CRISPR /Cas9‐mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse
2016; Springer Nature; Volume: 8; Issue: 5 Linguagem: Inglês
10.15252/emmm.201506039
ISSN1757-4684
AutoresYuting Guan, Yanlin Ma, Qi Li, Zhenliang Sun, Lie Ma, Lijuan Wu, Liren Wang, Li Zeng, Yanjiao Shao, Yuting Chen, Ning Ma, Wenqing Lü, Kewen Hu, Honghui Han, Yanhong Yu, Yuanhua Huang, Mingyao Liu, Dali Li,
Tópico(s)CAR-T cell therapy research
ResumoResearch Article10 March 2016Open Access Source DataTransparent process CRISPR/Cas9-mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse Yuting Guan Yuting Guan Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Search for more papers by this author Yanlin Ma Corresponding Author Yanlin Ma Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Hainan Reproductive Medical Center, the Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou, China Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, China Search for more papers by this author Qi Li Qi Li Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Hainan Reproductive Medical Center, the Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou, China Search for more papers by this author Zhenliang Sun Zhenliang Sun Fengxian Hospital affiliated to Southern Medical University, Shanghai, China Search for more papers by this author Lie Ma Lie Ma Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Search for more papers by this author Lijuan Wu Lijuan Wu Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Search for more papers by this author Liren Wang Liren Wang Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Search for more papers by this author Li Zeng Li Zeng Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Search for more papers by this author Yanjiao Shao Yanjiao Shao Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Search for more papers by this author Yuting Chen Yuting Chen Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Search for more papers by this author Ning Ma Ning Ma Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Hainan Reproductive Medical Center, the Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou, China Search for more papers by this author Wenqing Lu Wenqing Lu Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Search for more papers by this author Kewen Hu Kewen Hu Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Search for more papers by this author Honghui Han Honghui Han Bioray Laboratories Inc., Shanghai, China Search for more papers by this author Yanhong Yu Yanhong Yu Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, China Search for more papers by this author Yuanhua Huang Yuanhua Huang Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Hainan Reproductive Medical Center, the Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou, China Search for more papers by this author Mingyao Liu Corresponding Author Mingyao Liu Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Department of Molecular and Cellular Medicine, The Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, TX, USA Search for more papers by this author Dali Li Corresponding Author Dali Li Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Search for more papers by this author Yuting Guan Yuting Guan Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Search for more papers by this author Yanlin Ma Corresponding Author Yanlin Ma Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Hainan Reproductive Medical Center, the Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou, China Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, China Search for more papers by this author Qi Li Qi Li Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Hainan Reproductive Medical Center, the Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou, China Search for more papers by this author Zhenliang Sun Zhenliang Sun Fengxian Hospital affiliated to Southern Medical University, Shanghai, China Search for more papers by this author Lie Ma Lie Ma Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Search for more papers by this author Lijuan Wu Lijuan Wu Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Search for more papers by this author Liren Wang Liren Wang Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Search for more papers by this author Li Zeng Li Zeng Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Search for more papers by this author