In vivo genome editing for hemophilia B therapy by the combination of rebalancing and therapeutic gene knockin using a viral and non-viral vector.

Recent therapeutic strategies for hemophilia include long-term therapeutic gene expression using adeno-associated virus (AAV) and rebalancing therapy via the downregulation of anticoagulant pathways. However, these approaches have limitations in immune responses or insufficiency to control acute bleeding. Thus, we developed a therapeutic strategy for hemophilia B by a combined rebalancing and human factor 9 (hF9) gene knockin (KI) using a lipid nanoparticle (LNP) and AAV. Antithrombin (AT; Serpin Family C Member 1 [Serpinc1]) was selected as the target anticoagulation pathway for the gene KI. First, the combined use of LNP-clustered regularly interspaced short palindromic repeats (CRISPR) and AAV donor resulted in 20% insertions or deletions (indels) in Serpinc1 and 67% reduction of blood mouse AT concentration. Second, hF9 coding sequences were integrated into approximately 3% of the target locus. hF9 KI yielded approximately 1,000 ng/mL human factor IX (hFIX) and restored coagulation activity to a normal level. LNP-CRISPR injection caused sustained AT downregulation and hFIX production up to 63 weeks. AT inhibition and hFIX protein-production ability could be maintained by the proliferation of genetically edited hepatocytes in the case of partial hepatectomy. The co-administration of AAV and LNP showed no severe side effects except random integrations. Our results demonstrate hemophilia B therapy by a combination of rebalancing and hF9 KI using LNP and AAV.

Genome editing-mediated knock-in of therapeutic genes ameliorates the disease phenotype in a model of hemophilia.

Recently, clinical trials of adeno-associated virus-mediated replacement therapy have suggested long-term therapeutic effects for several genetic diseases of the liver, including hemophilia. However, there remain concerns regarding decreased therapeutic effects when the liver is regenerated or when physiological proliferation occurs. Although genome editing using the clustered regularly interspaced short palindromic repeats/Cas9 system provides an opportunity to solve this problem, low knock-in efficiency may limit its application for therapeutically relevant expression. Here, we identified a novel gene, APOC3, in which a strong promoter facilitated the expression of knocked-in genes in hepatocytes. We also investigated the effects of APOC3 editing using a small Cas9 protein derived from Campylobacter jejuni (CjCas9) in a hemophilic model. We demonstrated that adeno-associated virus-mediated delivery of CjCas9 and donor led to moderate levels of human factor 9 expression in APOC3-humanized mice. Moreover, knock-in-driven expression induced substantial recovery of clotting function in mice with hemophilia B. There was no evidence of off-target editing in vitro or in vivo. Collectively, our findings demonstrated therapeutically relevant expression using a precise and efficient APOC3-editing platform, providing insights into the development of further long-term therapeutics for diverse monogenic liver diseases.

In vivo delivery of CRISPR-Cas9 using lipid nanoparticles enables antithrombin gene editing for sustainable hemophilia A and B therapy.

Hemophilia is a hereditary disease that remains incurable. Although innovative treatments such as gene therapy or bispecific antibody therapy have been introduced, substantial unmet needs still exist with respect to achieving long-lasting therapeutic effects and treatment options for inhibitor patients. Antithrombin (AT), an endogenous negative regulator of thrombin generation, is a potent genome editing target for sustainable treatment of patients with hemophilia A and B. In this study, we developed and optimized lipid nanoparticles (LNPs) to deliver Cas9 mRNA along with single guide RNA that targeted AT in the mouse liver. The LNP-mediated CRISPR-Cas9 delivery resulted in the inhibition of AT that led to improvement in thrombin generation. Bleeding-associated phenotypes were recovered in both hemophilia A and B mice. No active off-targets, liver-induced toxicity, and substantial anti-Cas9 immune responses were detected, indicating that the LNP-mediated CRISPR-Cas9 delivery was a safe and efficient approach for hemophilia therapy.

Novel Severe Hemophilia A Mouse Model with Factor VIII Intron 22 Inversion.

