Featured
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| Open AccessRegulatory T cells expressing CD19-targeted chimeric antigen receptor restore homeostasis in Systemic Lupus Erythematosus
Systemic Lupus Erythematosus (SLE) is a chronic and progressive autoimmune disease characterized by abnormally activated B cells causing organ damage. Here authors introduce an adoptive cell therapy involving regulatory T cells overexpressing FoxP3 and harboring an anti-CD19 CAR to inhibit pathological B cells and thus tissue-harming autoimmunity in a humanized mouse model.
- M. Doglio
- , A. Ugolini
- & C. Bonini
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Article
| Open AccessBacterial protoplast-derived nanovesicles carrying CRISPR-Cas9 tools re-educate tumor-associated macrophages for enhanced cancer immunotherapy
CRISPR-Cas9 genome editing systems have great potential in cancer therapy. Here the authors report a gene-editing delivery system using functionalized nanovesicles derived from E. coli protoplasts to encapsulate Cas9-sgRNA ribonucleoprotein for the selective targeting of Pik3cg in tumor associated macrophages.
- Mingming Zhao
- , Xiaohui Cheng
- & Junfeng Zhang
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Article
| Open AccessHigh throughput intracellular delivery by viscoelastic mechanoporation
Scalability of mechanoporation strategies for intracellular delivery remains challenging. Here, the authors demonstrate a microfluidic approach for delivering mRNA and CRISPR-Cas systems to over 250 million cells per minute.
- Derin Sevenler
- & Mehmet Toner
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Article
| Open AccessA toolbox of astrocyte-specific, serotype-independent adeno-associated viral vectors using microRNA targeting sequences
Astrocytes, highly prevalent cells in the central nervous system, have been difficult to specifically target with adeno-associated viruses (AAVs). Here, the authors present a microRNA targeting cassette that de-targets other cells, increasing astrocyte specificity of AAVs to over 99%.
- Amy J. Gleichman
- , Riki Kawaguchi
- & S. Thomas Carmichael
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Article
| Open AccessLung SORT LNPs enable precise homology-directed repair mediated CRISPR/Cas genome correction in cystic fibrosis models
Roughly 10% of Cystic Fibrosis (CF) patients still have no effective medicine to take. Lung Selective Organ Targeting (SORT) Lipid Nanoparticles can efficiently deliver Cas9 mRNA, sgRNA, and donor ssDNA templates for precise homology-directed repair-mediated gene correction in ex vivo and in vivo CF models.
- Tuo Wei
- , Yehui Sun
- & Daniel J. Siegwart
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Article
| Open AccessmRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy
Large genes require dual adeno-associated viral (AAV) vectors for in vivo delivery/expression, but current methods have limitations. Here the authors develop and functionally evaluate REVeRT, an efficient and flexible dual AAV vector technology based on reconstitution via mRNA trans-splicing.
- Lisa Maria Riedmayr
- , Klara Sonnie Hinrichsmeyer
- & Elvir Becirovic
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Article
| Open AccessHuman OPRM1 and murine Oprm1 promoter driven viral constructs for genetic access to μ-opioidergic cell types
Tools for gaining long-term genetic access to mu-opioid receptor (MOR) neural cell types are limited. Here, the authors develop a suite of adeno-associated viral tools allowing selective genetic access to MOR cell types, and showcase their use across species.
- Gregory J. Salimando
- , Sébastien Tremblay
- & Gregory Corder
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Article
| Open AccessHigh-efficiency green management of potato late blight by a self-assembled multicomponent nano-bioprotectant
Unlike the other plant pathogens, Phytophtora infestans, the causative pathogen of potato late blight, can hardly take up dsRNA, which is a key obstacle in using dsRNA for disease control. Here, the authors design a self-assembled multicomponent nano-bioprotectant for potato late blight management.
- Yuxi Wang
- , Mingshan Li
- & Xiaodan Wang
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Article
| Open AccessCell type-specific delivery by modular envelope design
Targetable delivery vectors for genetic cargo are needed. Here the authors report a modular platform with separate fusion and targeting components—Delivery to Intended REcipient Cells Through Envelope Design (DIRECTED)—and show cell type-specific delivery.
