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1.
Nat Cell Biol ; 22(2): 246-256, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32015438

RESUMO

The Hippo and mammalian target of rapamycin complex 1 (mTORC1) pathways are the two predominant growth-control pathways that dictate proper organ development. We therefore explored potential crosstalk between these two functionally relevant pathways to coordinate their growth-control functions. We found that the LATS1 and LATS2 kinases, the core components of the Hippo pathway, phosphorylate S606 of Raptor, an essential component of mTORC1, to attenuate mTORC1 activation by impairing the interaction of Raptor with Rheb. The phosphomimetic Raptor-S606D knock-in mutant led to a reduction in cell size and proliferation. Compared with Raptor+/+ mice, RaptorD/D knock-in mice exhibited smaller livers and hearts, and a significant inhibition of elevation in mTORC1 signalling induced by Nf2 or Lats1 and Lats2 loss. Thus, our study reveals a direct link between the Hippo and mTORC1 pathways to fine-tune organ growth.

2.
Cold Spring Harb Protoc ; 2020(2): pdb.prot095596, 2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-32015001

RESUMO

The most commonly used method for production of recombinant adeno-associated virus (rAAVs) in research laboratories is by transient triple transfection of 293 cells with AAV cis and trans plasmids and an adenovirus helper plasmid. This protocol describes the processes required to prepare the transfected cell suspension for virus purification.

3.
Cold Spring Harb Protoc ; 2020(2): pdb.prot095612, 2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-32015002

RESUMO

This is a simple method for rapid preparation of recombinant adeno-associated virus (rAAV) stocks, which can be used for in vivo gene delivery. The purity of these vectors is considerably lower than that obtained by either CsCl gradient centrifugation or by combination of iodixanol gradient ultracentrifugation followed by column chromatography.

4.
Cold Spring Harb Protoc ; 2020(2): pdb.prot095638, 2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-32015003

RESUMO

This rapid and efficient method to prepare highly purified recombinant adeno-associated viruses (rAAVs) is based on binding of negatively charged rAAV capsids to an anion-exchange resin that is pH dependent.

5.
Hum Gene Ther ; 31(1-2): 4, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31967918
6.
Hum Gene Ther ; 31(1-2): 2-3, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31967921
7.
Neuropsychopharmacology ; 45(2): 384-393, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31277075

RESUMO

Dopamine (DA) signaling is critical for movement, motivation, and addictive behavior. The neuronal GTPase, Rit2, is enriched in DA neurons (DANs), binds directly to the DA transporter (DAT), and is implicated in several DA-related neuropsychiatric disorders. However, it remains unknown whether Rit2 plays a role in either DAergic signaling and/or DA-dependent behaviors. Here we leveraged the TET-OFF system to conditionally silence Rit2 in Pitx3IRES2-tTA mouse DANs. Following DAergic Rit2 knockdown (Rit2-KD), mice displayed an anxiolytic phenotype, with no change in baseline locomotion. Further, males exhibited increased acute cocaine sensitivity, whereas DAergic Rit2-KD suppressed acute cocaine sensitivity in females. DAergic Rit2-KD did not affect presynaptic TH and DAT protein levels in females, nor was TH was affected in males; however, DAT was significantly diminished in males. Paradoxically, despite decreased DAT levels in males, striatal DA uptake was enhanced, but was not due to enhanced DAT surface expression in either dorsal or ventral striatum. Finally, patch recordings in nucleus accumbens (NAcc) medium spiny neurons (MSNs) revealed reciprocal changes in spontaneous EPSP (sEPSP) frequency in male and female D1+ and D2+ MSNs following DAergic Rit2-KD. In males, sEPSP frequency was decreased in D1+, but not D2+, MSNs, whereas in females sEPSP frequency decreased in D2+, but not D1+, MSNs. Moreover, DAergic Rit2-KD abolished the ability of cocaine to reduce sEPSP frequency in D1+, but not D2+, male MSNs. Taken together, our studies are among the first to acheive AAV-mediated, conditional and inducible DAergic knockdown in vivo. Importantly, our results provide the first evidence that DAergic Rit2 expression differentially impacts striatal function and DA-dependent behaviors in males and females.