Yanjiao Shao Yanjiao Shao Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Search for more papers by this author Yuting Chen Yuting Chen Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Search for more papers by this author Ning Ma Ning Ma Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Hainan Reproductive Medical Center, the Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou, China Search for more papers by this author Wenqing Lu Wenqing Lu Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Search for more papers by this author Kewen Hu Kewen Hu Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Search for more papers by this author Honghui Han Honghui Han Bioray Laboratories Inc., Shanghai, China Search for more papers by this author Yanhong Yu Yanhong Yu Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, China Search for more papers by this author Yuanhua Huang Yuanhua Huang Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Hainan Reproductive Medical Center, the Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou, China Search for more papers by this author Mingyao Liu Corresponding Author Mingyao Liu Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Department of Molecular and Cellular Medicine, The Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, TX, USA Search for more papers by this author Dali Li Corresponding Author Dali Li Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China Search for more papers by this author Author Information Yuting Guan1,‡, Yanlin Ma 2,3,‡, Qi Li2, Zhenliang Sun4, Lie Ma1, Lijuan Wu1, Liren Wang1, Li Zeng1, Yanjiao Shao1, Yuting Chen1, Ning Ma2, Wenqing Lu1, Kewen Hu1, Honghui Han5, Yanhong Yu3, Yuanhua Huang2, Mingyao Liu 1,6 and Dali Li 1 1Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China 2Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Hainan Reproductive Medical Center, the Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou, China 3Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, China 4Fengxian Hospital affiliated to Southern Medical University, Shanghai, China 5Bioray Laboratories Inc., Shanghai, China 6Department of Molecular and Cellular Medicine, The Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, TX, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +86 898 66776091; E-mail: [email protected] *Corresponding author. Tel: +86 021 54345014; E-mail: [email protected] *Corresponding author. Tel: +86 021 24206824; E-mail: [email protected] EMBO Mol Med (2016)8:477-488https://doi.org/10.15252/emmm.201506039 See also: TH Nguyen & I Anegon (May 2016) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The X-linked genetic bleeding disorder caused by deficiency of coagulator factor IX, hemophilia B, is a disease ideally suited for gene therapy with genome editing technology. Here, we identify a family with hemophilia B carrying a novel mutation, Y371D, in the human F9 gene. The CRISPR/Cas9 system was used to generate distinct genetically modified mouse models and confirmed that the novel Y371D mutation resulted in a more severe hemophilia B phenotype than the previously identified Y371S mutation. To develop therapeutic strategies targeting this mutation, we subsequently compared naked DNA constructs versus adenoviral vectors to deliver Cas9 components targeting the F9 Y371D mutation in adult mice. After treatment, hemophilia B mice receiving naked DNA constructs exhibited correction of over 0.56% of F9 alleles in hepatocytes, which was sufficient to restore hemostasis. In contrast, the adenoviral delivery system resulted in a higher corrective efficiency but no therapeutic effects due to severe hepatic toxicity. Our studies suggest that CRISPR/Cas-mediated in situ genome editing could be a feasible therapeutic strategy for human hereditary diseases, although an efficient and clinically relevant delivery system is required for further clinical studies. Synopsis CRISPR/Cas9-mediated genome editing holds promise for the treatment of genetic disorders, but its potential for hemophilia treatment is unknown. This study shows that in genome correction via Cas9 is a feasible therapeutic strategy for hemophilia B. Identification a family with hemophilia B carrying a novel mutation, Y371D, in the human F9 gene. Generation of three distinct genetically modified mouse models and confirmation that the mouse harboring the novel Y371D mutation is a