Hemophilia A (HA) is an X-linked recessive blood coagulation disorder, and approximately 50% of severe HA patients are caused by F8 intron 22 inversion (F8I22I). However, the F8I22I mouse model has not been developed despite being a necessary model to challenge pre-clinical study. A mouse model similar to human F8I22I was developed through consequent inversion by CRISPR/Cas9-based dual double-stranded breakage (DSB) formation, and clinical symptoms of severe hemophilia were confirmed. The F8I22I mouse showed inversion of a 391 kb segment and truncation of mRNA transcription at the F8 gene. Furthermore, the F8I22I mouse showed a deficiency of FVIII activity (10.9 vs. 0 ng/mL in WT and F8I22I, p < 0.0001) and severe coagulation disorder phenotype in the activated partial thromboplastin time (38 vs. 480 s, p < 0.0001), in vivo bleeding test (blood loss/body weight; 0.4 vs. 2.1%, p < 0.0001), and calibrated automated thrombogram assays (Thrombin generation peak, 183 vs. 21.5 nM, p = 0.0012). Moreover, histological changes related to spontaneous bleeding were observed in the liver, spleen, and lungs. We present a novel HA mouse model mimicking human F8I22I. With a structural similarity with human F8I22I, the F8I22I mouse model will be applicable to the evaluation of general hemophilia drugs and the development of gene-editing-based therapy research.

High Homology-Directed Repair Using Mitosis Phase and Nucleus Localizing Signal.

In homology-directed repair, mediated knock-in single-stranded oligodeoxynucleotides (ssODNs) can be used as a homologous template and present high efficiency, but there is still a need to improve efficiency. Previous studies have mainly focused on controlling double-stranded break size, ssODN stability, and the DNA repair cycle. Nevertheless, there is a lack of research on the correlation between the cell cycle and single-strand template repair (SSTR) efficiency. Here, we investigated the relationship between cell cycle and SSTR efficiency. We found higher SSTR efficiency during mitosis, especially in the metaphase and anaphase. A Cas9 protein with a nuclear localization signal (NLS) readily migrated to the nucleus; however, the nuclear envelope inhibited the nuclear import of many nucleotide templates. This seemed to result in non-homologous end joining (NHEJ) before the arrival of the homologous template. Thus, we assessed whether NLS-tagged ssODNs and free NLS peptides could circumvent problems posed by the nuclear envelope. NLS-tagging ssODNs enhanced SSTR and indel efficiency by 4-fold compared to the control. Our results suggest the following: (1) mitosis is the optimal phase for SSTR, (2) the donor template needs to be delivered to the nucleus before nuclease delivery, and (3) NLS-tagging ssODNs improve SSTR efficiency, especially high in mitosis.

CRISPR-Cas9-mediated therapeutic editing of Rpe65 ameliorates the disease phenotypes in a mouse model of Leber congenital amaurosis.

Leber congenital amaurosis (LCA), one of the leading causes of childhood-onset blindness, is caused by autosomal recessive mutations in several genes including RPE65. In this study, we performed CRISPR-Cas9-mediated therapeutic correction of a disease-associated nonsense mutation in Rpe65 in rd12 mice, a model of human LCA. Subretinal injection of adeno-associated virus carrying CRISPR-Cas9 and donor DNA resulted in >1% homology-directed repair and ~1.6% deletion of the pathogenic stop codon in Rpe65 in retinal pigment epithelial tissues of rd12 mice. The a- and b-waves of electroretinograms were recovered to levels up to 21.2 ± 4.1% and 39.8 ± 3.2% of their wild-type mice counterparts upon bright stimuli after dark adaptation 7 months after injection. There was no definite evidence of histologic perturbation or tumorigenesis during 7 months of observation. Collectively, we present the first therapeutic correction of an Rpe65 nonsense mutation using CRISPR-Cas9, providing new insight for developing therapeutics for LCA.

In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni.

