RESUMEN
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.
Asunto(s)
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
Wild-type adeno-associated virus (AAV) can only replicate in the presence of helper factors, which can be provided by coinfecting helper viruses such as adenoviruses and herpesviruses. The AAV genome consists of a linear, single-stranded DNA (ssDNA), which is converted into different molecular structures within the host cell. Using high-throughput sequencing, we found that herpes simplex virus 1 (HSV-1) coinfection leads to a shift in the type of AAV genome end recombination. In particular, open-end inverted terminal repeat (ITR) recombination was enhanced, whereas open-closed ITR recombination was reduced in the presence of HSV-1. We demonstrate that the HSV-1 protein ICP8 plays an essential role in HSV-1-mediated interference with AAV genome end recombination, indicating that the previously described ICP8-driven mechanism of HSV-1 genome recombination may be underlying the observed changes. We also provide evidence that additional factors, such as products of true late genes, are involved. Although HSV-1 coinfection significantly changed the type of AAV genome end recombination, no significant change in the amount of circular AAV genomes was identified. IMPORTANCE Adeno-associated virus (AAV)-mediated gene therapy represents one of the most promising approaches for the treatment of genetic diseases. Currently, various GMP-compatible production methods can be applied to manufacture clinical-grade vector, including methods that employ helper factors derived from herpes simplex virus 1 (HSV-1). Yet, to date, we do not fully understand how HSV-1 interacts with AAV. We observed that HSV-1 modulates AAV genome ends similarly to the genome recombination events observed during HSV-1 replication and postulate that further improvements of the HSV-1 production platform may enhance packaging of the recombinant AAV particles.
Asunto(s)
Dependovirus/crecimiento & desarrollo , Dependovirus/genética , Genoma Viral/genética , Virus Helper/genética , Herpesvirus Humano 1/genética , Recombinación Genética/genética , Animales , Línea Celular , Chlorocebus aethiops , Coinfección/patología , Células HEK293 , Células HeLa , Herpes Simple/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Infecciones por Parvoviridae/patología , Secuencias Repetidas Terminales/genética , Células Vero , Interferencia Viral/genética , Replicación Viral/genéticaRESUMEN
The adeno-associated virus (AAV) non-structural Rep proteins catalyze all the DNA transactions required for virus viability including, DNA replication, transcription regulation, genome packaging, and during the latent phase, site-specific integration. Rep proteins contain two multifunctional domains: an Origin Binding Domain (OBD) and a SF3 helicase domain (HD). Studies have shown that Rep proteins have a dynamic oligomeric behavior where the nature of the DNA substrate molecule modulates its oligomeric state. In the presence of ssDNA, Rep68 forms a large double-octameric ring complex. To understand the mechanisms underlying AAV Rep function, we investigated the cryo-EM and X-ray structures of Rep68-ssDNA complexes. Surprisingly, Rep68 generates hybrid ring structures where the OBD forms octameric rings while the HD forms heptamers. Moreover, the binding to ATPγS promotes a large conformational change in the entire AAA+ domain that leads the HD to form both heptamer and hexamers. The HD oligomerization is driven by an interdomain linker region that acts as a latch to 'catch' the neighboring HD subunit and is flexible enough to permit the formation of different stoichiometric ring structures. Overall, our studies show the structural basis of AAV Rep's structural flexibility required to fulfill its multifunctional role during the AAV life cycle.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/genética , Dependovirus/genética , Proteínas Virales/genética , Adenosina Trifosfato/genética , Microscopía por Crioelectrón , ADN Helicasas/genética , ADN Helicasas/ultraestructura , ADN de Cadena Simple/ultraestructura , Proteínas de Unión al ADN/ultraestructura , Dependovirus/ultraestructura , Humanos , Proteínas Virales/ultraestructuraRESUMEN
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.
