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Therapeutic homology-independent targeted integration in retina and liver
Limits of AAV-mediated gene therapy include targeting dominant mutations and inducing long-term transgene expression. Here, the authors show that AAV-HITI results in efficient allele-independent integration of a donor DNA in both retina and liver providing therapeutic benefit in mouse models of either a genetic form of blindness or a lysosomal storage disease, respectively.
- Patrizia Tornabene
- , Rita Ferla
- & Alberto Auricchio
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Article
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In vivo base editing rescues cone photoreceptors in a mouse model of early-onset inherited retinal degeneration
Leber congenital amaurosis is caused by mutations in RPE65 and leads to retinal degeneration in children. Here, the authors show that in vivo base editing can prolong the survival of cone photoreceptors and rescue their function in a mouse model of the disease.
- Elliot H. Choi
- , Susie Suh
- & Krzysztof Palczewski
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Article
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FrCas9 is a CRISPR/Cas9 system with high editing efficiency and fidelity
Gene editing tools have tremendous potential for biomedical and basic research. Here the authors report a Cas9 from Faecalibaculum rodentium (FrCas9) that achieves efficient and specific gene editing in human cells with a NNTA palindrome PAMs for targeting optimal sites at TATA-boxes to enhance CRISPRa/i screening.
- Zifeng Cui
- , Rui Tian
- & Zheng Hu
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Article
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Choice 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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A systematic review and meta-analysis of gene therapy with hematopoietic stem and progenitor cells for monogenic disorders
Ex-vivo gene therapy with hematopoietic stem and progenitor cells (HSPCs) is a promising treatment for monogenic diseases. Here the authors report a systematic review and meta-analysis of available evidence assessing clinical outcomes of HSPC gene therapy from clinical trials.
- Francesca Tucci
- , Stefania Galimberti
- & Alessandro Aiuti
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Self-attenuating adenovirus enables production of recombinant adeno-associated virus for high manufacturing yield without contamination
Scalability, yield and quality of recombinant AAVs are a significant issue during manufacture. Here the authors describe a self-inhibiting helper adenovirus strategy that improves outcomes compared to helper-free approaches.
- Weiheng Su
- , Maria I. Patrício
- & Ryan Cawood
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Crosstalk between CRISPR-Cas9 and the human transcriptome
The off-target effects of CRISPR-Cas9 are thought to be mediated by its cognate guide RNA. Here the authors show that Cas9 independently interacts with the human transcriptome, correlating with elevated RNA editing even under guide RNA co-expression.
- Aaron A. Smargon
- , Assael A. Madrigal
- & Gene W. Yeo
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Mutation-specific reporter for optimization and enrichment of prime editing
While prime editing is a promising technique, some genomic sites remain difficult to edit. Here the authors present fluoPEER, fluorescent prime editing and enrichment reporter, to rank the efficiency of pegRNAs and prime editor variants.
- I. F. Schene
- , I. P. Joore
- & S. A. Fuchs
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Article
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Engineered bacterial voltage-gated sodium channel platform for cardiac gene therapy
In this in vitro, in silico, and in vivo study Nguyen and colleagues show that specific and stable viral gene delivery of engineered prokaryotic voltage-gated sodium channels (BacNav) to cardiomyocytes can directly augment cardiac tissue excitability and conduction.
- Hung X. Nguyen
- , Tianyu Wu
- & Nenad Bursac
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Article
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Inhibition of base editors with anti-deaminases derived from viruses
Anti-deaminases can inhibit APOBEC3, a component of cytosine base editors. Here Zhanjun Li and colleagues repurposed anti-deaminase proteins derived from viruses to inhibit base editors for use in efficient regulation of base editors’ activity in gene modification and therapeutic applications.
- Zhiquan Liu
- , Siyu Chen
- & Zhanjun Li
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Article
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Correction of a Factor VIII genomic inversion with designer-recombinases
Correction of disease-causing large genomic inversions remains challenging. Here, the authors developed a dual designer-recombinase system (RecF8) that efficiently corrects a 140 kb inversion frequently found in patients with severe Hemophilia A.
- Felix Lansing
- , Liliya Mukhametzyanova
- & Frank Buchholz
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Article
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Low immunogenicity of LNP allows repeated administrations of CRISPR-Cas9 mRNA into skeletal muscle in mice
In vivo delivery of CRISPR-Cas9 holds promise for treating muscular dystrophy, however, AAV delivery is known to be immunogenic. Here, the authors show that LNP delivery of CRISPR-Cas9 enables repeated injections into skeletal muscle and leads to restored dystrophin expression in multiple muscle groups.