Several CRISPR-Cas9 orthologues have been used for genome editing. Here, we present the smallest Cas9 orthologue characterized to date, derived from Campylobacter jejuni (CjCas9), for efficient genome editing in vivo. After determining protospacer-adjacent motif (PAM) sequences and optimizing single-guide RNA (sgRNA) length, we package the CjCas9 gene, its sgRNA sequence, and a marker gene in an all-in-one adeno-associated virus (AAV) vector and produce the resulting virus at a high titer. CjCas9 is highly specific, cleaving only a limited number of sites in the human or mouse genome. CjCas9, delivered via AAV, induces targeted mutations at high frequencies in mouse muscle cells or retinal pigment epithelium (RPE) cells. Furthermore, CjCas9 targeted to the Vegfa or Hif1a gene in RPE cells reduces the size of laser-induced choroidal neovascularization, suggesting that in vivo genome editing with CjCas9 is a new option for the treatment of age-related macular degeneration.

miR-374 promotes myocardial hypertrophy by negatively regulating vascular endothelial growth factor receptor-1 signaling.

Vascular endothelial growth factor (VEGF) is an essential cytokine that has functions in the formation of new blood vessels and regression of cardiac hypertrophy. VEGF/VEGF-receptor-1 (VEGFR1) signaling plays a key role in the regression of cardiac hypertrophy, whereas VEGF/VEGFR2 signaling leads to cardiac hypertrophy. In this study, we identified the prohypertrophic role of miR-374 using neonatal rat ventricular myocytes (NRVMs). Our results showed that overexpression of miR-374 activated G protein-coupled receptor-mediated prohypertrophic pathways by the inhibition of VEGFR1-dependent regression pathways. Luciferase assays revealed that miR-374 could directly target the 3'-untranslated regions of VEGFR1 and cGMP-dependent protein kinase-1. Collectively, these findings demonstrated that miR-374 was a novel pro-hypertrophic microRNA functioning to suppress the VEGFR1-mediated regression pathway. [BMB Reports 2017; 50(4): 208-213].

miR-185 inhibits endoplasmic reticulum stress-induced apoptosis by targeting Na+/H+ exchanger-1 in the heart.

Prolonged ER stress (ERS) can be associated with the induction of apoptotic cell death in various heart diseases. In this study, we searched for microRNAs affecting ERS in the heart using in silico and in vitro methods. We found that miR-185 directly targets the 3'-untranslated region of Na+/H+ exchanger-1 (NHE-1), a protein involved in ERS. Cardiomyocyte ERS-triggered apoptosis induced by 100 ng/ml tunicamycin (TM) or 1 µM thapsigargin (TG), ERS inducers, was significantly reduced by miR-185 overexpression. Protein expression of pro-apoptotic markers such as CCAAT/enhancer-binding protein homologous protein (CHOP) and cleaved-caspase-3 was also markedly reduced by miR-185 in a dose-dependent manner. Cariporide (20 µM), a pharmacological inhibitor of NHE-1, also attenuated ERS-induced apoptosis in cardiomyocytes and CHOP protein expression, suggesting that NHE-1 plays an important role in ERS-associated apoptosis in cardiomyocytes. Collectively, the present results demonstrate that miR-185 is involved in cardio- protection against ERS-mediated apoptotic cell death. [BMB Reports 2016; 49(4): 208-213].

The molecular interaction of heart LIM protein (HLP) with RyR2 and caveolin-3 is essential for Ca(2+)-induced Ca(2+) release in the heart.

The heart LIM protein (HLP) is a LIM-only protein family member that mediates protein-protein interactions. To date, no studies have yet been conducted regarding its function in the heart. In the present study, we have identified that HLP binds the cytosolic region of RyR2 in the heart using a bacterial two-hybrid system, LC-MS/MS, co-immunoprecipitation, and GST-pull down assays. Microscopy revealed that HLP forms a triple complex with RyR2 and caveolin-3. siRNA and adenovirus-mediated KD of HLP decreased the electrically evoked Ca(2+) release from the sarcoplasmic reticulum without directly affecting SERCA2 and RyR2 activities. Collectively, the HLP-RyR2 interaction in the cell surface caveolae region may be essential for efficient excitation-contraction coupling in the heart.

miR-185 plays an anti-hypertrophic role in the heart via multiple targets in the calcium-signaling pathways.