Asunto(s)
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
Adeno-associated virus (AAV) is a small human Dependovirus whose low immunogenicity and capacity for long-term persistence have led to its widespread use as vector for gene therapy. Despite great recent successes in AAV-based gene therapy, further improvements in vector technology may be hindered by an inadequate understanding of various aspects of basic AAV biology. AAV is unique in that its replication is largely dependent on a helper virus and cellular factors. In the absence of helper virus coinfection, wild-type AAV establishes latency through mechanisms that are not yet fully understood. Challenging the currently held model for AAV latency, we show here that the corepressor Krüppel-associated box domain-associated protein 1 (KAP1) binds the latent AAV2 genome at the rep ORF, leading to trimethylation of AAV2-associated histone 3 lysine 9 and that the inactivation of KAP1 repression is necessary for AAV2 reactivation and replication. We identify a viral mechanism for the counteraction of KAP1 in which interference with the KAP1 phosphatase protein phosphatase 1 (PP1) by the AAV2 Rep proteins mediates enhanced phosphorylation of KAP1-S824 and thus relief from KAP1 repression. Furthermore, we show that this phenomenon involves recruitment of the NIPP1 (nuclear inhibitor of PP1)-PP1α holoenzyme to KAP1 in a manner dependent upon the NIPP1 FHA domain, identifying NIPP1 as an interaction partner for KAP1 and shedding light on the mechanism through which PP1 regulates cellular KAP1 activity.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Dependovirus/metabolismo , Receptores de Neuropéptido Y/antagonistas & inhibidores , Proteína 28 que Contiene Motivos Tripartito/metabolismo , Proteínas Virales/metabolismo , Línea Celular , Replicación del ADN/fisiología , ADN Viral/genética , Proteínas de Unión al ADN/genética , Dependovirus/genética , Epigénesis Genética , Genoma Viral , Células HEK293 , Células HeLa , Humanos , Infecciones por Parvoviridae/metabolismo , Infecciones por Parvoviridae/virología , Receptores de Neuropéptido Y/metabolismo , Proteínas Virales/genética , Virión/metabolismo , Latencia del Virus , Replicación Viral/fisiologíaRESUMEN
Niemann-Pick type C disease (NP-C) is a fatal neurodegenerative lysosomal storage disorder. It is caused in 95% of cases by a mutation in the NPC1 gene that encodes NPC1, an integral transmembrane protein localized to the limiting membrane of the lysosome. There is no cure for NP-C but there is a disease-modifying drug (miglustat) that slows disease progression but with associated side effects. Here, we demonstrate in a well-characterized mouse model of NP-C that a single administration of AAV-mediated gene therapy to the brain can significantly extend lifespan, improve quality of life, prevent or ameliorate neurodegeneration, reduce biochemical pathology and normalize or improve various indices of motor function. Over-expression of human NPC1 does not cause adverse effects in the brain and correctly localizes to late endosomal/lysosomal compartments. Furthermore, we directly compare gene therapy to licensed miglustat. Even at a low dose, gene therapy has all the benefits of miglustat but without adverse effects. On the basis of these findings and on-going ascendency of the field, we propose intracerebroventricular gene therapy as a potential therapeutic option for clinical use in NP-C.
Asunto(s)
Adenoviridae/genética , Proteínas Portadoras/administración & dosificación , Modelos Animales de Enfermedad , Trastornos Neurológicos de la Marcha/prevención & control , Terapia Genética , Longevidad/genética , Glicoproteínas de Membrana/administración & dosificación , Enfermedad de Niemann-Pick Tipo C/prevención & control , Animales , Proteínas Portadoras/fisiología , Trastornos Neurológicos de la Marcha/genética , Trastornos Neurológicos de la Marcha/patología , Humanos , Inflamación/genética , Inflamación/patología , Inflamación/prevención & control , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Transgénicos , Mutación , Proteína Niemann-Pick C1 , Enfermedad de Niemann-Pick Tipo C/genética , Enfermedad de Niemann-Pick Tipo C/patologíaRESUMEN
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.
