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| Open AccessImproving the safety of human pluripotent stem cell therapies using genome-edited orthogonal safeguards
Human pluripotent stem cell derived therapies can have serious safety risks. Here the authors design two drug inducible genetic safeguards to deplete undifferentiated hPSCs and hPSC-derived cell types.
- Renata M. Martin
- , Jonas L. Fowler
- & Kyle M. Loh
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Article
| Open AccessChemical modifications of adenine base editor mRNA and guide RNA expand its application scope
Cas9 base editors are promising tools for correcting pathogenic single nucleotide mutations. Here the authors chemically modify mRNA encoding the editor and the gRNA to enhance editing and broaden its application.
- Tingting Jiang
- , Jordana M. Henderson
- & Wen Xue
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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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| Open AccessNaturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA
Endosomal sequestration of lipid-based nanoparticles is a barrier to delivery of nucleic acids. Here the authors test an array of cholesterol variants and perform in-depth investigation of nanoparticle shape, internal structure and intracellular trafficking.
- Siddharth Patel
- , N. Ashwanikumar
- & Gaurav Sahay
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| Open AccessExpanding the genome-targeting scope and the site selectivity of high-precision base editors
Base editors can be limited by precision and the size of the target window. Here the authors test Cas9s that recognise alternative PAMs to obtain a series of high-precision editors.
- Junjie Tan
- , Fei Zhang
- & Ralph Bock
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Article
| Open AccessSingle AAV-mediated mutation replacement genome editing in limited number of photoreceptors restores vision in mice
Replacing mutant genes with wildtype copies using adeno-associated virus (AAV) has been explored for the treatment of inherited retinopathies, but the low cargo limit restricts its use. Here the authors describe a single AAV platform that allows local replacement of a mutated sequence with its wildtype counterpart, based on combined CRISPR-Cas9 and micro-homology-mediated end joining.
- Koji M. Nishiguchi
- , Kosuke Fujita
- & Toru Nakazawa
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Article
| Open AccessDevelopment of a forward-oriented therapeutic lentiviral vector for hemoglobin disorders
Vectors used in gene therapy for hemoglobin disorders carry globin in a reverse-orientation to prevent the loss of key regulatory elements by RNA splicing, but this limits their efficiency. Here, the authors develop a vector carrying β-globin in a forward orientation and show that it has improved titers and transduction efficiency in humanized mice and nonhuman primates.
- Naoya Uchida
- , Matthew M. Hsieh
- & John F. Tisdale
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Article
| Open AccessHigh levels of AAV vector integration into CRISPR-induced DNA breaks
In-depth characterization of adeno-associated virus (AAV)-mediated CRISPR delivery is still lacking. Here, the authors show high levels of integration into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in vivo.
- Killian S. Hanlon
- , Benjamin P. Kleinstiver
- & Bence György
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Article
| Open AccessCRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation
CRISPR-Cas9 genome editing is presumed to knock out gene function by generating a frameshift during NHEJ repair. Here, the authors investigate mRNA and protein expression in edited lines and find genome editing can generate internal ribosome entry sites or alternatively spliced variants.
- Rubina Tuladhar
- , Yunku Yeu
- & Lawrence Lum
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Article
| Open AccessHuman genome-edited hematopoietic stem cells phenotypically correct Mucopolysaccharidosis type I
Mucopolysaccharidosis type I (MPSI) is a lysosomal storage disease caused by insufficient iduronidase (IDUA) activity. Here, the authors use an ex vivo genome editing approach to overexpress IDUA in human hematopoietic stem and progenitor cells and show it can phenotypically correct MSPI in mouse model.
- Natalia Gomez-Ospina
- , Samantha G. Scharenberg
- & Matthew H. Porteus
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Article
| Open AccessMultifunctional cationic nanosystems for nucleic acid therapy of thoracic aortic dissection
Thoracic aortic dissection has a high fatality rate and no effective treatment. Here, the authors develop cationic nanoparticles for the delivery of miR-145 and show that they stabilize vascular structures and prevent further deterioration of the aorta in mouse models of the disease.
- Chen Xu
- , Yanzhenzi Zhang
- & Fu-Jian Xu
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Article
| Open AccessBone-targeting AAV-mediated silencing of Schnurri-3 prevents bone loss in osteoporosis
The adaptor protein SHN3 suppresses new bone formation by controlling osteoblast activity. Here, the authors show that ablation of SHN3 function, either genetically or by delivering an artificial miRNA via AAV9, rescues bone loss in osteoporotic mice, and show that engineering of the AAV9 capsid improves targeting to bone
- Yeon-Suk Yang
- , Jun Xie
- & Jae-Hyuck Shim
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Article
| Open AccessA high-throughput screening and computation platform for identifying synthetic promoters with enhanced cell-state specificity (SPECS)
Synthetic promoters can be superior to native ones but the design is challenging without knowledge of gene regulation. Here the authors develop a pipeline that allows for screening a synthetic promoter library to identify high performance promoters in potentially any given cell state of interest.