MicroRNA (miRNA) is an endogenous non-coding RNA species that either inhibits RNA translation or promotes degradation of target mRNAs. miRNAs often regulate cellular signaling by targeting multiple genes within the pathways. In the present study, using Gene Set Analysis, a useful bioinformatics tool to identify miRNAs with multiple target genes in the same pathways, we identified miR-185 as a key candidate regulator of cardiac hypertrophy. Using a mouse model, we found that miR-185 was significantly down-regulated in myocardial cells during cardiac hypertrophy induced by transverse aortic constriction. To confirm that miR-185 is an anti-hypertrophic miRNA, genetic manipulation studies such as overexpression and knock-down of miR-185 in neonatal rat ventricular myocytes were conducted. The results showed that up-regulation of miR-185 led to anti-hypertrophic effects, while down-regulation led to pro-hypertrophic effects, suggesting that miR-185 has an anti-hypertrophic role in the heart. Our study further identified Camk2d, Ncx1, and Nfatc3 as direct targets of miR-185. The activity of Nuclear Factor of Activated T-cell (NFAT) and calcium/calmodulin-dependent protein kinase II delta (CaMKIIδ) was negatively regulated by miR-185 as assessed by NFAT-luciferase activity and western blotting. The expression of phospho-phospholamban (Thr-17), a marker of CaMKIIδ activity, was also significantly reduced by miR-185. In conclusion, miR-185 effectively blocked cardiac hypertrophy signaling through multiple targets, rendering it a potential drug target for diseases such as heart failure.

The miR-19a/b family positively regulates cardiomyocyte hypertrophy by targeting atrogin-1 and MuRF-1.

Progressive cardiac hypertrophy owing to pathological stimuli, such as pressure overload, is frequently associated with the development of heart failure, a major cause of morbidity and mortality worldwide. Growing evidence has shown that miRNAs are extensively involved in the pathogenesis of cardiac hypertrophy. In the present study, we examined the hypothesis that the miR-19a/b family acts as a key regulator of cardiac hypertrophy and apoptosis. Forced overexpression of miR-19a/b was sufficient to induce hypertrophy in rat neonatal cardiomyocytes. Luciferase assays revealed that miR-19a/b directly target the anti-hypertrophic genes atrogin-1 and MuRF-1 (muscle RING-finger protein-1). The endogenous expressions of the target genes were down-regulated by miR-19a/b. Pro-hypertrophic calcineurin/NFAT (nuclear factor of activated T-cells) signalling was elevated markedly in the presence of miR-19b, and the calcineurin inhibitor CsA (cyclosporin A) and the PKC (protein kinase C) inhibitor GF10923X significantly attenuated the miR-19b-mediated increase in cell size and expression of hypertrophic markers. Furthermore, miR-19b led to increased cell survival through up-regulation of the NFAT target gene encoding α-crystallin-B and repression of the pro-apoptotic gene Bim (Bcl-2-interacting mediator of cell death) under ER (endoplasmic reticulum) stress conditions. Taken together, the results of the present study demonstrate that the miR-19a/b family regulates phenotypes of cardiomyocytes via suppression of multiple direct target genes.

Both basic and acidic amino acid residues of IpTx(a) are involved in triggering substate of RyR1.

Imperatoxin A (IpTx(a)) is known to modify the gating of skeletal ryanodine receptor (RyR1). In this paper, the ability of charged aa residues of IpTx(a) to induce substate of native RyR1 in HSR was examined. Our results show that the basic residues (e.g., Lys¹9, Lys²°, Lys²², Arg²³, and Arg²4) are important for producing substate of RyR1. In addition, other basic residues (e.g., Lys³°, Arg³¹, and Arg³³ near the C-terminus and some acidic residues (e.g., Glu²9, Asp¹³, and Asp²) are also involved in the generation of substate. Residues such as Lys8 and Thr²6 may be involved in the self-regulation of substate of RyR1, since alanine substitution of the aa residues led to a drastic conversion to the substate. The modifications of the channel gating by the wild-type and mutant toxins were similar in purified RyR1. Taken together, the specific charge distributions on the surface of IpTx(a) are essential for regulation of the channel gating of RyR1.