new hemophilia B model. Hepatic in situ correction of the point mutation in the F9 allele via CRISPR/Cas9-mediated genome editing was sufficient to restore hemostasis in hemophilia B mice. Introduction Hemophilia B (HB), an X-linked genetic bleeding disorder caused by deficiency of coagulator factor IX (FIX), affects 1 of every 25,000 to 30,000 males worldwide (Thompson & Chen, 1993). Based on the FIX plasma procoagulant levels, the disease is classified as mild (5–40% of normal activity), moderate (1–5% of normal activity), and severe (< 1% of normal activity; White et al, 2001). As solely increasing the plasma FIX levels as low as 1% results in significant restoration of clotting activity, HB is considered a good model for evaluating the efficacy of distinct gene therapy strategies. Introducing the F9 gene cDNA into the liver (the natural source of FIX secretion) of HB animal models and patients through recombinant adeno-associated virus (rAAV) has proven to be efficacious (Kay et al, 1993, 2000; Snyder et al, 1997; Nathwani et al, 2014), but two major concerns remain: the duration of expression and the safety issue of AAV-mediated random insertion of the transgene into the host genome. Sleeping Beauty transposon-induced random insertion of the FIX transgene in HB mice has shown that integration into the host genome is feasible for long-term and high-level FIX expression (Yant et al, 2000). Recent studies demonstrated that site-specific integration of human F9 cDNA into transcriptionally highly active genomic loci in hepatocytes through either a nuclease (zinc finger nuclease) dependent or independent manner successfully ameliorated the disease in mice (Li et al, 2011; Anguela et al, 2013; Barzel et al, 2015). These strategies theoretically overcome the significant concerns of transgene expression duration and random insertion-induced safety issue, but whether direct correction of the F9 mutation by targeting the endogenous locus is sufficient for restoration of clotting activity through somatic genome editing is still not determined. The CRISPR/Cas9 system developed from an RNA-mediated adaptive immune system identified in bacteria is a revolutionary technology for gene editing in cells and organisms (Jinek et al, 2012; Cong et al, 2013; Hwang et al, 2013; Li et al, 2013; Mali et al, 2013; Shen et al, 2013; Wang et al, 2013; Doudna & Charpentier, 2014; Niu et al, 2014). Cas9/sgRNA induces site-specific DNA double-strand breaks (DSBs) which then initiate either error-prone non-homologous end joining (NHEJ) or homology-directed repair (HDR) in the presence of donor DNA templates (Cong et al, 2013; Mali et al, 2013). Pioneer studies have shown potential applications of the CRISPR/Cas9 system for correction of genetic disorders in cells or mouse embryos (Schwank et al, 2013; Wu et al, 2013; Long et al, 2014), as well as prevention of cardiovascular disease in adult mice through disruption of the PCSK9 gene (Ding et al, 2014). Recently, a mouse model of hereditary tyrosinemia type I(HT1)caused by a point mutation of fumarylacetoacetate hydrolase (FAH) was phenotypically restored via Cas9-mediated gene repair in vivo (Yin et al, 2014). In this particular model, the repaired cells had a survival advantage and expanded to replace the mutant hepatocytes when the pharmacological inhibitor NTBC was withdrawn (Yin et al, 2014). As the initial correction rate in the model is lower to 0.4%, it is essential to investigate the efficacy of Cas9-mediated in vivo genetic correction of other heritable diseases in which the repaired cells cannot be easily selected for repopulation. Here, we identify a family with HB carrying a novel FIX mutation which is confirmed as an HB causative mutation through generation of mice with an identical mutation via the CRISPR/Cas9 system. Delivery of the Cas9 component in vivo resulted in correction of over 0.56% of endogenous F9 alleles in hepatocytes and restored hemostasis in mice. Our data strongly demonstrate that correction of genetic disorders through repair of mutations in situ via CRISPR/Cas9-mediated genome editing is feasible. Results Identification of novel F9 mutation in HB patients A 9-year-old male proband (Fig 1A IV:2) was diagnosed HB with an abnormal activated partial thromboplastin time (aPTT) of 84 s (reference values: 25 s ~35 s) with a normal factor VIII activity. His clotting activity was remarkably