Asunto(s)
Antiparkinsonianos/administración & dosificación , GTP Ciclohidrolasa/administración & dosificación , Vectores Genéticos/uso terapéutico , Levodopa/biosíntesis , Trastornos Parkinsonianos/terapia , Putamen/metabolismo , Tirosina 3-Monooxigenasa/administración & dosificación , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/efectos adversos , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/análogos & derivados , Animales , Antiparkinsonianos/uso terapéutico , Dependovirus/genética , Evaluación Preclínica de Medicamentos , Femenino , GTP Ciclohidrolasa/análisis , GTP Ciclohidrolasa/genética , GTP Ciclohidrolasa/metabolismo , Genes Reporteros , Genes Sintéticos , Vectores Genéticos/administración & dosificación , Humanos , Macaca mulatta , Masculino , Actividad Motora/efectos de los fármacos , Trastornos Parkinsonianos/inducido químicamente , Porción Compacta de la Sustancia Negra/química , Porción Compacta de la Sustancia Negra/patología , Prueba de Estudio Conceptual , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/análisis , Proteínas Recombinantes/uso terapéutico , Tirosina 3-Monooxigenasa/análisis , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Recombinant adeno-associated viruses (AAVs) are popular in vivo gene transfer vehicles. However, vector doses needed to achieve therapeutic effect are high and some target tissues in the central nervous system remain difficult to transduce. Gene therapy trials using AAV for the treatment of neurological disorders have seldom led to demonstrated clinical efficacy. Important contributing factors are low transduction rates and inefficient distribution of the vector. To overcome these hurdles, a variety of capsid engineering methods have been utilized to generate capsids with improved transduction properties. Here we describe an alternative approach to capsid engineering, which draws on the natural evolution of the virus and aims to yield capsids that are better suited to infect human tissues. We generated an AAV capsid to include amino acids that are conserved among natural AAV2 isolates and tested its biodistribution properties in mice and rats. Intriguingly, this novel variant, AAV-TT, demonstrates strong neurotropism in rodents and displays significantly improved distribution throughout the central nervous system as compared to AAV2. Additionally, sub-retinal injections in mice revealed markedly enhanced transduction of photoreceptor cells when compared to AAV2. Importantly, AAV-TT exceeds the distribution abilities of benchmark neurotropic serotypes AAV9 and AAVrh10 in the central nervous system of mice, and is the only virus, when administered at low dose, that is able to correct the neurological phenotype in a mouse model of mucopolysaccharidosis IIIC, a transmembrane enzyme lysosomal storage disease, which requires delivery to every cell for biochemical correction. These data represent unprecedented correction of a lysosomal transmembrane enzyme deficiency in mice and suggest that AAV-TT-based gene therapies may be suitable for treatment of human neurological diseases such as mucopolysaccharidosis IIIC, which is characterized by global neuropathology.
Asunto(s)
Cápside/fisiología , Terapia Genética/métodos , Ingeniería de Proteínas/métodos , Animales , Dependovirus/genética , Femenino , Vectores Genéticos , Masculino , Ratones , Ratones Endogámicos C57BL , Mucopolisacaridosis III/genética , Mucopolisacaridosis III/terapia , Células Fotorreceptoras/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Retina/fisiología , Distribución Tisular , Transducción GenéticaRESUMEN
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.
Asunto(s)
Replicación del ADN , ADN Viral/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , ADN Viral/química , ADN Viral/metabolismo , Proteínas de Unión al ADN/genética , Humanos , Mutagénesis Sitio-Dirigida , Mutación/genética , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Proteínas Virales/genéticaRESUMEN
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.
Asunto(s)
Secuencias de Aminoácidos , Proteínas de Unión al ADN/metabolismo , Dependovirus/fisiología , Proteínas Virales/metabolismo , Integración Viral , Línea Celular , Células Epiteliales/virología , Fibroblastos/virología , HumanosRESUMEN
Adeno-associated virus (AAV) nonstructural proteins Rep78 and Rep68 carry out all DNA transactions that regulate the AAV life cycle. They share two multifunctional domains: an N-terminal origin binding/nicking domain (OBD) from the HUH superfamily and a SF3 helicase domain. A short linker of â¼20 amino acids that is critical for oligomerization and function connects the two domains. Although X-ray structures of the AAV5 OBD and AAV2 helicase domains have been determined, information about the full-length protein and linker conformation is not known. This article presents the solution structure of AAV2 Rep68 using small-angle X-ray scattering (SAXS). We first determined the X-ray structures of the minimal AAV2 Rep68 OBD and of the OBD with the linker region. These X-ray structures reveal novel features that include a long C-terminal α-helix that protrudes from the core of the protein at a 45° angle and a partially structured linker. SAXS studies corroborate that the linker is not extended, and we show that a proline residue in the linker is critical for Rep68 oligomerization and function. SAXS-based rigid-body modeling of Rep68 confirms these observations, showing a compact arrangement of the two domains in which they acquire a conformation that positions key residues in all domains on one face of the protein, poised to interact with DNA.
