scholarly journals The position of the target site for engineered nucleases improves the aberrant mRNA clearance in in vivo genome editing

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jae Hoon Lee ◽  
Sungsook Yu ◽  
Tae Wook Nam ◽  
Jae-il Roh ◽  
Young Jin ◽  
...  
Keyword(s):  
Genome Editing ◽  
Target Site ◽  
Engineered Nucleases

Robust Factor IX Expression Following ZFN-Mediated Genome Editing in An Adult Mouse Model of Hemophilia B

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 668-668
Author(s):  
Xavier M Anguela ◽  
Rajiv Sharma ◽  
Hojun Li ◽  
Virginia Haurigot ◽  
Anand Bhagwat ◽  
...  
Keyword(s):  
Mouse Model ◽  
Genome Editing ◽  
Partial Hepatectomy ◽  
Transgene Expression ◽  
Target Site ◽  
Factor Ix ◽  
Regulatory Control ◽  
Neonatal Mice ◽  
Proliferation Of Hepatocytes

Abstract Abstract 668 As a therapeutic strategy, site-specific modification of the genome has the potential to avoid some of the disadvantages of traditional gene replacement approaches such as insertional mutagenesis and lack of endogenous regulatory control of expression. We have recently reported that zinc finger nuclease (ZFN) driven gene correction can be achieved in vivo in a neonatal mouse model of hemophilia by combining AAV-mediated delivery of both the ZFNs and a Factor IX donor template with homology to the targeted F.IX gene (Li et al., Nature, 2011). The mouse model carries a mutant human F.IX mini-gene (hF9mut) knocked into the ROSA26 locus and ZFN-mediated cleavage followed by donor-dependent repair results in restoration of functional F.IX expression. AAV-ZFN and AAV-Donor vectors were administered to neonatal mice, where the rapid proliferation of hepatocytes in the growing animal may promote genome editing through homology directed repair (HDR). Here we sought to investigate whether ZFN-mediated genome editing is feasible in adult animals with predominantly quiescent hepatocytes. Tail vein injection of the AAV-ZFN and AAV-Donor, containing a promoterless wild type factor IX insert flanked by arms of homology to the target site, into adult (8 week old) mice (n=17) resulted in stable (>10wk) circulating F.IX levels of 730–1900 ng/mL (15-38% of normal), whereas mice receiving ZFN alone (n=9) exhibited F.IX levels below detection (<15 ng/mL). Co-delivery of AAV-Mock (luciferase expressing) & AAV-Donor (n=9), yielded <65 ng/mL F.IX. Importantly, mice lacking the hF9mut gene averaged less than 100 ng/mL after receiving AAV-ZFN and AAV-Donor (n=8), suggesting that F.IX expression was derived from on-target genome editing. To eliminate the potential for hF.IX expression resulting from episomal (non-integrated) AAV genomes we performed a two-thirds partial hepatectomy two days after AAV administration. Liver regeneration following hepatectomy is known to substantially reduce expression from non-integrated AAV genomes yet no significant differences in transgene expression were observed compared to non-hepatectomized mice: circulating F.IX levels in the AAV-ZFN + AAV-Donor group (n=13) ranged between 678–1240 ng/mL, whereas mice receiving ZFN alone (n=8) or Mock + AAV-Donor (n=8) had no detectable F.IX expression, or <100 ng/mL F.IX, respectively. Taken together, these data suggest that the F.IX expression in ZFN + Donor treated mice was derived from stable correction of the genome at the intended target site. In summary, we have shown that synchronized cell proliferation of hepatocytes, either in neonatal mice or following partial hepatectomy, is not necessary to achieve highly efficient genome editing and resultant high levels of transgene expression in vivo. These findings substantially expand the potential of ZFN-mediated genome editing as a therapeutic modality. Disclosures: Doyon: Sangamo Biosciences: Employment. Gregory:Sangamo Biosciences: Employment. Holmes:Sangamo Biosciences: Employment.

In Vivo Genome Editing in Neonatal Mouse Liver Preferentially Utilizes Homology Directed Repair

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4422-4422 ◽  
Author(s):  
Xavier M. Anguela ◽  
Rajiv Sharma ◽  
Yannick Doyon ◽  
Thomas Wechsler ◽  
David Paschon ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mouse Liver ◽  
Neonatal Mouse ◽  
Target Site ◽  
Hepatocyte Proliferation ◽  
Post Injection ◽  
Employment Equity ◽  
Homology Directed Repair ◽  
Equity Ownership