- Daniel Strebinger
- , Chris J. Frangieh
- & Feng Zhang
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Article
| Open AccessA mosaic adeno-associated virus vector as a versatile tool that exhibits high levels of transgene expression and neuron specificity in primate brain
Viral vectors with intense and long-lasting transgene expression are essential for manipulating and imaging of neuronal activity in the primate brain. Here, the authors created a mosaic vector composed of capsid proteins derived from AAV1 and AAV2 that exhibits improved transgene expression and neuronal specificity.
- Kei Kimura
- , Yuji Nagai
- & Masahiko Takada
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Article
| Open AccessMultiomics analysis of naturally efficacious lipid nanoparticle coronas reveals high-density lipoprotein is necessary for their function
ApoE is known to be important for lipid nanoparticle function. Here, the authors shows that efficacious coronal ApoE originates from high-density lipoprotein (HDL) particles and, enhances hepatic delivery, making HDL a superior biomarker for lipid nanoparticle potency.
- Kai Liu
- , Ralf Nilsson
- & Alan Sabirsh
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Article
| Open AccessFunctional gene delivery to and across brain vasculature of systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates
Delivering genes to and across the brain vasculature efficiently and specifically across species remains challenging. Here, the authors show that endothelial-specific AAVs with serotype flexibility enable redosing and transform the brain vasculature into an in vivo biofactory in genetically diverse rodents. In primates, these vectors cross the blood-brain-barrier and show broad tropism.
- Xinhong Chen
- , Damien A. Wolfe
- & Viviana Gradinaru
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Article
| Open AccessThe AAV capsid can influence the epigenetic marking of rAAV delivered episomal genomes in a species dependent manner
rAAV vectors vary in their effectiveness between species, making it difficult to predict clinical outcomes. Here authors show that AAV capsid proteins influence the vector epigenomic state in cells, and that a single amino acid change in the vector can alter the vector epigenome and hence transgene expression levels between species.
- Adriana Gonzalez-Sandoval
- , Katja Pekrun
- & Mark A. Kay
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Article
| Open AccessEngineering tumor-specific gene nanomedicine to recruit and activate T cells for enhanced immunotherapy
Intratumoral abundance of chemokines, such as CXCL9, is an important driver of T cell infiltration in tumors. Here the authors describe the design of a tumor-specific expression strategy to drive secretion of CXCL9 and an anti-PD-L1 scFv (αPD-L1) in the tumor microenvironment, promoting T cell recruitment and anti-tumor immune response in preclinical cancer models.
- Yue Wang
- , Shi-Kun Zhou
- & Jun Wang
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Article
| Open AccessLantern-shaped flexible RNA origami for Smad4 mRNA delivery and growth suppression of colorectal cancer
mRNA delivery has shown great potential in the treatment of various diseases. Here, the authors develop a lantern-shaped flexible origami for nanolization of single mRNA molecules and demonstrate efficient delivery of Smad4 mRNA, achieving suppression of colorectal cancer tumour growth.
- Muren Hu
- , Chang Feng
- & Xiaoli Zhu
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Article
| Open AccessIdentification of a physiologic vasculogenic fibroblast state to achieve tissue repair
Here, the authors report on the discovery of physiological vasculogenic fibroblasts capable of forming functional blood vessels. In vivo tissue reprogramming triggered by topical tissue nanotransfection (TNT) of a single anti-miR-200b oligonucleotide achieved therapeutic tissue vascularization.
- Durba Pal
- , Subhadip Ghatak
- & Chandan K. Sen
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Article
| Open AccessA cooperative nano-CRISPR scaffold potentiates immunotherapy via activation of tumour-intrinsic pyroptosis
Delivery of immune therapy drugs to tumours might be hampered by their limited bioavailability and the difficulty of targeting complex exogenous compounds. Here authors trigger immunologic cell death, via activating tumour-cell-intrinsic pathways via CRISPR-based nanotechnology to enable efficient anti-tumour immune response in mouse models of melanoma.
- Ning Wang
- , Chao Liu
- & Changyang Gong
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Article
| Open AccessSelf-cleaving guide RNAs enable pharmacological selection of precise gene editing events in vivo
Here, authors show that precise integration of an rAAV vector into a chromosomal target locus results in expression of a self-cleaving guide RNA. This recombination event creates genetic resistance to a hepatotoxic compound and enables in vivo expansion of gene-edited hepatocytes using a selection drug.