Asunto(s)
Proteínas de Unión al ADN/química , Dependovirus/química , Proteínas Virales/química , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Infecciones por Parvoviridae/virología , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Dependovirus/fisiología , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Regiones Promotoras Genéticas , Proteína Fosfatasa 1/genética , Proteínas Virales/metabolismo , Integración Viral , Línea Celular , Humanos , Latencia del VirusRESUMEN
Retinitis pigmentosa, other inherited retinal diseases, and age-related macular degeneration lead to untreatable blindness because of the loss of photoreceptors. We have recently shown that transplantation of mouse photoreceptors can result in improved vision. It is therefore timely to develop protocols for efficient derivation of photoreceptors from human pluripotent stem (hPS) cells. Current methods for photoreceptor derivation from hPS cells require long periods of culture and are rather inefficient. Here, we report that formation of a transient self-organized neuroepithelium from human embryonic stem cells cultured together with extracellular matrix is sufficient to induce a rapid conversion into retinal progenitors in 5 days. These retinal progenitors have the ability to differentiate very efficiently into Crx(+) photoreceptor precursors after only 10 days and subsequently acquire rod photoreceptor identity within 4 weeks. Directed differentiation into photoreceptors using this protocol is also possible with human-induced pluripotent stem (hiPS) cells, facilitating the use of patient-specific hiPS cell lines for regenerative medicine and disease modeling.
Asunto(s)
Colágeno/farmacología , Fibroblastos/citología , Laminina/farmacología , Células Madre Pluripotentes/citología , Proteoglicanos/farmacología , Células Fotorreceptoras Retinianas Bastones/citología , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Colágeno/química , Combinación de Medicamentos , Matriz Extracelular/química , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Factores de Crecimiento de Fibroblastos/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Expresión Génica/efectos de los fármacos , Vectores Genéticos , Proteínas Hedgehog/farmacología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Laminina/química , Lentivirus/genética , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Proteoglicanos/química , Células Fotorreceptoras Retinianas Bastones/metabolismo , Imagen de Lapso de Tiempo , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
The functional heart is comprised of distinct mesoderm-derived lineages including cardiomyocytes, endothelial cells and vascular smooth muscle cells. Studies in the mouse embryo and the mouse embryonic stem cell differentiation model have provided evidence indicating that these three lineages develop from a common Flk-1(+) (kinase insert domain protein receptor, also known as Kdr) cardiovascular progenitor that represents one of the earliest stages in mesoderm specification to the cardiovascular lineages. To determine whether a comparable progenitor is present during human cardiogenesis, we analysed the development of the cardiovascular lineages in human embryonic stem cell differentiation cultures. Here we show that after induction with combinations of activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF, also known as FGF2), vascular endothelial growth factor (VEGF, also known as VEGFA) and dickkopf homolog 1 (DKK1) in serum-free media, human embryonic-stem-cell-derived embryoid bodies generate a KDR(low)/C-KIT(CD117)(neg) population that displays cardiac, endothelial and vascular smooth muscle potential in vitro and, after transplantation, in vivo. When plated in monolayer cultures, these KDR(low)/C-KIT(neg) cells differentiate to generate populations consisting of greater than 50% contracting cardiomyocytes. Populations derived from the KDR(low)/C-KIT(neg) fraction give rise to colonies that contain all three lineages when plated in methylcellulose cultures. Results from limiting dilution studies and cell-mixing experiments support the interpretation that these colonies are clones, indicating that they develop from a cardiovascular colony-forming cell. Together, these findings identify a human cardiovascular progenitor that defines one of the earliest stages of human cardiac development.