8.
Immunity ; 52(1): 167-182.e7, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31883839

RESUMO

Multiple sclerosis (MS) is a demyelinating, autoimmune disease of the central nervous system. While work has focused on myelin and axon loss in MS, less is known about mechanisms underlying synaptic changes. Using postmortem human MS tissue, a preclinical nonhuman primate model of MS, and two rodent models of demyelinating disease, we investigated synapse changes in the visual system. Similar to other neurodegenerative diseases, microglial synaptic engulfment and profound synapse loss were observed. In mice, synapse loss occurred independently of local demyelination and neuronal degeneration but coincided with gliosis and increased complement component C3, but not C1q, at synapses. Viral overexpression of the complement inhibitor Crry at C3-bound synapses decreased microglial engulfment of synapses and protected visual function. These results indicate that microglia eliminate synapses through the alternative complement cascade in demyelinating disease and identify a strategy to prevent synapse loss that may be broadly applicable to other neurodegenerative diseases. VIDEO ABSTRACT.


Assuntos
Complemento C3/imunologia , Encefalomielite Autoimune Experimental/patologia , Microglia/patologia , Esclerose Múltipla/patologia , Sinapses/patologia , Tálamo/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Callithrix , Linhagem Celular Tumoral , Complemento C3/antagonistas & inibidores , Modelos Animais de Doenças , Feminino , Gliose/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Receptores de Complemento 3b/metabolismo
9.
Mol Ther ; 28(2): 422-430, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31843447

RESUMO

Short hairpin RNAs that are delivered by recombinant adeno-associated virus (rAAV) have the potential to elicit long-term RNAi therapy for human disease. However, the discovery that short hairpin sequences can cause truncation of the rAAV genome calls into question the efficiency and gene-silencing specificity of this strategy in humans. Here, we report that embedding the guide strand of a small silencing RNA into an artificial microRNA (miRNA) scaffold derived from mouse miRNA-33 ensures rAAV genomic integrity and reduces off-targeting by 10-fold, while maintaining effective in vivo target gene repression in mice.

10.
Front Immunol ; 10: 2449, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31824476

RESUMO

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) characterized by mucosa damage associated with an uncontrolled inflammatory response. This immunological impairment leads to altered inflammatory mediators such as IL-33, which is shown to increase in the mucosa of active UC (aUC) patients. MicroRNAs present a distorted feature in inflamed colonic mucosa and are potential IL-33 regulating candidates in UC. Therefore, we studied the microRNA and mRNA profiles in inflamed colonic samples of UC patients, evaluating the effect of a microRNA (selected by in silico analysis and its expression in UC patients), on IL-33 under inflammatory conditions. We found that inflamed mucosa (n = 8) showed increased expression of 40 microRNAs and 2,120 mRNAs, while 49 microRNAs and 1,734 mRNAs were decreased, as determined by microarrays. In particular, IL-33 mRNA showed a 3.8-fold increase and eight members of a microRNA family (miR-378), which targets IL-33 mRNA in the 3'UTR, were decreased (-3.9 to -3.0 times). We selected three members of the miR-378 family (miR-378a-3p, miR-422a, and miR-378c) according to background information and interaction energy analysis, for further correlation analyses with IL-33 expression through qPCR and ELISA, respectively. We determined that aUC (n = 24) showed high IL-33 levels, and decreased expression of miR-378a-3p and miR-422a compared to inactive UC (n = 10) and controls (n = 6). Moreover, both microRNAs were inversely correlated with IL-33 expression, while miR-378c does not show a significant difference. To evaluate the effect of TNFα on the studied microRNAs, aUC patients with anti-TNF therapy were compared to aUC receiving other treatments. The levels of miR-378a-3p and miR-378c were higher in aUC patients with anti-TNF. Based on these findings, we selected miR-378a-3p to exploring the molecular mechanism involved by in vitro assays, showing that over-expression of miR-378a-3p decreased the levels of an IL-33 target sequence ß-gal-reporter gene in HEK293 cells. Stable miR-378a-3p over-expression/inhibition inversely modulated IL-33 content and altered viability of HT-29 cells. Additionally, in an inflammatory context, TNFα decreased miR-378a-3p levels in HT-29 cells enhancing IL-33 expression. Together, our results propose a regulatory mechanism of IL-33 expression exerted by miR-378a-3p in an inflammatory environment, contributing to the understanding of UC pathogenesis.