- Ming-Ru Wu
- , Lior Nissim
- & Timothy K. Lu
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Article
| Open AccessNear physiological spectral selectivity of cochlear optogenetics
Cochlear implant spectral resolution is limited by current spread from each stimulation electrode. Here the authors compare optogenetic, electric and acoustic stimulation in gerbils and demonstrate improved spectral resolution of optogenetic over conventional electric stimulation.
- Alexander Dieter
- , Carlos J. Duque-Afonso
- & Tobias Moser
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Article
| Open AccessNext-generation muscle-directed gene therapy by in silico vector design
Adeno-associated viral vectors (AAV) are being developed for gene therapy of skeletal muscle, but it is a challenge to achieve robust gene expression. Here, the authors identify muscle-specific cisregulatory elements that lead to a substantial increase in micro-dystrophin and follistatin expression, resulting in a safe and sustainable rescue of the dystrophic phenotype in mouse models.
- S. Sarcar
- , W. Tulalamba
- & M. K. Chuah
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Article
| Open AccessThe functional synergism of microRNA clustering provides therapeutically relevant epigenetic interference in glioblastoma
The delivery of single therapeutic microRNAs in brain cancers is challenging. Here, the authors engineer three neuronal microRNAs (miR-124, 128 and 137) into a cluster that, targeting oncogenic chromatin repressors, increases survival of GBM-bearing mice when combined with chemotherapy.
- Vivek Bhaskaran
- , Michal O. Nowicki
- & Pierpaolo Peruzzi
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Article
| Open AccessEngineering of high-precision base editors for site-specific single nucleotide replacement
Base editors can target multiple bases within a window around the target site, reducing their specificity. Here the authors engineer the connection between the deaminase and Cas domain to narrow the window of activity.
- Junjie Tan
- , Fei Zhang
- & Ralph Bock
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Article
| Open AccessHematopoietic PBX-interacting protein mediates cartilage degeneration during the pathogenesis of osteoarthritis
Osteoarthritis (OA) is associated with cartilage degeneration, and no effective therapy exists. Here, the authors show that the HPIP protein modulates OA progression by regulating Wnt signaling, and that its knockdown in joints via AAV-mediated gene silencing attentuates pathology.
- Quanbo Ji
- , Xiaojie Xu
- & Yan Wang
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Article
| Open AccessOrthogonal Cas9–Cas9 chimeras provide a versatile platform for genome editing
Therapeutic genome engineering relies on the development of reliable, robust and versatile tools. Here the authors develop Cas9-Cas9 chimeras with high target site activity that generate predictable deletions.
- Mehmet Fatih Bolukbasi
- , Pengpeng Liu
- & Scot A. Wolfe
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| Open AccessAntigen-selective modulation of AAV immunogenicity with tolerogenic rapamycin nanoparticles enables successful vector re-administration
Immunogenicity of AAV vectors renders repeated AAV dosing ineffective. Here the authors show that coadministration of nanoparticle-encapsulated rapamycin overcomes AAV immunogenicity through Treg induction, enabling efficient AAV redosing in mice and nonhuman primates.
- Amine Meliani
- , Florence Boisgerault
- & Federico Mingozzi
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| Open AccessHighly efficient genome editing by CRISPR-Cpf1 using CRISPR RNA with a uridinylate-rich 3′-overhang
Cpf1 is a promising alternative to Cas9 though indel generation efficiency is target dependent. Here the authors show that the addition of a polyU 3′ overhang can improve the efficiency of low efficiency guide RNAs.
- Su Bin Moon
- , Jeong Mi Lee
- & Yong-Sam Kim
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Article
| Open AccessArgininosuccinic aciduria fosters neuronal nitrosative stress reversed by Asl gene transfer
Patients with mutations in the ASL gene present with argininosuccinic aciduria characterised by hyperammonaemia and cognitive impairment. Here, the authors show that cerebral disease involves neuronal nitrosative/oxidative stress that is not induced by hyperammonaemia, and that it can be reversed using AAV-ASL directed to liver and brain in mice.
- Julien Baruteau
- , Dany P. Perocheau
- & Simon N. Waddington
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| Open AccessHigh-fidelity CRISPR/Cas9- based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis
Suppression of gene expression due to aberrant promoter methylation contributes to organ fibrosis. Here, the authors couple a deactivated Cas9 to the TET3 catalytic domain to induce expression of four antifibrotic genes, and show that lentiviral-mediated delivery is effective in reducing kidney fibrosis in mouse models.