- Eriya Kenjo
- , Hiroyuki Hozumi
- & Akitsu Hotta
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Find and cut-and-transfer (FiCAT) mammalian genome engineering
Mammalian genome engineering has advanced tremendously over the last decade, however there is still a need for robust gene writing with size scaling capacity. Here the authors present Find Cut-and-Transfer (FiCAT) technology to delivery large targeted payload insertion in cell lines and in vivo in mouse models.
- Maria Pallarès-Masmitjà
- , Dimitrije Ivančić
- & Marc Güell
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A general theoretical framework to design base editors with reduced bystander effects
Base editors can edit target nucleotides, and identical ones that are within the editing window. Here the authors build an analytical model to propose general principles of editor design to reduce bystander effects.
- Qian Wang
- , Jie Yang
- & Anatoly B. Kolomeisky
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Hepatic expression of GAA results in enhanced enzyme bioavailability in mice and non-human primates
Pompe disease is currently treated with enzyme replacement therapy (ERT) with recombinant human acid alpha-glucosidase (GAA). Here, the authors show hepatic-directed gene therapy with AAV vectors enhances GAA bioavailability compared with ERT, resulting in improved rescue of the disease phenotype in mice and broad enzyme distribution in mice and non-human primates.
- Helena Costa-Verdera
- , Fanny Collaud
- & Federico Mingozzi
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Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo
Long-term expression of Cas9 following precision genome editing in vivo may lead to undesirable consequences. Here we show that a single-vector, self-inactivating AAV system containing Cas9 nuclease, guide, and DNA donor can use homology-directed repair to correct disease mutations in vivo.
- Raed Ibraheim
- , Phillip W. L. Tai
- & Erik J. Sontheimer
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Exosome-mediated stable epigenetic repression of HIV-1
A strategy to control HIV-1 infection is to stably repress HIV-1 and induce “deep latency”. Here the authors show that a recombinant anti-HIV-1-1 protein can be packaged as mRNA into exosomes and delivered systemically to repress HIV-1-1 within the context of virus infected mice and achieve long term silencing of HIV-1-1 expression.
- Surya Shrivastava
- , Roslyn M. Ray
- & Kevin V. Morris
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Article
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Treatment of skeletal and non-skeletal alterations of Mucopolysaccharidosis type IVA by AAV-mediated gene therapy
Mucopolysaccharidosis type IVA (MPSIVA) is a lysosomal storage disorder causing severe skeletal and non-skeletal alterations in patients. Here, the authors generate a MPSIVA rat model that mimics the disabling human pathology and develop an AAV9-Galns gene therapy to treat the disease.
- Joan Bertolin
- , Víctor Sánchez
- & Fatima Bosch
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Mirtron-mediated RNA knockdown/replacement therapy for the treatment of dominant retinitis pigmentosa
Rhodopsin-related dominant retinitis pigmentosa is a degenerative disease of the retina of the eye for which there is no current treatment. In this study, the authors use a novel form of RNA-interference- artificial mirtrons- to slow retinal degeneration in a mouse model of the disease.
- Harry O. Orlans
- , Michelle E. McClements
- & Robert E. MacLaren
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CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells
CRISPR-Cas9 generates double-strand breaks, which pose a threat to genome integrity. Here the authors show loss of heterozygosity in edited hematopoietic progenitor cells.
- J. Boutin
- , J. Rosier
- & A. Bedel
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Disrupting the LINC complex by AAV mediated gene transduction prevents progression of Lamin induced cardiomyopathy
Mutations in the LaminA gene are the second most common inherited cause of Dilated Cardiomyopathy, a major form of heart failure. Here the authors show that disruption of the nuclear protein SUN1 in cardiomyocytes, by AAV mediated transduction of a SUN1 inhibitor, significantly suppress cardiomyopathy progression, providing a potential therapeutic route to treat this disease.
- Ruth Jinfen Chai
- , Hendrikje Werner
- & Colin L. Stewart
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Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases
Small CRISPR Cas9 proteins have potential in gene therapies but generally have poor editing efficiency or specificity and often recognize sub-optimal PAM sequences. Here the authors characterise four small nucleases and use protein engineering to create effective in vivo editors.
- Moritz J. Schmidt
- , Ashish Gupta
- & Wayne M. Coco
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AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines
The utility of AsCas12a can be limited to poor editing efficiency. Here the authors identify a variant, “AsCas12a Ultra”, that has high on-target specificity demonstrated through editing of clinically relevant T cell genes.