11.
J Virol ; 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31826994

RESUMO

Adeno-associated viruses (AAVs) from clade E are often used as vectors in gene delivery applications. This clade includes rhesus isolate 10 (AAVrh.10) and 39 (AAVrh.39) which, unlike representative AAV8, are capable of crossing the blood-brain barrier (BBB) thereby enabling the delivery of therapeutic genes to the CNS. Here the capsid structures of AAV8, AAVrh.10 and AAVrh.39 have been determined by cryo-electron microscopy and three-dimensional image reconstruction to 3.08, 2.75, and 3.39 Šresolution, respectively, to enable a direct structural comparison. AAVrh.10 and AAVrh.39 are 98% identical in amino acid sequence but only ∼93.5% to AAV8. However, the capsid structures of all three viruses are similar with only minor differences observed in the previously described surface variable regions suggesting that the specific residues S269 and N472, absent in AAV8, may confer the ability to cross the BBB in AAVrh.10 and AAVrh.39. Head-to-head comparison of empty and genome-containing particles showed DNA ordered in the previously described nucleotide-binding pocket supporting the suggested role of this pocket in DNA packaging for the Dependoparvovirus The structural characterization of these viruses provides a platform for future vector engineering efforts towards improved gene delivery success with respect to specific tissue targeting, transduction efficiency, antigenicity or receptor retargeting.Importance Recombinant adeno-associated virus vectors (rAAVs), based on AAV8 and AAVrh.10, have been utilized in multiple clinical trials to treat different monogenetic diseases. Closely related AAVrh.39 has also shown promise in vivo As recently attained for other AAV biologics, e.g. Luxturna and Zolgensma, based on AAV2 and AAV9, respectively, the vectors in this study will likely gain FDA approval for commercialization in the near future. The study characterized the capsid structures of these clinical vectors, at atomic resolution using cryo-EM and image reconstruction, for comparative analysis. The analysis suggested two key residues, S269 and N472, as determinants of BBB crossing for AAVrh.10 and AAVrh.39, a feature utilized for CNS delivery of therapeutic genes. The structure information thus provides a platform for engineering to improve receptor retargeting or tissue specificity. These are important challenges in the field that need attention. Capsid structure information also provides knowledge potentially applicable for regulatory product approval.

12.
Cold Spring Harb Protoc ; 2019(12): pdb.prot095554, 2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31792137

RESUMO

The easiest way to confirm the structure and identity of genomic DNA isolated from purified adenoviral recombinants is restriction enzyme digestion and gel electrophoresis. This analysis entails comparing the restriction patterns of the adenoviral vector DNA with that plasmid that was used to initiate the entire rescue and expansion process. The integrity of the viral backbone and the presence of both the transgene and viral ITRs are assessed.

13.
Cold Spring Harb Protoc ; 2019(12): pdb.prot095588, 2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31792138

RESUMO

The sensitivity of an assay for replication-competent adenoviruses (RCAs) can often be enhanced by biological amplification of the RCAs with serial passage. Here, we describe an extension of this technique, termed "concentration passage," in which RCA replicated during the first plating of the vector is collected and concentrated onto one-tenth of the original number of cells. This significantly increases the chances of detecting the RCAs. Combining this approach with the use of quantitative polymerase chain reaction (qPCR) for sensitive detection of the RCA E1 gene, we are able to reach levels of sensitivity of 1 IU of RCAs in 1011 vector particles. The protocol described here is tailored for HuAd5 vectors using wild-type HuAd5 as the RCA surrogate. However, we have also adapted this technique with similar sensitivity to vectors based on other adenovirus serotypes. If other adenovirus serotypes are assayed, careful consideration should be given to the appropriate RCA surrogate. Strictly speaking, if the vector is propagated in HEK-293 or similar cell lines, the RCA surrogate should be a hybrid virus containing the HuAd5 E1 gene.

15.
Genome Biol ; 20(1): 276, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31843008

RESUMO

BACKGROUND: Hemophilia A, a bleeding disorder resulting from F8 mutations, can only be cured by gene therapy. A promising strategy is CRISPR-Cas9-mediated precise insertion of F8 in hepatocytes at highly expressed gene loci, such as albumin (Alb). Unfortunately, the precise in vivo integration efficiency of a long insert is very low (~ 0.1%). RESULTS: We report that the use of a double-cut donor leads to a 10- to 20-fold increase in liver editing efficiency, thereby completely reconstituting serum F8 activity in a mouse model of hemophilia A after hydrodynamic injection of Cas9-sgAlb and B domain-deleted (BDD) F8 donor plasmids. We find that the integration of a double-cut donor at the Alb locus in mouse liver is mainly through non-homologous end joining (NHEJ)-mediated knock-in. We then target BDDF8 to multiple sites on introns 11 and 13 and find that NHEJ-mediated insertion of BDDF8 restores hemostasis. Finally, using 3 AAV8 vectors to deliver genome editing components, including Cas9, sgRNA, and BDDF8 donor, we observe the same therapeutic effects. A follow-up of 100 mice over 1 year shows no adverse effects. CONCLUSIONS: These findings lay the foundation for curing hemophilia A by NHEJ knock-in of BDDF8 at Alb introns after AAV-mediated delivery of editing components.