- Amita Tiyaboonchai
- , Anne Vonada
- & Markus Grompe
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Article
| Open AccessCross-species evolution of a highly potent AAV variant for therapeutic gene transfer and genome editing
Gonzalez et al. use a viral evolutionary approach to generate cross-species compatible AAV (ccAAVs) vectors. They describe a highly potent new variant, AAV.cc47, with enhanced transduction efficiency over AAV serotype 9 and show its efficacy in different mouse models, pigs and non-human primates.
- Trevor J. Gonzalez
- , Katherine E. Simon
- & Aravind Asokan
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Article
| Open AccessCell-specific regulation of gene expression using splicing-dependent frameshifting
Precise and reliable gene delivery remains technically challenging. Here, the authors show that rationally designed frameshifting splicing can be used to express genes only in targeted cell types, with the potential to enhance the specificity AAV gene delivery.
- Jonathan P. Ling
- , Alexei M. Bygrave
- & Seth Blackshaw
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Article
| Open AccessPiperazine-derived lipid nanoparticles deliver mRNA to immune cells in vivo
Next-generation lipid nanoparticles that target non-hepatocytes could be important clinical tools. Using in vivo DNA barcoding, the authors identify piperazine-containing lipids deliver mRNA to immune cells without targeting ligands.
- Huanzhen Ni
- , Marine Z. C. Hatit
- & James E. Dahlman
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Article
| Open AccessMulti-step screening of DNA/lipid nanoparticles and co-delivery with siRNA to enhance and prolong gene expression
Plasmid DNA offers extended transgene expression duration compared to mRNA technologies. Here, using a multi-step screening platform, the authors report the best performing nanoparticle formulations for liver-targeted plasmid DNA expression in vivo.
- Yining Zhu
- , Ruochen Shen
- & Hai-Quan Mao
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Article
| Open AccessEfficient spatially targeted gene editing using a near-infrared activatable protein-conjugated nanoparticle for brain applications
Spatial control of gene expression allows precise control over biological processes. Here, the authors develop an efficient light-responsive formulation based on upconversion nanoparticles, and demonstrate on-demand genetic manipulation in deep brain tissue.
- Catarina Rebelo
- , Tiago Reis
- & Lino Ferreira
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Article
| Open AccessLiver-directed lentiviral gene therapy corrects hemophilia A mice and achieves normal-range factor VIII activity in non-human primates
“Lentiviral gene therapy to the liver establishes stable long-term normal to supra-normal coagulation factor VIII activity in mouse models of hemophilia A and in non-human primates, representing a potential new treatment option for people with hemophilia A.”.
- Michela Milani
- , Cesare Canepari
- & Alessio Cantore
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Article
| Open AccessSubthalamic nucleus stabilizes movements by reducing neural spike variability in monkey basal ganglia
Chemogenetic inactivation of the subthalamic nucleus in monkeys increases spike train variability in the pallidum and prolongs movement time, suggesting its role in stabilizing pallidal spike trains to achieve stable motor control.
- Taku Hasegawa
- , Satomi Chiken
- & Atsushi Nambu
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Article
| Open AccessLight triggered nanoscale biolistics for efficient intracellular delivery of functional macromolecules in mammalian cells
Ballistic delivery with micro/nano-particles has been successfully used to transfect plant cells, however, has failed in mammalian cells due to toxic effects. Here, the authors report on a self-assembled nano-ballistic delivery system for the delivery of functional macromolecules and demonstrate efficient transfection of mammalian cells.
- Juan C. Fraire
- , Elnaz Shaabani
- & Kevin Braeckmans
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Article
| Open AccessChoice of vector and surgical approach enables efficient cochlear gene transfer in nonhuman primate
Gene therapy using Adeno-associated viral vectors (AAV) rescues hearing and balance deficits in mouse models of human disorders. Here, the authors show that AAVAnc80L65 allows efficient cochlear gene transfer in nonhuman primates, and motivate future studies to evaluate gene therapy for hearing and balance disorders.
- Eva Andres-Mateos
- , Lukas D. Landegger
- & Luk H. Vandenberghe
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Article
| Open AccessCas9-derived peptides presented by MHC Class II that elicit proliferation of CD4+ T-cells
There have been reports of immune responses against Cas9 which may impair clinical use. Here the authors scan a cohort comparable to the North American population vis-à-vis distribution of MHC-II variants to identify Cas9 peptides presented by MHC-II proteins and can stimulate CD4 + T-cells.