Abstract Genome editing has the potential to provide long-term therapeutic gene expression in vivo. We have previously demonstrated efficient editing in a mouse model of hemophilia B through liver-directed adeno-associated viral vector (AAV) delivery of a zinc finger nuclease (ZFN) pair and a corrective donor. We determined that homology is not necessary to achieve efficient levels of genome editing in adult mice, consistent with the fact that quiescent cells, including adult hepatocytes, are not thought to be amenable to homology directed repair (HDR). As a consequence of the donor containing a splice acceptor, both HDR and homology independent vector integration are capable of driving human factor 9 (hF.IX) expression. In this study we sought to determine whether hF.IX expression in mice treated as neonates, undergoing substantial hepatocyte proliferation, is predominantly the result of HDR or homology independent genome editing. Provided the efficacy is not substantially reduced, an HDR dependent approach would impose additional constraints on targeting. Treatment of neonatal hF9mut mice (harboring the ZFN target site) with 1x1011 vg AAV8-ZFN and 5x1011 vg AAV8-Donor via retro-orbital injection resulted in a drastic difference in hF.IX expression between donors with and without homology 10 weeks post injection (Homology: 1531 ± 174.5 ng/mL vs. No-homology: 146.1 ± 5.8 ng/mL; n=12 and 7, respectively). We next asked whether HDR could be stimulated even more specifically through the induction of DNA single strand breaks at the target site. We treated neonatal mice with homologous or non-homologous donors, as well as ZFNs or ZFNickases (in which one FokI nuclease domain was inactivated with the D450A mutation). ZFNickases were indeed active, resulting in ~250 ng/mL hF.IX 4 weeks post injection (Figure 1). Interestingly, we could not detect hF.IX in mice treated with ZFNickase and no-homology donor (LOD: 15ng/mL). To rule out the possibility that this was simply due to the lower efficacy of ZFNickases compared to ZFNs, we increased the ZFNickase dose 4 fold. Four weeks post treatment, we observed substantial levels of hF.IX in mice treated with homologous donor (2041 ± 269 ng/mL) and were again unable to detect hF.IX in mice treated with the non-homologous donor (n=10 and 7, respectively). These data point to homology directed repair as the primary mechanism of protein production for genome editing in neonatal mouse liver, and suggest improvements in both efficacy and specificity can be made through deeper understanding of the molecular requirements of this approach. Figure 1. Figure 1. Disclosures Anguela: Spark Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Doyon:Sangamo BioSciences: Employment. Wechsler:Sangamo BioSciences: Employment. Paschon:Sangamo BioSciences: Employment. Davidson:Spark Therapeutics: Consultancy. Gregory:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. High:Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties.

scholarly journals A therapeutic genome editing primer for cardiologist

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Martina Caiazza ◽  
Daniele Masarone ◽  
Giuseppe Limongelli
Keyword(s):  
Lipid Metabolism ◽  
Animal Models ◽  
Genome Editing ◽  
Genome Engineering ◽  
Living Organism ◽  
Cardiovascular Disorder ◽  
Cardiovascular Disorders ◽  
New Class ◽  
Engineered Nucleases

Genome editing, or genome engineering is a type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of a living organism using engineered nucleases, or molecular scissors. Genome editing is being rapidly adopted into all fields of biomedical research, including the cardiovascular field, where it has facilitated a greater understanding of lipid metabolism, electrophysiology, cardiomyopathies, and other cardiovascular disorders, has helped to create a wider variety of cellular and animal models, and has opened the door to a new class of therapies. In this review, we discuss the applications of in vivo genome-editing therapies for cardiovascular disorder.

scholarly journals Points of View on the Tools for Genome/Gene Editing

2021 ◽  
Vol 22 (18) ◽  
pp. 9872
Author(s):  
Chin-Kai Chuang ◽  
Wei-Ming Lin
Keyword(s):  
Genome Editing ◽  
Dna Sequence ◽  
Nuclease Activity ◽  
Target Site ◽  
Homing Endonucleases ◽  
Null Mutant ◽  
Complementary Strand ◽  
Guide Rna ◽  
Strand Breakage

Theoretically, a DNA sequence-specific recognition protein that can distinguish a DNA sequence equal to or more than 16 bp could be unique to mammalian genomes. Long-sequence-specific nucleases, such as naturally occurring Homing Endonucleases and artificially engineered ZFN, TALEN, and Cas9-sgRNA, have been developed and widely applied in genome editing. In contrast to other counterparts, which recognize DNA target sites by the protein moieties themselves, Cas9 uses a single-guide RNA (sgRNA) as a template for DNA target recognition. Due to the simplicity in designing and synthesizing a sgRNA for a target site, Cas9-sgRNA has become the most current tool for genome editing. Moreover, the RNA-guided DNA recognition activity of Cas9-sgRNA is independent of both of the nuclease activities of it on the complementary strand by the HNH domain and the non-complementary strand by the RuvC domain, and HNH nuclease activity null mutant (H840A) and RuvC nuclease activity null mutant (D10A) were identified. In accompaniment with the sgRNA, Cas9, Cas9(D10A), Cas9(H840A), and Cas9(D10A, H840A) can be used to achieve double strand breakage, complementary strand breakage, non-complementary strand breakage, and no breakage on-target site, respectively. Based on such unique characteristics, many engineered enzyme activities, such as DNA methylation, histone methylation, histone acetylation, cytidine deamination, adenine deamination, and primer-directed mutation, could be introduced within or around the target site. In order to prevent off-targeting by the lasting expression of Cas9 derivatives, a lot of transient expression methods, including the direct delivery of Cas9-sgRNA riboprotein, were developed. The issue of biosafety is indispensable in in vivo applications; Cas9-sgRNA packaged into virus-like particles or extracellular vesicles have been designed and some in vivo therapeutic trials have been reported.

scholarly journals In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Menglong Chen ◽  
Hui Shi ◽  
Shixue Gou ◽  
Xiaomin Wang ◽  
Lei Li ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Mouse Model ◽  
Genome Editing ◽  
Large Scale ◽  
Gene Editing ◽  
High Efficiency ◽  
Muscle Fibers ◽  
Muscle Cells

Abstract Background Mutations in the DMD gene encoding dystrophin—a critical structural element in muscle cells—cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. Methods In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46–54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. Results Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan. Conclusions We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

scholarly journals An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

2021 ◽  
Author(s):  
Eugene V. Gasanov ◽  
Justyna Jędrychowska ◽  
Michal Pastor ◽  
Malgorzata Wiweger ◽  
Axel Methner ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Target Site ◽  
Proof Of Concept ◽  
Intervention Site ◽  
End Joining ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Non Homologous End Joining

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

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