decreased to about 2% of the normal level (Table 1). By taking a detailed family history, five patients were identified in the pedigree (Fig 1A). A single missense mutation in exon 8 of F9 was identified in all tested patients. The mutation causes a thymidine-to-guanine transversion at nucleotide position 31094, replacing tyrosine with aspartate at amino acid 371 (Fig 1B). This mutation is a novel variation which has not been reported either in the Hemophilia B Mutation Database (http://www.factorix.org/; Thompson & Chen, 1993) or the Human Gene Mutation Database (HGMD, http://www.hgmd.cf.ac.uk/ac/index.php), although a 31095A>C variation has been reported in HGMD leading to a Y371S mutation causing mild HB (20% of normal FIX activity). As residue 371 is located in the highly conserved serine protease domain (Fig 1C), we decided to generate mouse models containing the corresponding mutations to further confirm the phenotype as well as to explore the potential of CRISPR/Cas9-mediated genome editing for amelioration of HB in mice. Figure 1. Characterization of a F9 gene mutation in a family with a history of bleeding diathesis Family pedigree. Blackened symbols indicate the patients with hemophilia B; white circles with dots show carriers of the mutation; open symbols indicate healthy individuals. Circles represent females; squares represent males. The proband is labeled with an arrow. The partial sequences of the F9 gene from a healthy subject (left), the patient (middle), and a carrier (right). Amino acid alignment of partial sequence of the serine protease domain of FIX in 10 species. An asterisk (*) indicates positions which have a single, fully conserved residue. A colon (:) indicates conservation between groups of strongly similar properties. A period (.) indicates conservation between groups of weakly similar properties. Download figure Download PowerPoint Table 1. Factor IX clotting activity of members in the hemophilia B family Subject FIX functional activity (% of normal level) Proband (IV2) 2 Proband's twin brother (IV3) 2 Proband's non-twin brother (IV1) 70 Proband's grandfather (II1) 3 Proband's aunt (III3) 55 Normal FIX activity range (%): > 45 Generation and characterization of F9 mutant mouse strains We employed the Cas9 system as previously described (Shao et al, 2014) to introduce a precise point mutation in the mouse F9 locus corresponding to human amino acid residue 371 (position 381 in mouse FIX, Fig 1C). After injection of Cas9 components and distinct donor templates into mouse zygotes (Fig 2A), three mouse strains, respectively, bearing the novel mutant site F9Y381D and two previously reported sites (F9Y381S and F9383STOP) were generated (Fig 2B and Appendix Fig S1). No off-target cleavage was detected through T7E1 analysis and sequencing of the 10 most likely potential off-target sites (Appendix Fig S2). The F9 mRNA level in liver tissue was not impaired in F9Y381D and F9Y381S mice but significantly decreased in F9383STOP mice since the premature stop codon dramatically affects mRNA stability (Fig 2C). Consistently, the protein level was almost invisible in F9383STOP mice but not in F9Y381D mice compared to wild-type controls (Fig 2C), suggesting that the Y381D mutation does not influence actual FIX levels and stability. The average aPTT for 8-week-old wild-type mice was 22.04 ± 0.77 s (n = 5), but aPTTs were prolonged to 41.78 ± 1.18 s and 45.684 ± 1.10 s for the F9383STOP (n = 5) and the F9Y381D (n = 5) mouse strains, respectively (Fig 2D). The aPTT for the F9Y381S strain was not significantly increased (aPTT = 22.71 ± 1.17 s; n = 5; Fig 2D) which was consistent with observations in humans. No significant difference of average prothrombin time (PT) was observed in 8-week-old wild-type, F9Y381S, F9Y381D, and F9383STOP mouse strains (Fig 2E). The prolonged aPTT and the normal PT demonstrated that the F9Y381D and F9383STOP mouse strains generated in this study are models for HB. Additionally, these strains were subjected to a tail-clip challenge for further confirmation of defects in hemostasis. The blood loss volume of the 4 strains of mice in 5 min after tail-clip was recorded as shown in Fig 2F. F9383STOP mice (n = 5) lost more than 10 times the volume of blood lost by wild-type controls (n = 5), but no significant difference was observed between wild-type controls and F9Y381S (n = 6) or F9Y381D mice (n = 