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Miocitos Cardíacos/citología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Activinas/farmacología , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Linaje de la Célula/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/trasplante , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Proteínas Proto-Oncogénicas c-kit/genética , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/deficiencia , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Estrogen receptor α is commonly used in synthetic biology to control the activity of genome editing tools. The activating ligands, estrogens, however, interfere with various cellular processes, thereby limiting the applicability of this receptor. Altering its ligand preference to chemicals of choice solves this hurdle but requires adaptation of unspecified ligand-interacting residues. Here, we provide a solution by combining rational protein design with multi-site-directed mutagenesis and directed evolution of stably integrated variants in Saccharomyces cerevisiae. This method yielded an estrogen receptor variant, named TERRA, that lost its estrogen responsiveness and became activated by tamoxifen, an anti-estrogenic drug used for breast cancer treatment. This tamoxifen preference of TERRA was maintained in mammalian cells and mice, even when fused to Cre recombinase, expanding the mammalian synthetic biology toolbox. Not only is our platform transferable to engineer ligand preference of any steroid receptor, it can also profile drug-resistance landscapes for steroid receptor-targeted therapies.
Asunto(s)
Estradiol , Receptor alfa de Estrógeno , Animales , Ratones , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/química , Receptor alfa de Estrógeno/metabolismo , Estradiol/química , Estradiol/metabolismo , Ligandos , Tamoxifeno/farmacología , Tamoxifeno/metabolismo , Receptores de Estrógenos/genética , Receptores de Estrógenos/química , Receptores de Estrógenos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , MamíferosRESUMEN
A variety of viruses establish latency by integrating their genome into the host genome. The integration event generally occurs in a nonspecific manner, precluding the prediction of functional consequences from resulting disruptions of affected host genes. The nonpathogenic adeno-associated virus (AAV) is unique in its ability to stably integrate in a site-specific manner into the human MBS85 gene. To gain a better understanding of the integration mechanism and the consequences of MBS85 disruption, we analyzed the molecular structure of AAV integrants in various latently infected human cell lines. Our study led to the observation that AAV integration causes an extensive but partial duplication of the target gene. Intriguingly, the molecular organization of the integrant leaves the possibility that a functional copy of the disrupted target gene could potentially be preserved despite the resulting rearrangements. A latently infected, Mbs85-targeted mouse ES cell line was generated to study the functional consequences of the observed duplication-based integration mechanism. AAV-modified ES cell lines continued to self-renew, maintained their multilineage differentiation potential and contributed successfully to mouse development when injected into blastocysts. Thus, our study reveals a viral strategy for targeted genome addition with the apparent absence of functional consequences.
Asunto(s)
Dependovirus/genética , Marcación de Gen/métodos , Provirus/genética , Integración Viral , Latencia del Virus , Animales , Línea Celular , Células Madre Embrionarias/metabolismo , Expresión Génica , Humanos , Ratones , Proteína Fosfatasa 1/genéticaRESUMEN
Elucidation of pathways involved in mouse strain-dependent variation in the hematopoietic stem cell (HSC) compartment may reveal novel mechanisms relevant in vivo. Here, we demonstrate genetically determined variation in the proliferation of lin-Sca1++kit+ (LSK) primitive hematopoietic progenitor cells in response to transforming growth factor-beta (TGF-beta) 2, the dose response of which was biphasic with a stimulatory effect at low concentrations. In contrast, the dose responses of TGF-beta1 or -beta3 were inhibitory and did not show mouse strain-dependent variation. A quantitative trait locus (QTL) for the effect of TGF-beta2 was identified on chromosome 4 overlapping with a QTL regulating the frequency of LSK cells. These overlapping QTL were corroborated by the observation that the frequency of LSK cells is lower in adult Tgfb2+/- mice than in wild-type littermates, indicating that TGF-beta2 is a genetically determined positive regulator LSK number in vivo. Furthermore, adult Tgfb2+/- mice have a defect in competitive repopulation potential that becomes more pronounced upon serial transplantation. In fetal TGF-beta2-deficient HSCs, a defect only appears after serial reconstitution. These data suggest that TGF-beta2 can act cell autonomously and is important for HSCs that have undergone replicative stress. Thus, TGF-beta2 is a novel, genetically determined positive regulator of adult HSCs.