- Vijaya L. Simhadri
- , Louis Hopkins
- & Zuben E. Sauna
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Article
| Open AccessA synthetic circuit for buffering gene dosage variation between individual mammalian cells
Transient plasmid transfection produces a wide distribution of copy numbers and expression heterogeneity. Here the authors present synthetic circuits called Equalizers that buffer copy number variation at the single-cell level and minimize cell-to-cell expression variability.
- Jin Yang
- , Jihwan Lee
- & François St-Pierre
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Article
| Open AccessNon-conditioned bone marrow chimeric mouse generation using culture-based enrichment of hematopoietic stem and progenitor cells
Bone marrow chimaeric mice are a valuable tool in research, but require myeloablative conditioning. Here the authors demonstrate efficient FACS-free enrichment of haematopoietic stem and progenitor cells for transplantation into unconditioned recipient mice, as well as for genetic engineering using polyvinyl alcohol based media.
- Kiyosumi Ochi
- , Maiko Morita
- & Satoshi Yamazaki
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Article
| Open AccessChemogenetic ON and OFF switches for RNA virus replication
Therapeutic application of RNA viruses requires tight control over viral activity. Here the authors design a regulatory switch that enables control over activity with clinically approved HIV protease inhibitors.
- E. Heilmann
- , J. Kimpel
- & D. von Laer
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Article
| Open AccessLentivirus-mediated gene therapy for Fabry disease
Treatments for Fabry disease, an inherited lysosomal disorder caused by the deficiency of the enzyme alpha-galactosidase A, are not fully efficacious. Here the authors report a single-arm phase I trial of gene therapy with autologous, lentivirus-transduced, hematopoietic cells that express alpha-galactosidase A to demonstrate that this approach is safe in five patients with Fabry disease.
- Aneal Khan
- , Dwayne L. Barber
- & Jeffrey A. Medin
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Article
| Open AccessUniversal toxin-based selection for precise genome engineering in human cells
Genome engineering in cell lines or human stem cells often has poor efficiency, limiting the development of research and therapeutic applications. Here, the authors use a toxin-based selection system for precise bi-allelic engineering in cells and in vivo.
- Songyuan Li
- , Nina Akrap
- & Marcello Maresca
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Comment
| Open AccessThe once and future gene therapy
Gene therapy is at an inflection point. Recent successes in genetic medicine have paved the path for a broader second wave of therapies and laid the foundation for next-generation technologies. This comment summarizes recent advances and expectations for the near future.
- Karen Bulaklak
- & Charles A. Gersbach
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Article
| Open AccessIdentification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants
Adeno-associated virus is the basis of many gene therapies and gene transfer vectors. Here the authors report a pipeline to enable side-by-side comparison of pre-selected capsids in a high throughput manner.
- Jonas Weinmann
- , Sabrina Weis
- & Dirk Grimm
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Article
| Open AccessIn situ reprogramming of gut bacteria by oral delivery
It is difficult to precisely target bacterial populations in the mammalian gut. Here the authors use encapsulated phages to deliver dCas9 to E. coli in the mouse gut to modulate RFP expression.
- Bryan B. Hsu
- , Isaac N. Plant
- & Pamela A. Silver
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Article
| Open AccessA protein-independent fluorescent RNA aptamer reporter system for plant genetic engineering
Fluorescent RNA aptamers could potentially be used as protein-independent reporters of transgene expression in plants. Here, the authors report that an optimized RNA aptamer, developed from Broccoli, can be used to detect transgene expression in stable and transiently transformed plant tissue.
- Jiuyuan Bai
- , Yao Luo
- & Yun Zhao
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Article
| Open AccessEngineering monocyte/macrophage−specific glucocerebrosidase expression in human hematopoietic stem cells using genome editing
Gaucher disease is a lysosomal storage disorder caused by insufficient glucocerebrosidase expression. Here, the authors describe a CRISPR/Cas9-based gene-editing approach to re-express this enzyme in human blood stem cells and show that they can engraft in NSG mice and differentiate into functional macrophages.
- Samantha G. Scharenberg
- , Edina Poletto
- & Natalia Gomez-Ospina
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Article
| Open AccessSystemic nanoparticle delivery of CRISPR-Cas9 ribonucleoproteins for effective tissue specific genome editing
Therapeutic targets of CRISPR-Cas can often not be accessed due to lack of carriers to deliver RNPs systematically. Here, the authors engineer modified lipid nanoparticles for delivery of gene editing proteins to specific tissues.