13), suggesting that the Y381D mutation does not cause very severe HB in mice. However, the survival rate of F9383STOP and F9Y381D mice was significantly decreased in the two days after the tail-clip challenge (Fig 2G). Taken together, these data indicate the successful generation of HB mouse models and suggest that the Y381D and 383STOP mutations dramatically disrupted FIX activity. Figure 2. Generation and characterization of variant F9 mutant mouse strains Schematic diagram of the strategy to generate F9Y381D and F9Y381S mouse strains. Introduced mutant oligonucleotide is in red, and the corresponding amino acids are in green. Sanger sequencing showing the point mutations in F9 mutant strains. Mutated oligonucleotides are indicated by arrows, and the premature stop codon is underlined. Expression of F9 mRNA and protein in hepatic tissue of indicated mouse strains. Data represent means ± SE. The significant effect was obtained using the two-tailed unpaired Student's t-test to determine the P?value. M: Marker; arrowhead: FIX protein; Note: the bands of FIX in F9383STOP lanes are faint with small molecular weight smears. The gel is representative of three independent experiments. Measurement of coagulation activity by aPTT in mice at 8 weeks of age. n = 5 for each group, data represent means ± SE. P?value was determined using two-tailed unpaired Student's t-test. Measurement of coagulation activity by PT in mice at 8 weeks of age. n = 5 for each group, data represent means ± SE. Measurement of blood loss over a 5-min period after tail transection in mice at 8 weeks of age. Wild-type, n = 5; F9Y381S, n = 6; F9Y381D, n = 13; F9383STOP, n = 5. P-value was determined using two-tailed unpaired Student's t-test. Survival rate of mice after the tail-clip challenge. The mice were monitored for 2 days after tail clipping. Wild-type, n = 5; F9Y381S, n = 6; F9Y381D, n = 13; F9383STOP, n = 5. Source data are available online for this figure. Source Data for Figure 2 [emmm201506039-sup-0003-SDataFigure2.pdf] Download figure Download PowerPoint Restoration of hemostasis in HB mice through naked DNA injection of Cas9 components To explore whether correction of a mutated F9 gene in situ can restore clotting activity in adult HB mice, we employed CRISPR/Cas9 system-mediated genome editing. As DNA-based vectors do not have viral contaminants and almost no immunogenicity (Kay, 2011), we first explored hydrodynamic tail vein (HTV) injection, a sophisticated strategy for in vivo gene delivery in animals. The pX458 plasmid containing Cas9-2A-GFP components (Fig 3A) was delivered through our modified HTV injection procedure to test the transgenic efficiency. Twenty hours after pX458 plasmid injection, liver tissue was obtained and eGFP expression was detected in 18.2 ± 3.1% of hepatocytes on average (Appendix Fig S3). Donor templates as either a 120-nt ssODN or a plasmid were delivered with pX458 through HTV injection (Fig 3A). To prevent donor DNA cleavage by Cas9/sgRNA, the HDR donor plasmid contains the G>T corrected nucleotide and 10 synonymous single-nucleotide exchanges which are flanked by about 400-bp homologous arms on each side (Fig 3A and E). The average aPTTs for WT and F9Y381D mutant mice were 22.1 ± 1.75 s and 51.88 ± 2.71 s, respectively (Fig 3B). Eight weeks after injection of Cas9/ssODN or Cas9/donor plasmids, the average PT was not affected but aPTTs significantly dropped to 32.7 ± 2.02 s (P = 0.0004 compared to F9Y381D) and 31.6 ± 4.99 s (P = 0.0046 compared to F9Y381D), respectively (Fig 3B). Additionally, the aPTT of Cas9/donor plasmid (px458 + donor)-treated F9Y381D mice was significantly shortened compared to the mock-treated (mock + donor) group (P = 0.0306), but had no significant difference compared to the WT group (P = 0.0892). Similar results were also obtained when we used ssODN as donor templates (Fig 3B). To further confirm the therapeutic effect, tail-clip challenge assay was performed. The survival rate was increased from 38% (5 out of 13) in the untreated group to 86% (12 out of 14) in F9Y381D mice which had received Cas9/donor plasmid injection (Fig 3C). Our data suggest that Cas9-mediated hepatic genome editing corrected the F9 mutation in situ and significantly restored the coagulation activity of adult F9Y381D mice. Sequencing analysis of 177 TA-clones suggested that the indel rate and HDR rate both were 0.56% in the ssODN