16.
Adv Sci (Weinh) ; 6(21): 1900440, 2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31728271

RESUMO

Delivery of genome editing tools to mammalian zygotes has revolutionized animal modeling. However, the mechanical delivery method to introduce genes and proteins to zygotes remains a challenge for some animal species that are important in biomedical research. Here, an approach to achieve gene delivery and genome editing in nonhuman primate embryos is presented by infecting zygotes with recombinant adeno-associated viruses (rAAVs). Together with previous reports from the authors of this paper and others, this approach is potentially applicable to a broad range of mammals. In addition to genome editing and animal modeling, this rAAV-based method can facilitate gene function studies in early-stage embryos.

17.
Mol Ther ; 27(12): 2123-2133, 2019 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-31543414

RESUMO

Symptoms of spinal muscular atrophy (SMA) disease typically begin in the late prenatal or the early postnatal period of life. The intrauterine (IU) correction of gene expression, fetal gene therapy, could offer effective gene therapy approach for early onset diseases. Hence, the overall goal of this study was to investigate the efficacy of human survival motor neuron (hSMN) gene expression after IU delivery in SMA mouse embryos. First, we found that IU-intracerebroventricular (i.c.v.) injection of adeno-associated virus serotype-9 (AAV9)-EGFP led to extensive expression of EGFP protein in different parts of the CNS with a great number of transduced neural stem cells. Then, to implement the fetal gene therapy, mouse fetuses received a single i.c.v. injection of a single-stranded (ss) or self-complementary (sc) AAV9-SMN vector that led to a lifespan of 93 (median of 63) or 171 (median 105) days for SMA mice. The muscle pathology and number of the motor neurons also improved in both study groups, with slightly better results coming from scAAV treatment. Consequently, fetal gene therapy may provide an alternative therapeutic approach for treating inherited diseases such as SMA that lead to prenatal death or lifelong irreversible damage.

18.
Cold Spring Harb Protoc ; 2019(8): pdb.prot095562, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31371467

RESUMO

Traditionally, adenovirus and recombinant adenovirus infectious titers have been measured by plaque assay, in which the cells are infected with serially diluted adenovirus stock and then overlaid with agar; a plaque will form as the result of a single infectious event. Although this method gives a quantitative readout (number of plaques corrected for the dilution), there can be issues with sensitivity and reproducibility, especially when adenovirus serotypes are used that infect standard cell lines with poor efficiency. An alternative approach is to plate serial dilutions of the cells growing in the wells of a 96-well tissue culture plate and determine the dilution at which 50% of the wells are infected. This ancient and reliable technique known as the "tissue culture infection dose 50%" (TCID50) end-point dilution method has been used for titering a number of viruses, especially those that do not readily form plaques. Usually, infected wells are determined by direct examination for cytopathic effect (CPE) or cell viability. However, by combining a 96-well TCID50 format and the power of quantitative polymerase chain reaction (qPCR) for detection, a large increase in sensitivity-in our hands 10-fold, with a range of both transgenes and adenovirus serotypes-can be achieved. This protocol uses a 96-well TCID50 format, in conjunction with qPCR for sensitive and quantitative positive-well calling, to determine infectious titer of adenovirus vectors.

19.
Cold Spring Harb Protoc ; 2019(8): pdb.prot095570, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31371468

RESUMO

Here we describe the preparation of quantitative polymerase chain reaction (qPCR) standards.

20.
Sci Transl Med ; 11(502)2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31341061

RESUMO

A number of simian and simian human immunodeficiency viruses (SIV and SHIV, respectively) have been used to assess the efficacy of HIV-1 vaccine strategies. Among these, SIVmac239 is considered among the most stringent because, unlike SHIV models, its full genome has coevolved in its macaque host and its tier 3 envelope glycoprotein (Env) is exceptionally hard to neutralize. Here, we investigated the ability of eCD4-Ig, an antibody-like entry inhibitor that emulates the HIV-1 and SIV receptor and coreceptor, to prevent SIVmac239 infection. We show that rh-eCD4-IgI39N expressed by recombinant adeno-associated virus (AAV) vectors afforded four rhesus macaques complete protection from high-dose SIVmac239 challenges that infected all eight control macaques. However, rh-eCD4-IgI39N-expressing macaques eventually succumbed to serial escalating challenge doses that were 2, 8, 16, and 32 times the challenge doses that infected the control animals. Despite receiving greater challenge doses, these macaques had significantly lower peak and postpeak viral loads than the control group. Virus isolated from three of four macaques showed evidence of strong immune pressure from rh-eCD4-IgI39N, with mutations located in the CD4-binding site, which, in one case, exploited a point-mutation difference between rh-eCD4-IgI39N and rhesus CD4. Other escape pathways associated with clear fitness costs to the virus. Our data report effective protection of rhesus macaques from SIVmac239.

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