirus Humano 1/fisiología , Proteínas Nucleares/metabolismo , Integración Viral , Replicación Viral , Ácido Anhídrido Hidrolasas , Proteínas de Ciclo Celular/genética , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Proteína Homóloga de MRE11 , Proteínas Nucleares/genéticaRESUMEN
Skeletal muscle remodelling and contractile dysfunction occur through both acute and chronic disease processes. These include the accumulation of insoluble aggregates of misfolded amyloid proteins that is a pathological feature of Huntington's disease (HD). While HD has been described primarily as a neurological disease, HD patients' exhibit pronounced skeletal muscle atrophy. Given that huntingtin is a ubiquitously expressed protein, skeletal muscle fibres may be at risk of a cell autonomous HD-related dysfunction. However the mechanism leading to skeletal muscle abnormalities in the clinical and pre-clinical HD settings remains unknown. To unravel this mechanism, we employed the R6/2 transgenic and HdhQ150 knock-in mouse models of HD. We found that symptomatic animals developed a progressive impairment of the contractile characteristics of the hind limb muscles tibialis anterior (TA) and extensor digitorum longus (EDL), accompanied by a significant loss of motor units in the EDL. In symptomatic animals, these pronounced functional changes were accompanied by an aberrant deregulation of contractile protein transcripts and their up-stream transcriptional regulators. In addition, HD mouse models develop a significant reduction in muscle force, possibly as a result of a deterioration in energy metabolism and decreased oxidation that is accompanied by the re-expression of the HDAC4-DACH2-myogenin axis. These results show that muscle dysfunction is a key pathological feature of HD.
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Enfermedad de Huntington/patología , Músculo Esquelético/patología , Animales , Atrofia , Técnicas de Sustitución del Gen , Histona Desacetilasas/metabolismo , Humanos , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Ratones , Ratones Transgénicos , Músculo Esquelético/metabolismo , Miogenina/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismoRESUMEN
The inability of Adeno-Associated Virus (AAV) to replicate on its own is a strong argument in favor of the use of recombinant AAV vectors for in vivo gene transfer. However, some previous studies suggested that AAV may become replication competent in cells exposed to a genotoxic stress even in the absence of co-infection with a helper virus. To comprehensively explore this phenomenon, we examined AAV genome replication in several human cell lines exposed to different genotoxic conditions. We found that all treatments induced only negligible levels of AAV replication never exceeding ten fold above background. Further investigation indicated that induction of helper-independent AAV replication relied on the synergistic contribution of several extrinsic factors linked to the origin of the cell line and the quality of the AAV preparation. These results further support the notion that helper independent AAV replication cannot occur at significant levels in vivo.
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Daño del ADN , Dependovirus/fisiología , Replicación Viral , Línea Celular , Virus Helper/fisiología , HumanosRESUMEN
Adeno-associated virus type 2 (AAV2) is a human parvovirus that relies on a helper virus for efficient replication. Herpes simplex virus 1 (HSV-1) supplies helper functions and changes the environment of the cell to promote AAV2 replication. In this study, we examined the accumulation of cellular replication and repair proteins at viral replication compartments (RCs) and the influence of replicating AAV2 on HSV-1-induced DNA damage responses (DDR). We observed that the ATM kinase was activated in cells coinfected with AAV2 and HSV-1. We also found that phosphorylated ATR kinase and its cofactor ATR-interacting protein were recruited into AAV2 RCs, but ATR signaling was not activated. DNA-PKcs, another main kinase in the DDR, was degraded during HSV-1 infection in an ICP0-dependent manner, and this degradation was markedly delayed during AAV2 coinfection. Furthermore, we detected phosphorylation of DNA-PKcs during AAV2 but not HSV-1 replication. The AAV2-mediated delay in DNA-PKcs degradation affected signaling through downstream substrates. Overall, our results demonstrate that coinfection with HSV-1 and AAV2 provokes a cellular DDR which is distinct from that induced by HSV-1 alone.
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Coinfección/genética , Daño del ADN , Dependovirus/fisiología , Herpes Simple/genética , Herpesvirus Humano 1/fisiología , Infecciones por Parvoviridae/genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Coinfección/enzimología , Coinfección/virología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dependovirus/genética , Herpes Simple/enzimología , Herpes Simple/virología , Herpesvirus Humano 1/genética , Interacciones Huésped-Patógeno , Humanos , Infecciones por Parvoviridae/enzimología , Infecciones por Parvoviridae/virología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Replicación ViralRESUMEN
Initially thought as being non-immunogenic, recombinant AAVs have emerged as efficient vector candidates for treating monogenic diseases. It is now clear however that they induce potent immune responses against transgene products which can lead to destruction of transduced cells. Therefore, developing strategies to circumvent these immune responses and facilitate long-term expression of transgenic therapeutic proteins is a main challenge in gene therapy. We evaluated herein a strategy to inhibit the undesirable immune activation that follows muscle gene transfer by administration of CTLA-4/Ig to block the costimulatory signals required early during immune priming and by using gene transfer of PD-1 ligands to inhibit T cell functions at the tissue sites. We provide the proof of principle that this combination immunoregulatory therapy targeting two non-redundant checkpoints of the immune response, i.e., priming and effector functions, can improve persistence of transduced cells in experimental settings where cytotoxic T cells escape initial blockade. Therefore, CTLA-4/Ig plus PD-L1/2 combination therapy represents a candidate approach to circumvent the bottleneck of immune responses directed toward transgene products.
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Adeno-associated virus (AAV) is a human parvovirus that replicates only in cells coinfected with a helper virus, such as adenovirus or herpes simplex virus type 1 (HSV-1). We previously showed that nine HSV-1 factors are able to support AAV rep gene expression and genome replication. To elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis of cellular and HSV-1 factors associated with Rep proteins and thus potentially recruited within AAV replication compartments (AAV RCs). This study resulted in the identification of approximately 60 cellular proteins, among which factors involved in DNA and RNA metabolism represented the largest functional categories. Validation analyses indicated that the cellular DNA replication enzymes RPA, RFC, and PCNA were recruited within HSV-1-induced AAV RCs. Polymerase delta was not identified but subsequently was shown to colocalize with Rep within AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, we found that AAV replication is associated with the recruitment of components of the Mre11/Rad50/Nbs1 complex, Ku70 and -86, and the mismatch repair proteins MSH2, -3, and -6. Finally, several HSV-1 factors were also found to be associated with Rep, including UL12. We demonstrated for the first time that this protein plays a role during AAV replication by enhancing the resolution of AAV replicative forms and AAV particle production. Altogether, these analyses provide the basis to understand how AAV adapts its replication strategy to the nuclear environment induced by the helper virus.