PbSe nanocrystal-based infrared-to-visible up-conversion device.

Low-cost hybrid up-conversion devices with infrared sensitivity to 1.5 µm were obtained by integrating a colloidal PbSe nanocrystal near-infrared sensitizing layer on a green phosphorescent organic light emitting diode. A ZnO nanocrystal hole blocking layer is incorporated in the devices for keeping the device off in the absence of IR excitation. The maximum photon (1.3 µm)-to-photon (0.52 µm) conversion efficiency is 1.3%. The extension (until 1.5 µm) of the near-infrared wavelengths, which can be converted to visible light, may be able to improve night vision.

Ryanodine receptor assembly: a novel systems biology approach to 3D mapping.

Ryanodine receptors (RyRs) are intracellular Ca(2+) release channels (CRCs) that play a pivotal role in cellular Ca(2+) signaling. In striated muscles, RyR-mediated Ca(2+) release from the sarcoplasmic reticulum (SR) induces elevation of cytosolic Ca(2+) concentration and subsequent muscle contraction. Evidence from various sources suggests that RyRs in homo-tetrameric conformation form a large conductance Ca(2+) permeable channel in the central pore and large cytoplasmic domains. RyRs form a large assembly with various cytosolic and luminal proteins. A number of papers have been published concerning the functions of RyRs and the regulation of the associated proteins, but the three dimensional (3D) structure of the assembly has not been addressed in detail. In this paper, we have attempted to establish a 3D-map for the assembly of RyRs by considering published cryo-EM data, available X-ray crystallographic information and molecular modeling methods.

Overexpression and purification of the RyR1 pore-forming region.

Ryanodine receptor 1 (RyR1) is a large homotetrameric calcium channel that plays a pivotal role in skeletal muscle contraction. Sequence comparison and mutagenesis studies indicate that the pore architecture of RyR1, including the last two transmembrane helices and the luminal loop linking them, is similar to that of the bacterial KcsA K(+) channel. Here, we describe the overexpression and purification of the C-terminal polyhistidine-tagged RyR1 pore-forming region. The nonionic detergent lauryldimethylamine oxide (LDAO) was selected for solubilization of the protein based on its ability to extract the protein from the membrane and to maintain it in a monodisperse state. The protein was then purified using nickel-affinity chromatography and gel filtration. Gel filtration analysis confirmed that the RyR1 fragment containing the pore-forming region (amino acids 4829-5037) is sufficient to form a tetramer.

Occurrence of atypical Ca2+ transients in triadin-binding deficient-RYR1 mutants.

Triadin in the junctional sarcoplasmic reticulum (SR) of skeletal muscle cells has been suggested to interact with ryanodine receptor 1 (RYR1) via its KEKE motifs. Recently, we showed that amino acid residues D4878, D4907, and E4908 in RYR1 are critical for triadin-binding in vitro [J.M. Lee, S.H. Rho, D.W. Shin, C. Cho, W.J. Park, S.H. Eom, J. Ma, D.H. Kim, Negatively charged amino acids within the intraluminal loop of ryanodine receptor are involved in the interaction with triadin, J. Biol. Chem. 279 (2004) 6994-7000]. In order to test whether a disruption of the triadin-binding site(s) in RYR1 affects SR Ca(2+) release, alanine-substituted single (D4878A, D4907A, and E4908A) and triple (RYR1-TM) mutants of D4878, D4907, and E4908 were expressed in RYR1-null myotubes. Co-immunoprecipitation experiments showed a 50-60% decrease of triadin brought down in the D4907A and RYR1-TM complexes compared to the triadin-wtRYR1 complex. Ca(2+) imaging experiments using Fluo-4-AM showed atypical caffeine responses in myotubes expressing D4907A and RYR1-TM characterized by either a lack of or slower activation and faster inactivation of Ca(2+) transients. The results suggest that disruption of interaction between triadin and RYR1 impairs RYR1 function and SR Ca(2+) release.