- Liyang Zhang
- , John A. Zuris
- & Christopher A. Vakulskas
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Article
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Efficient precise in vivo base editing in adult dystrophic mice
Base editing is one approach used to correct mutations causing cause Duchenne muscular dystrophy (DMD), but limitations are in the requirement for a specific PAM motif and the large size beyond the packaging capacity of adeno-associated virus (AAV). Here, the authors modify the NG-targeting adenine base editor to recognize a broader PAM, devise an intein split strategy to package the otherwise oversized adenine base editor into AAV, and show it efficiently restores dystrophin expression in muscle and heart when systemically injected in a mouse model of DMD
- Li Xu
- , Chen Zhang
- & Renzhi Han
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Article
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Overexpression of human BAG3P209L in mice causes restrictive cardiomyopathy
An amino acid exchange (P209L) in the human co-chaperone BAG3 gives rise to severe childhood restrictive cardiomyopathy. Here the authors describe humanized transgenic mouse models which phenocopy the disease and provide insight into the pathogenic mechanisms.
- Kenichi Kimura
- , Astrid Ooms
- & Michael Hesse
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Mitochondrial targeted meganuclease as a platform to eliminate mutant mtDNA in vivo
Heteroplasmic mitochondrial DNA mutations lack effective treatments. Here the authors adapt I-CreI meganuclease to target the mitochondria and specifically-eliminate mtDNA with a m.5024C>T mutation in the mttRNA Ala gene.
- Ugne Zekonyte
- , Sandra R. Bacman
- & Carlos T. Moraes
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CRISPECTOR provides accurate estimation of genome editing translocation and off-target activity from comparative NGS data
The control of off-target activity is a challenge for adapting CRISPR to therapeutic use. Here the authors present CRISPECTOR, a software tool to detect, evaluate and quantify editing activity, including translocations, from NGS data.
- Ido Amit
- , Ortal Iancu
- & Zohar Yakhini
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Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice
Prime editors use a template sequence within their pegRNA to facilitate nucleotide substitutions or local indels. Here the authors use AAVs to deliver a split-intein prime editor in vivo to correct a pathogenic mutation.
- Pengpeng Liu
- , Shun-Qing Liang
- & Wen Xue
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Article
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Gene therapy via canalostomy approach preserves auditory and vestibular functions in a mouse model of Jervell and Lange-Nielsen syndrome type 2
Jervell and Lange-Nielsen syndrome is characterised by congenital deafness and vestibular dysfunction, and is caused by mutations in KCNE1 or KCNQ1. Here, the authors show that gene therapy via canalostomy at early postnatal stage can preserve the morphology of inner ear and auditory and vestibular functions in a mouse model of human JLNS2.
- Xuewen Wu
- , Li Zhang
- & Xi Lin
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Article
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Cas9-AAV6 gene correction of beta-globin in autologous HSCs improves sickle cell disease erythropoiesis in mice
CRISPR mediated gene correction of sickle cell disease (SCD) in patient-derived hematopoietic stem cells is a promising avenue for therapy. Here the authors use a humanized SCD mouse model to study gene editing in the context of autologous transplantation.
- Adam C. Wilkinson
- , Daniel P. Dever
- & Matthew H. Porteus
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The TRACE-Seq method tracks recombination alleles and identifies clonal reconstitution dynamics of gene targeted human hematopoietic stem cells
Genetic barcoding has been used to track clonal dynamics of cells. Here, the authors develop a Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), to barcode repaired alleles by introducing silent mutations or outside of coding regions, to show clonal complexity of edited CD34 + cells following engraftment.
- Rajiv Sharma
- , Daniel P. Dever
- & Ravindra Majeti
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Article
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MiCas9 increases large size gene knock-in rates and reduces undesirable on-target and off-target indel edits
Cas9 fused to DNA damage repair proteins can improve rates of gene knock-in but the chimeric protein is often large. Here the authors fuse Cas9 to a minimal Brex27 motif from BRCA2 consisting of thirty-six amino acids to enhance the efficacy and safety of gene editing.
- Linyuan Ma
- , Jinxue Ruan
- & Jie Xu
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CRISPR based editing of SIV proviral DNA in ART treated non-human primates
Removal of integrated HIV DNA remains a roadblock for HIV cure. Here, Mancuso et al. show that intravenous administration of an adeno-associated virus-based CRISPR/Cas9 gene editing construct to SIV-infected macaques results in excision of integrated proviral DNA from infected blood cells and tissues known to be viral reservoirs.
