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AAV expression,cells

Moumita Paul-Heng, Mario Leong, Eithne Cunningham, Daniel L J Bunker, Katherine Bremner, Zane Wang, Chuanmin Wang, Szun Szun Tay, Claire McGuffog, Grant J Logan, Ian E Alexander, Min Hu, Stephen I Alexander, Tim D Sparwasser, Patrick Bertolino, David G Bowen, G Alex Bishop, Alexandra Sharland
Adeno-associated viral vector-mediated (AAV-mediated) expression of allogeneic major histocompatibility complex class I (MHC class I) in recipient liver induces donor-specific tolerance in mouse skin transplant models in which a class I allele (H-2Kb or H-2Kd) is mismatched between donor and recipient. Tolerance can be induced in mice primed by prior rejection of a donor-strain skin graft, as well as in naive recipients. Allogeneic MHC class I may be recognized by recipient T cells as an intact molecule (direct recognition) or may be processed and presented as an allogeneic peptide in the context of self-MHC (indirect recognition)...
August 9, 2018: JCI Insight
Niansheng Ju, Rundong Jiang, Stephen L Macknik, Susana Martinez-Conde, Shiming Tang
Whereas optogenetic techniques have proven successful in their ability to manipulate neuronal populations-with high spatial and temporal fidelity-in species ranging from insects to rodents, significant obstacles remain in their application to nonhuman primates (NHPs). Robust optogenetics-activated behavior and long-term monitoring of target neurons have been challenging in NHPs. Here, we present a method for all-optical interrogation (AOI), integrating optical stimulation and simultaneous two-photon (2P) imaging of neuronal populations in the primary visual cortex (V1) of awake rhesus macaques...
August 8, 2018: PLoS Biology
Tsukasa Ohmori
Hemophilia is a congenital hemorrhagic disease caused by genetic abnormalities in coagulation factor VIII or factor IX. Current conventional therapy to prevent bleeding requires frequent intravenous injections of coagulation factor concentrates from early childhood. Accordingly, gene therapy for hemophilia remains an exciting future prospect for patients and their families, due to its potential to cure the disease through a one-time treatment. After a series of successes in basic research, recent clinical trials have demonstrated clear efficacy of gene therapy for hemophilia using adeno-associated virus (AAV) vectors...
August 6, 2018: International Journal of Hematology
Kota Sato, Yukihiro Shiga, Yurika Nakagawa, Kosuke Fujita, Koji M Nishiguchi, Hiroshi Tawarayama, Namie Murayama, Shigeto Maekawa, Takeshi Yabana, Kazuko Omodaka, Shota Katayama, Qiwei Feng, Satoru Tsuda, Toru Nakazawa
Purpose: To assess the therapeutic potential of endothelin-converting enzyme-like 1 (Ecel1) in a mouse model of optic nerve crush. Methods: Ecel1 expression was evaluated with real time quantitative (qRT)-PCR, Western blotting, and immunohistochemistry in mouse retinas after optic nerve crush. Vinblastine administration to the optic nerve and the intravitreal injection of N-methyl-d-aspartate (NMDA) were used to assess Ecel1 gene expression. Ecel1 was deleted with an adeno-associated viral (AAV) clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9 system, and retinal ganglion cell (RGC) survival was investigated with retrograde labeling, qRT-PCR, and visual evoked potential...
August 1, 2018: Investigative Ophthalmology & Visual Science
Chandra S Boosani, Palanikumar Gunasekar, Megan Block, Wanlin Jiang, Zefu Zhang, Mohamed Radwan, Devendra K Agrawal
Increased expression of DNA methyltransferase-1 (DNMT1) associates with the progression of many human diseases. Since DNMT1 induces cell proliferation, drugs that inhibit DNMT1 have been used to treat proliferative diseases. Since these drugs are non-specific inhibitors of DNMT1, subsidiary events or the compensatory mechanisms that are activated in the absence of DNMT1 limit their therapeutic application. Here, we studied the molecular mechanisms that occur during angioplasty-induced restenosis and found that DNMT1 inhibition in both in-vitro and in-vivo approaches resulted in the induction of DNMT3a expression...
August 1, 2018: Canadian Journal of Physiology and Pharmacology
Shih-Yun Chen, Hsiao-Ying Kuo, Fu-Chin Liu
Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows...
July 10, 2018: Journal of Visualized Experiments: JoVE
Chiara Milanese, Silvia Cerri, Ayse Ulusoy, Simona V Gornati, Audrey Plat, Sylvia Gabriels, Fabio Blandini, Donato A Di Monte, Jan H Hoeijmakers, Pier G Mastroberardino
The involvement of DNA damage and repair in aging processes is well established. Aging is an unequivocal risk factor for chronic neurodegenerative diseases, underscoring the relevance of investigations into the role that DNA alterations may have in the pathogenesis of these diseases. Consistently, even moderate impairment of DNA repair systems facilitates the onset of pathological features typical of PD that include derangement of the dopaminergic system, mitochondrial dysfunction, and alpha-synuclein stress...