- Xingbo Xu
- , Xiaoying Tan
- & Michael Zeisberg
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Article
| Open AccessAAV vector-mediated in vivo reprogramming into pluripotency
In vivo reprogramming of somatic cells is hampered by the need for vectors to express the OKSM factors in selected organs. Here the authors report new AAV-based vectors capable of in vivo reprogramming at low doses.
- Elena Senís
- , Lluc Mosteiro
- & Dirk Grimm
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Article
| Open AccessIn utero nanoparticle delivery for site-specific genome editing
The correction of genetic defects in utero could allow for improved outcomes of gene therapy. Here, the authors demonstrate safe delivery of nanoparticles to fetal mouse tissues, and show that nanoparticles containing peptide nucleic acids to edit the beta-globin gene are effective in a mouse model of beta-thalassemia.
- Adele S. Ricciardi
- , Raman Bahal
- & W. Mark Saltzman
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Article
| Open AccessEfficient RNA drug delivery using red blood cell extracellular vesicles
RNA delivery for disease treatment often has low uptake efficiencies and cytotoxicity. Here the authors produce extracellular vesicles from red blood cells for in vivo cargo delivery.
- Waqas Muhammad Usman
- , Tin Chanh Pham
- & Minh T. N. Le
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Article
| Open AccessCRISPR-LbCpf1 prevents choroidal neovascularization in a mouse model of age-related macular degeneration
The CRISPR endonuclease LbCpf1 is reported to have greater efficiency and specificity than Cas9. Here, the authors use LbCpf1 to target the angiogenesis-related genes VEGF and HIF1a, and show that delivery of the nuclease using AAV9 is effective in mouse models of macular degeneration.
- Taeyoung Koo
- , Sung Wook Park
- & Jin-Soo Kim
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Article
| Open AccessCtIP fusion to Cas9 enhances transgene integration by homology-dependent repair
The integration of exogenous DNA into the genome using CRISPR–Cas9 often presents a challenge to researchers. Here the authors fuse CtIP to Cas9 to stimulate recombination at target loci.
- M. Charpentier
- , A. H. Y. Khedher
- & J. P. Concordet
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Article
| Open AccessLong-term microdystrophin gene therapy is effective in a canine model of Duchenne muscular dystrophy
Duchenne muscular dystrophy is a progressive degenerative disease of muscles caused by mutations in the dystrophin gene. Here the authors use AAV vectors to deliver microdystrophin to dogs with muscular dystrophy, and show restoration of dystrophin expression and reduction of symptoms up to 26 months of age.
- Caroline Le Guiner
- , Laurent Servais
- & George Dickson
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Article
| Open AccessGenome editing abrogates angiogenesis in vivo
Abnormal angiogenesis causes many ocular diseases. Here the authors employ CRISPR/Cas9 gene editing technology to silence VEGFR2, a major regulator of angiogenesis, in retinal endothelium and abrogate angiogenesis in the mouse models of oxygen-induced retinopathy and laser-induced choroid neovascularization.
- Xionggao Huang
- , Guohong Zhou
- & Hetian Lei
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Article
| Open AccessDual loss of human POLQ and LIG4 abolishes random integration
Homologous recombination mediated gene targeting is highly inefficient in human cells due to random integration events, Here the authors show that dual repression of polymerase θ and DNA ligase IV eliminate random integration events.
- Shinta Saito
- , Ryo Maeda
- & Noritaka Adachi
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Article
| Open AccessAptazyme-embedded guide RNAs enable ligand-responsive genome editing and transcriptional activation
CRISPR-Cas9 is a powerful technique for manipulating the human genome, however temporal and spatial control of activity would facilitate safer, more selective use. Here the authors incorporate aptazymes into guide RNAs to allow small molecule activation of CRISPR-Cas9.
- Weixin Tang
- , Johnny H. Hu
- & David R. Liu
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Article
| Open AccessHit and go CAS9 delivered through a lentiviral based self-limiting circuit
While CRISPR-Cas9 is a powerful technology, it’sin vivoapplication can be limited by unwanted off-target editing events. Here the authors present SLiCES, a self-limiting Cas9 circuit to enhance editing by preventing residual nuclease activity.
- Gianluca Petris
- , Antonio Casini
- & Anna Cereseto
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Article
| Open AccessPABPN1 gene therapy for oculopharyngeal muscular dystrophy
Oculopharyngeal muscular dystrophy is caused by trinucleotide repeat expansions in thePABPN1gene. Here the authors use AAV-based gene therapy to knockdown the mutant gene and replace it with a wild-type allele, and show effectiveness in mice and in patient cells.