- Pietro Mancuso
- , Chen Chen
- & Kamel Khalili
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Docking sites inside Cas9 for adenine base editing diversification and RNA off-target elimination
Current SpCas9 adenine base editors that minimise RNA off-target activities have constrained editing windows. Here the authors use domain insertion of TadA into Cas9 to narrow, expand or shift the editing window with RNA off-target minimization simultaneously
- Shuo Li
- , Bo Yuan
- & Tian-Lin Cheng
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Article
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Vaccine elicitation of HIV broadly neutralizing antibodies from engineered B cells
A vaccine to generate durable HIV broadly neutralizing antibodies (bnAb) from engineered B cells holds promise as an HIV functional cure. Here, the authors show that CRISPR/Cas-modified B cells expressing bnAbs as functional antigen receptors can be immunized to generate long-lived, germinal centre matured bnAb memory and plasma cells in mice.
- Deli Huang
- , Jenny Tuyet Tran
- & James E. Voss
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Comment
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Turning genes into medicines—what have we learned from gene therapy drug development in the past decade?
Gene and cell therapy products approved over the past decade in Europe and North America have provided new therapeutic options for single gene disorders and for hematologic malignancies. Lessons learned, and limitations identified, are reviewed.
- Katherine A. High
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Comment
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The 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
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Identification 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
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Prime editing for functional repair in patient-derived disease models
Prime editing uses Cas9 nickase fused to a reverse transcriptase to edit genetic information. Here, the authors prime edit primary adult stem cells in 3D organoid cultures to show functional correction of pathogenic mutations without genome-wide off-target effects.
- Imre F. Schene
- , Indi P. Joore
- & Sabine A. Fuchs
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Article
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In vivo CRISPR/Cas9 targeting of fusion oncogenes for selective elimination of cancer cells
Fusion oncogenes (FO) are common in cancers, but specific targeting of these chimeric genes are challenging. Here the authors report a CRISPR/Cas9 strategy that targets two intronic regions to disrupt the FOs in cancer cells and show that this approach reduces tumour growth and prolongs survival in animal models of cancer.
- M. Martinez-Lage
- , R. Torres-Ruiz
- & S. Rodriguez-Perales
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Article
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Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing
Off-target effects and the feasibility for AAV-mediated delivery are the major barriers impeding the clinical in vivo application of base editors. Here, the authors report the small size AAV-deliverable Cas9-ABE variant that has improved on-target editing efficiency and reduced RNA-off target footprint.
- Minh Thuan Nguyen Tran
- , Mohd Khairul Nizam Mohd Khalid
- & Alex W. Hewitt
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Article
| Open Access
CRISPR GUARD protects off-target sites from Cas9 nuclease activity using short guide RNAs
Off-target editing remains a concern for therapeutic applications of CRISPR-Cas9. Here the authors present CRISPR GUARD, which uses very short non-cleaving gRNAs to prevent editing at off-target sites.
- Matthew A. Coelho
- , Etienne De Braekeleer
- & Benjamin J. M. Taylor
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Article
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Targeted gene correction of human hematopoietic stem cells for the treatment of Wiskott - Aldrich Syndrome
In recent years, hematopoietic stem cells gene editing has emerged as a promising tool to treat blood disorders. Here the authors develop a CRISPR/Cas9-based genome editing strategy that allows the precise correction of Wiskott-Aldrich Syndrome in vitro and in vivo with high efficiency.
- Rajeev Rai
- , Marianna Romito
- & Alessia Cavazza
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Article
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A lung tropic AAV vector improves survival in a mouse model of surfactant B deficiency
Surfactant protein B (SP-B) deficiency is a genetic lung disease that results in lethal respiratory distress within months of birth. Here, the authors describe a gene therapy strategy using a rationally designed AAV6 capsid that restores surfactant homeostasis, prevents lung injury, and improves survival in a mouse model of SP-B deficiency.
- Martin H. Kang
- , Laura P. van Lieshout
- & Bernard Thébaud
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Article
| Open Access
Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins
A platform for systemic therapeutic transgene expression independent of patient mutations needs a safe and highly transcribed locus. Here the authors ex vivo edit HPSCs using CRISPR-Cas9 to integrate transgenes under the α-globin promoter to achieve erythroid specific expression.
- Giulia Pavani
- , Marine Laurent
- & Mario Amendola
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Article
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Engineering 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
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Structural characterization of a novel human adeno-associated virus capsid with neurotropic properties
Adeno-associated viruses (AAVs) are vehicles for gene therapy in humans, but currently only a limited amount of AAV serotypes is available. Here, the authors identify a novel AAV, AAVv66, and demonstrate enhanced production yields, virion stability, and CNS transduction compared to the clinically approved serotype AAV2.
- Hung-Lun Hsu
- , Alexander Brown
- & Guangping Gao
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Article
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Improving 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 Access
Chemical 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
ZNF117 regulates glioblastoma stem cell differentiation towards oligodendroglial lineage