Asunto(s)
Células Madre Hematopoyéticas/citología , Sitios de Carácter Cuantitativo/genética , Células Madre/citología , Factor de Crecimiento Transformador beta/genética , Animales , Mapeo Cromosómico , Células Madre Hematopoyéticas/fisiología , Hígado/citología , Hígado/embriología , Ratones , Ratones Endogámicos , Ratones Noqueados , Células Madre/fisiología , Factor de Crecimiento Transformador beta/deficiencia , Factor de Crecimiento Transformador beta/fisiología , Factor de Crecimiento Transformador beta2RESUMEN
The nonpathogenic human adeno-associated virus type 2 (AAV-2) has adopted a unique mechanism to site-specifically integrate its genome into the human MBS85 gene, which is embedded in AAVS1 on chromosome 19. The fact that AAV has evolved to integrate into this ubiquitously transcribed region and that the chromosomal motifs required for integration are located a few nucleotides upstream of the translation initiation start codon of MBS85 suggests that the transcriptional activity of MBS85 might influence site-specific integration and thus might be involved in the evolution of this mechanism. In order to begin addressing this question, we initiated the characterization of the human MBS85 promoter region and compared its transcriptional activity to that of the AAV-2 p5 promoter. Our results clearly indicate that AAVS1 is defined by a complex transcriptional environment and that the MBS85 promoter shares key regulatory elements with the viral p5 promoter. Furthermore, we provide evidence for bidirectional MBS85 promoter activity and demonstrate that the minimal motifs required for AAV site-specific integration are present in the 5' untranslated region of the gene and play a posttranscriptional role in the regulation of MBS85 expression. These findings should provide a framework to further elucidate the complex interactions between the virus and its cellular host in this unique pathway to latency.
Asunto(s)
Dependovirus/genética , Dependovirus/fisiología , Perfilación de la Expresión Génica , Regiones Promotoras Genéticas , Proteína Fosfatasa 1/genética , Integración Viral , Regiones no Traducidas 5' , Secuencia de Bases , Línea Celular , Humanos , Datos de Secuencia Molecular , Proteína Fosfatasa 1/biosíntesis , Alineación de SecuenciaRESUMEN
We have previously designed a library of lentiviral vectors to generate somatic-transgenic rodents to monitor signalling pathways in diseased organs using whole-body bioluminescence imaging, in conscious, freely moving rodents. We have now expanded this technology to adeno-associated viral vectors. We first explored bio-distribution by assessing GFP expression after neonatal intravenous delivery of AAV8. We observed widespread gene expression in, central and peripheral nervous system, liver, kidney and skeletal muscle. Next, we selected a constitutive SFFV promoter and NFκB binding sequence for bioluminescence and biosensor evaluation. An intravenous injection of AAV8 containing firefly luciferase and eGFP under transcriptional control of either element resulted in strong and persistent widespread luciferase expression. A single dose of LPS-induced a 10-fold increase in luciferase expression in AAV8-NFκB mice and immunohistochemistry revealed GFP expression in cells of astrocytic and neuronal morphology. Importantly, whole-body bioluminescence persisted up to 240 days. We have validated a novel biosensor technology in an AAV system by using an NFκB response element and revealed its potential to monitor signalling pathway in a non-invasive manner in a model of LPS-induced inflammation. This technology complements existing germline-transgenic models and may be applicable to other rodent disease models.
Asunto(s)
Dependovirus/genética , Vectores Genéticos/genética , Ratones Transgénicos/genética , Animales , Técnicas Biosensibles/métodos , Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Inflamación/genética , Luciferasas de Luciérnaga/genética , Ratones , FN-kappa B/genética , Regiones Promotoras Genéticas/genética , Transducción de Señal/genética , Virus Formadores de Foco en el Bazo/genética , Transcripción Genética/genéticaRESUMEN
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.