- A. Malerba
- , P. Klein
- & G. Dickson
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Article
| Open AccessEnhancing titres of therapeutic viral vectors using the transgene repression in vector production (TRiP) system
The maximum titre of therapeutic viral vectors can be adversely affected by the encoded transgene. Here the authors repress transgene expression in producing cells by employing the tryptophan RNA-binding attenuation protein and show that it improves titre of RNA- and DNA-based viral vectors expressing toxic transgenes.
- H. E. Maunder
- , J. Wright
- & D. C. Farley
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Article
| Open AccessSemi-automated closed system manufacturing of lentivirus gene-modified haematopoietic stem cells for gene therapy
Current methods for haematopoietic stem cell gene therapy are laborious and require special licensed facilities. Here the authors develop a semi-automated protocol using a commercially available device to allow for benchtop generation of gene-modified blood cell products for transplantation, that meet current standards.
- Jennifer E. Adair
- , Timothy Waters
- & Hans-Peter Kiem
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Article
| Open AccessEngineering prokaryotic channels for control of mammalian tissue excitability
Restoring lost excitability of injured tissue is a paramount of regenerative medicine. By using a combined expression of bacterial voltage-gated Na+ channel, Kir2.1, and connexin-43 in non-excitable human fibroblasts, here the authors generate excitable cells that rescue action potential conduction in an in vitromodel of cardiac fibrosis.
- Hung X. Nguyen
- , Robert D. Kirkton
- & Nenad Bursac
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Article
| Open AccessRationally engineered Troponin C modulates in vivo cardiac function and performance in health and disease
Heart contraction, which is decreased in disease, is determined by Ca2+binding to troponin C. Here, the authors combine a protein engineering approach with gene therapy to modulate heart contractility in mice with the use of rationally designed Troponin C variants, suggesting a new therapy for diseased hearts.
- Vikram Shettigar
- , Bo Zhang
- & Jonathan P. Davis
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Article
| Open AccessDevelopment and rescue of human familial hypercholesterolaemia in a xenograft mouse model
Familial hypercholesterolemia (FH) is a congenital disease associated with high plasma cholesterol levels. Here, the authors recapitulate FH in chimeric mice, in which livers are repopulated with hepatocytes from an FH patient, and successfully correct the disease using adenovirus-mediated gene therapy.
- Beatrice Bissig-Choisat
- , Lili Wang
- & Karl-Dimiter Bissig
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Article
| Open AccessGene therapy restores vision in rd1 mice after removal of a confounding mutation in Gpr179
The rd1 mouse is the most widely used model to study retinal degeneration. Here, the authors identify a wide-spread mutation in these mice that may explain the failure of previous gene therapeutic approaches and show that long-lasting restoration of vision is possible in rd1 mice without this mutation.
- Koji M. Nishiguchi
- , Livia S. Carvalho
- & Robin R. Ali
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Article |
Mybpc3 gene therapy for neonatal cardiomyopathy enables long-term disease prevention in mice
Hereditary hypertrophic cardiomyopathy (HCM) is caused by mutations in cardiomyocyte genes, such as MYBPC3. Here, the authors use virus-mediated gene therapy to correct Mycbpc3mutations in 1-day-old mice and, by administering just a single dose, prevent development of HCM over a period of 34 weeks.
- Giulia Mearini
- , Doreen Stimpel
- & Lucie Carrier
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Cardiac arrhythmia induced by genetic silencing of ‘funny’ (f) channels is rescued by GIRK4 inactivation
The ‘funny’ current (If) is important for the generation and regulation of the heart’s automaticity. Here the authors show that If silencing through genetic modification of the f-channel component HCN4 causes heart arrhythmia by altering Ca2+handling in pacemaker myocytes.
- Pietro Mesirca
- , Jacqueline Alig
- & Matteo E. Mangoni
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Article
| Open AccessPlatelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A
Haemophilia is a genetic bleeding disorder associated with a deficiency in the coagulation factor VIII. Here, the authors use gene therapy to achieve stable overexpression of factor VIII in platelets of dogs with haemophilia A, preventing the occurrence of severe bleeding episodes for over 2.5 years.
- Lily M. Du
- , Paquita Nurden
- & David A. Wilcox
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An ex vivo gene therapy approach to treat muscular dystrophy using inducible pluripotent stem cells
Patient-specific induced pluripotent stem (iPS) cells hold great potential for regenerative cell therapies. Here Filareto et al. genetically correct iPS cells from mice with muscular dystrophy and use these cells to treat the same animals, providing a proof-of-principle for autologous iPS cell therapy.
- Antonio Filareto
- , Sarah Parker
- & Rita C. R. Perlingeiro