July 26, 2018: Cell Death & Disease
Laura P van Lieshout, Jakob M Domm, Tara N Rindler, Kathy L Frost, Debra L Sorensen, Sarah J Medina, Stephanie A Booth, James P Bridges, Sarah K Wootton
Gene therapy for the treatment of genetic disorders has demonstrated considerable therapeutic success in clinical trials. Among the most effective and commonly used gene delivery vectors are those based on adeno-associated virus (AAV). Despite these advances in clinical gene therapy, further improvements in AAV vector properties such as rapid intracellular processing and transgene expression, targeted transduction of therapeutically relevant cell types, and longevity of transgene expression, will render extension of such successes to many other human diseases...
June 15, 2018: Molecular Therapy. Methods & Clinical Development
Wen-Tao Deng, Saravanan Kolandaivelu, Astra Dinculescu, Jie Li, Ping Zhu, Vince A Chiodo, Visvanathan Ramamurthy, William W Hauswirth
Rod and cone phosphodiesterase 6 (PDE6) are key effector enzymes of the vertebrate phototransduction pathway. Rod PDE6 consists of two catalytic subunits PDE6α and PDE6β and two identical inhibitory PDE6γ subunits, while cone PDE6 is composed of two identical PDE6α' catalytic subunits and two identical cone-specific PDE6γ' inhibitory subunits. Despite their prominent function in regulating cGMP levels and therefore rod and cone light response properties, it is not known how each subunit contributes to the functional differences between rods and cones...
2018: Frontiers in Molecular Neuroscience
Gabriela Mercado, Nélida López, Alexis Martínez, Sergio P Sardi, Claudio Hetz
Parkinson's disease (PD) compromises motor control due to the loss of dopaminergic neurons in the substantia nigra pars compacta. At the histopathological level, PD is characterized by the accumulation of Lewy bodies, large protein inclusions containing aggregated αSynuclein (αSyn). The progression of PD involves the spreading of αSyn misfolding through the brain mediated by a prion-like mechanism, where the protein is transferred between cells. Here we report that αSyn internalization is a dynamic process, where the protein transits through different sub-cellular compartments...
July 16, 2018: Biochemical and Biophysical Research Communications
Kazuo Kunisawa, Takeshi Shimizu, Itaru Kushima, Branko Aleksic, Daisuke Mori, Yasuyuki Osanai, Kenta Kobayashi, Anna M Taylor, Manzoor A Bhat, Akiko Hayashi, Hiroko Baba, Norio Ozaki, Kazuhiro Ikenaka
Myelinated axons segregate the axonal membrane into four defined regions: the node of Ranvier, paranode, juxtaparanode and internode. The paranodal junction consists of specific component proteins, such as neurofascin155 (NF155) on the glial side, and Caspr and Contactin on the axonal side. Although paranodal junctions are thought to play crucial roles in rapid saltatory conduction and nodal assembly, the role of their interaction with neurons is not fully understood. In a previous study, conditional NF155 knockout in oligodendrocytes led to disorganization of the paranodal junctions...
July 19, 2018: Journal of Neurochemistry
Kelvin Yi Chong Teo, Shu Yen Lee, Amutha Veluchamy Barathi, Sai Bo Bo Tun, Licia Tan, Ian Jeffery Constable
Purpose: To determine if the surgical removal of the internal limiting membrane (ILM) in nonhuman primates (NHPs) will result in safe and effective transfection of adeno-associated viral (AAV2) vectors using green fluorescent protein (GFP) as a reporter. Methods: Six Macaca fascicularis NHP eyes underwent vitrectomy, ILM peel with layering of 1.7 × 1013 genome copies per milliliter of AAV2-GFP under air. Four control eyes underwent only vitrectomy and pooling under air...
July 2, 2018: Investigative Ophthalmology & Visual Science
So Young Yoo, Su-Nam Jeong, Jeong-In Kang, Seung-Wuk Lee
Here, we demonstrated chimeric adeno-associated virus (chimeric AAV), AAV-DJ-mediated cardiovascular reprogramming strategy to generate new cardiomyocytes and limit collagen deposition in cardiac fibroblasts by inducing synergism of chimeric AAV-expressing Gata4, Mef2c, Tbx5 (AAV-GMT)-mediated heart reprogramming and chimeric AAV-expressing thymosin β4 (AAV-Tβ4)-mediated heart regeneration. AAV-GMT promoted a gradual increase in expression of cardiac-specific genes, including Actc1, Gja1, Myh6, Ryr2, and cTnT, with a gradual decrease in expression of a fibrosis-specific gene, procollagen type I and here AAV-Tβ4 help to induce GMT expression, providing a chimeric AAV-mediated therapeutic cell reprogramming strategy for ischemic heart diseases...
May 31, 2018: ACS Omega
Yang Li, Xiao-Lin Tang, Fei Yu, Hua-Jun Li, Wei Yuan
OBJECTIVE: To determine the expression of miR-30b in hair cells of mice,and its regulatory effect on the target gene DNM 1 and expression of Dynamin,the key protein of synaptic endocytosis in inner hair cells. METHODS: The basilar membrane of cochlear in adult C57 mice was obtained. The expression of miR-30b in the hair cells was detected by in situ hybridization. Luciferase vector was constructed and transfected into 293T cells with miR-30b. Changes in luciferase activity were measured to verify whether DNM 1 was the target gene of miR-30b...