group (Fig 3D). Through deep sequencing analysis, the modification efficiency was determined in the group that received plasmid donor and Cas9/sgRNA. About 4.39% of F9 alleles were modified, including 2.84% indel mutations and 1.55% G>T corrections (Fig 3D and E). In three randomly selected individual mice, the highest HDR rate was 2.84% with 2.77% indel mutations (Fig 3F), suggesting that the plasmid donor exhibits a high fidelity of corrective repair. The majority of HDRs resulted in the desired G>T correction and total or partial synonymous substitutions (Fig 3E). These data suggested that the presence of the corrected gene in about 0.56% of endogenous F9 alleles is sufficient to restore clotting activity. No significant difference in blood aspartate transaminase (AST) and alanine transaminase (ALT) levels between control and naked DNA-injected group was observed at 8 weeks (Fig 3G), and liver tissues were histologically normal despite a mild increase of inflammatory cytokine mRNA levels (Fig 3H), suggesting that HTV injection of naked DNA vectors was well tolerated in accordance with a previous report (Yin et al, 2014). Figure 3. Amelioration of HB in F9 mutant mice through naked DNA injection of Cas9 components Schematic diagram of plasmids used for treatment in vivo. Cas9 protein and sgRNA were from pX458 vector. The plasmid donor was 800 bp in length in pEASY vector. The ssODN donor was 120 oligonucleotides. Test of clotting activity by aPTT and PT at 8 weeks following hydrodynamic tail vein injection of 120 μg pX458 and 120 μg donor plasmids (or 120 μg ssODN) per mouse. Data are presented as mean ± SE. The experiment was replicated three times. P?value was determined using two-tailed unpaired Student's t-test. Survival rate of mice after the tail-clip challenge. F9Y381D mice were treated with or without Cas9/sgRNA/donor DNA for 3 months. After tail-clip challenge, the survival rate of each group over 2 days was determined. Wild-type, n = 9; untreated F9Y381D, n = 13; DNA-treated F9Y381D, n = 14. Frequency of genetic modification in F9Y381D hepatocytes was determined either by deep sequencing (Cas9/donor vector-treated group) or by TA-clone sequencing (Cas9/ssODN group). HDR donor design for correction of Y381D and representative Illumina sequencing reads (rn) in DNA-treated HB mice. Red text indicates the correction of mutation, whereas blue text indicates the synonymous mutations. The genome editing efficiency of individual mice is presented. Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) level was determined in F9Y381D mice 8 weeks after HTV injection (n = 5). Data are presented as mean ± SE. P?value was determined using two-tailed unpaired Student's t-test. mRNA levels of inflammatory cytokines from liver tissue of naked DNA-treated mice were determined by real-time PCR. Data represent means ± SE. The experiment was replicated three times. P?value was determined using two-tailed unpaired Student's t-test. Download figure Download PowerPoint Genetic correction of F9 mutation via Cas9 system delivered through recombinant adenovirus Next, we sought to increase the transduction efficiency of the Cas9 components to achieve a higher HDR rate in mouse hepatocytes. An adenoviral (Adv) gene delivery system was employed due to its large DNA capacity, high efficiency, and non-integration into the host genome (Crystal, 2014). More importantly, genome editing using a protein-capped AdV was more accurate than using other vectors (Holkers et al, 2014). We generated adenoviral Cas9 (AdvCas9) and a vector containing the corrective donor template following a sgRNA target (AdvG/T; Fig 4A). To test the infection and editing efficiency, 1 × 1010 and 7 × 1010 vector genomes of AdvCas9 and AdvG/T were delivered per mouse via tail vein injection. Four days later, almost all hepatocytes were infected (Fig 4B) and caused an about 19% mutation frequency detected through T7E1 assays (Fig 4C and Appendix Table S5). Additionally, no off-target mutations were detected in hepatocytes (Appendix Fig S4). To our surprise, the aPTTs were not shortened in F9Y381D mice 8 weeks after treatment in both the low-dose group (1 × 1010 and 1 × 1010 vector genomes of AdvCas9 and AdvG/T) and the high-dose group (1 × 1010 and 7 × 1010 vector genomes of AdvCas9 and AdvG/T) compared with untreated F9Y381D mice (Fig 4D). The indel rate and HDR rate of t
Referência(s)