- Tuo Wei
- , Qiang Cheng
- & Daniel J. Siegwart
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Article
| Open AccessExtracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping
Expression of Cas9 and gRNA from viral vectors in vivo may cause off-target activity. Here the authors present NanoMEDIC, which uses nanovesicles to transiently deliver editing machinery to hard-to-transfect cells.
- Peter Gee
- , Mandy S. Y. Lung
- & Akitsu Hotta
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Article
| Open AccessCaliciviral protein-based artificial translational activator for mammalian gene circuits with RNA-only delivery
Synthetic RNA-based circuits allow for gene regulation without the risk of mutagenesis. Here the authors demonstrate a Caliciviral VPg-based Translational activator which allows translational activation of synthetic mRNAs.
- Hideyuki Nakanishi
- & Hirohide Saito
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Article
| Open AccessGene therapy for progressive familial intrahepatic cholestasis type 3 in a clinically relevant mouse model
Progressive familial intrahepatic cholestasis type 3 is a disease caused by mutations in the gene encoding the multidrug resistance protein 3, and has limited treatment options. Here they show that adeno-associated virus mediated gene therapy prevents disease progression in a mouse disease model.
- Nicholas D. Weber
- , Leticia Odriozola
- & Cristian Smerdou
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Article
| Open AccessAn anionic, endosome-escaping polymer to potentiate intracellular delivery of cationic peptides, biomacromolecules, and nanoparticles
Most reagents designed to deliver cargo into cells are cationic and so cannot deliver cationic cargo. Here the authors show that pretreating cells with the anionic polymer poly(propylacrylic acid) facilitates the uptake and endosomal escape of a wide variety of cationic cargo in numerous cell types.
- Brian C. Evans
- , R. Brock Fletcher
- & Craig L. Duvall
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Article
| Open AccessSplit selectable markers
Selectable markers are widely used in cell engineering but there is only a limited variety to choose from. Here the authors split markers using inteins, allowing up to six transgene integration events to be selected for with one marker.
- Nathaniel Jillette
- , Menghan Du
- & Albert Wu Cheng
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Article
| Open AccessLinkage between endosomal escape of LNP-mRNA and loading into EVs for transport to other cells
Lipid nanoparticles (LNPs) are potential platforms for RNA-based therapeutics, but the fate of LNP-RNAs upon internalization into the cell is unclear. Here, the authors show that LNP-mRNAs and ionizable lipids escape the endosomes and are re-released via extracellular vesicles which could deliver the functional mRNA to other cells.
- Marco Maugeri
- , Muhammad Nawaz
- & Hadi Valadi
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Article
| Open AccessAAV-ie enables safe and efficient gene transfer to inner ear cells
There are currently few AAV vectors that can effectively target the diverse cell types of the inner ear. Here the authors design AAV-ie for gene delivery to the mouse cochlea, targeting cochlear supporting cells.
- Fangzhi Tan
- , Cenfeng Chu
- & Guisheng Zhong
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Article
| Open AccessAn anaerobic bacterium host system for heterologous expression of natural product biosynthetic gene clusters
Anaerobic bacteria represent a rich source of biological and chemical diversity but are difficult to cultivate and there is a lack of heterologous expression systems. Here the authors develop an expression system based on S. mutans UA159 for biosynthetic gene clusters from anaerobic bacteria.
- Tingting Hao
- , Zhoujie Xie
- & Yihua Chen
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Article
| Open AccessImproved TMC1 gene therapy restores hearing and balance in mice with genetic inner ear disorders
Mutations in the mechanotransduction channel component TMC1/2 cause deafness. Here, the authors use a synthetic AAV to replace TMC1 and 2 in the inner ear and show restoration of cochlear and vestibular function, of neuronal reponses in the auditory cortex and of hearing and balance in mice.
- Carl A. Nist-Lund
- , Bifeng Pan
- & Jeffrey R. Holt
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Article
| Open AccessA RNA producing DNA hydrogel as a platform for a high performance RNA interference system
Interfering RNA have a range of therapeutic and research based applications, issues with delivery have made systems that make siRNA in situ of interest. Here, the author report on the creation of a DNA hydrogel with improved stability and transcription efficiency over plasmid DNA.
- Jaejung Song
- , Minhyuk Lee
- & Nokyoung Park