May 2018: Sichuan da Xue Xue Bao. Yi Xue Ban, Journal of Sichuan University. Medical Science Edition
Yang Wu, Liangyu Jiang, Hao Geng, Tian Yang, Zengpeng Han, Xiaobing He, Kunzhang Lin, Fuqiang Xu
Current large-scale recombinant adeno-associated virus (rAAV) production systems based on the baculovirus expression vector (BEV) remain complicated and cost-intensive, and they lack versatility and flexibility. Here we present a novel recombinant baculovirus integrated with all packaging elements for the production of rAAV. To optimize BEV construction, ribosome leaky-scanning mechanism was used to express AAV Rep and Cap proteins downstream of the PH and P10 promoters in the pFast.Bac.Dual vector, respectively, and the rAAV genome was inserted between the two promoters...
September 21, 2018: Molecular Therapy. Methods & Clinical Development
Elena Senís, Lluc Mosteiro, Stefan Wilkening, Ellen Wiedtke, Ali Nowrouzi, Saira Afzal, Raffaele Fronza, Henrik Landerer, Maria Abad, Dominik Niopek, Manfred Schmidt, Manuel Serrano, Dirk Grimm
In vivo reprogramming of somatic cells into induced pluripotent stem cells (iPSC) holds vast potential for basic research and regenerative medicine. However, it remains hampered by a need for vectors to express reprogramming factors (Oct-3/4, Klf4, Sox2, c-Myc; OKSM) in selected organs. Here, we report OKSM delivery vectors based on pseudotyped Adeno-associated virus (AAV). Using the AAV-DJ capsid, we could robustly reprogram mouse embryonic fibroblasts with low vector doses. Swapping to AAV8 permitted to efficiently reprogram somatic cells in adult mice by intravenous vector delivery, evidenced by hepatic or extra-hepatic teratomas and iPSC in the blood...
July 9, 2018: Nature Communications
Anne-Kathrin Herrmann, Stefanie Grosse, Kathleen Börner, Chiara Krämer, Ellen Wiedtke, Manuel Gunkel, Dirk Grimm
Over the last decade, others and we have started to dissect the role of the assembly-activating protein AAP for the formation of Adeno-associated virus (AAV) capsids based on different viral serotypes. Recently, our group has specifically studied AAP's relevance during production of AAV gene therapy vectors in mammalian or insect cells, and found AAP to be essential for capsid protein stabilization and generation of functional vector particles. Here, we additionally addressed the lingering question whether molecular AAV evolution via DNA family shuffling of viral capsid genes would perturb AAP functionality due to concurrent and inadvertent recombination of the AAP open reading frame...
July 6, 2018: Human Gene Therapy
Yoichiro Ikeda, Zhao Sun, Xiao Ru, Luk H Vandenberghe, Benjamin D Humphreys
BACKGROUND: After injury, mesenchymal progenitors in the kidney interstitium differentiate into myofibroblasts, cells that have a critical role in kidney fibrogenesis. The ability to deliver genetic material to myofibroblast progenitors could allow new therapeutic approaches to treat kidney fibrosis. Preclinical and clinical studies show that adeno-associated viruses (AAVs) efficiently and safely transduce various tissue targets in vivo ; however, protocols for transduction of kidney mesenchymal cells have not been established...
July 5, 2018: Journal of the American Society of Nephrology: JASN
Adrien Savy, Minna U Kaikkonen, Adrien Léger, Yohann Dickx, Lionel Galibert, Otto-Wilhelm Merten
The human Adeno-Associated Virus serotype 2 (wtAAV2) is a common non-pathological virus and its recombinant form (rAAV) is widely used as gene therapy vector. Although rAAVs are routinely produced in the Baculovirus/Sf9 cell system, wtAAV2 has never been studied in this context. We tried to produce wtAAV2 in the baculovirus/Sf9 cell system hypothesizing that the wtAAV2 may be considered as a normal recombinant AAV transgene. Through our attempts to produce wtAAV2 in Baculovirus/Sf9, we found that wtAAV2 p5 promoter, which controls the expression of large Rep proteins in mammalian cells, was active in this system...
2018: PloS One
Hanchao Li, Qian Li, Xueting Zhang, Xiaoyan Zheng, Qiannan Zhang, Zhiming Hao
AIM: To clarify the effects of thymosin β4 (Tβ4) on CCl4 -induced hepatic fibrosis in mice and further explore the underlying mechanisms. METHODS: Expression of Tβ4 in fibrotic liver tissues was assessed by real-time quantitative reverse transcriptase PCR (RQ-PCR) and immunohistochemistry (IHC). The effects of intraperitoneal adeno-associated virus-Tβ4 (AAV-Tβ4) on CCl4 -induced hepatic fibrosis were observed through the evaluation of collagen deposition, hepatic stellate cell (HSC) activation and pro-fibrotic cytokine expression...
July 4, 2018: Journal of Gene Medicine
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