Journal articles on the topic 'Neonatal genome editing'
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Anguela, Xavier M., Rajiv Sharma, Hojun Li, Virginia Haurigot, Anand Bhagwat, Robert J. Davidson, Shangzhen Zhou, et al. "Robust Factor IX Expression Following ZFN-Mediated Genome Editing in An Adult Mouse Model of Hemophilia B." Blood 118, no. 21 (November 18, 2011): 668. http://dx.doi.org/10.1182/blood.v118.21.668.668.
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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.
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Anguela, Xavier M., Rajiv Sharma, Yannick Doyon, Thomas Wechsler, David Paschon, Robert J. Davidson, Shangzhen Zhou, Philip D. Gregory, Michael C. Holmes, and Katherine A. High. "In Vivo Genome Editing in Neonatal Mouse Liver Preferentially Utilizes Homology Directed Repair." Blood 126, no. 23 (December 3, 2015): 4422. http://dx.doi.org/10.1182/blood.v126.23.4422.4422.
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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.
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Zhang, Shengwen, Amy Bastille, Susana Gordo, Nikhil Ramesh, Jenisha Vora, Elizabeth McCarthy, Xiaohan Zhang, et al. "Novel AAV-mediated genome editing therapy improves health and survival in a mouse model of methylmalonic acidemia." PLOS ONE 17, no. 9 (September 20, 2022): e0274774. http://dx.doi.org/10.1371/journal.pone.0274774.
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Methylmalonic acidemia (MMA) is an inborn error of metabolism mostly caused by mutations in the mitochondrial methylmalonyl-CoA mutase gene (MMUT). MMA patients suffer from frequent episodes of metabolic decompensation, which can be life threatening. To mimic both the dietary restrictions and metabolic decompensation seen in MMA patients, we developed a novel protein-controlled diet regimen in a Mmut deficient mouse model of MMA and demonstrated the therapeutic benefit of mLB-001, a nuclease-free, promoterless recombinant AAV GeneRideTM vector designed to insert the mouse Mmut into the endogenous albumin locus via homologous recombination. A single intravenous administration of mLB-001 to neonatal or adult MMA mice prevented body weight loss and mortality when challenged with a high protein diet. The edited hepatocytes expressed functional MMUT protein and expanded over time in the Mmut deficient mice, suggesting a selective growth advantage over the diseased cells. In mice with a humanized liver, treatment with a human homolog of mLB-001 resulted in site-specific genome editing and transgene expression in the transplanted human hepatocytes. Taken together, these findings support the development of hLB-001 that is currently in clinical trials in pediatric patients with severe forms of MMA.
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Weyand, Angela C., Steve J. Grzegorski, and Jordan A. Shavit. "Genome Editing of Factor V in Zebrafish Embryos Results in a Severe Hemostatic Defect without Spontaneous Hemorrhage." Blood 128, no. 22 (December 2, 2016): 2565. http://dx.doi.org/10.1182/blood.v128.22.2565.2565.
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Abstract The cessation of bleeding following trauma is a crucial element in vertebrate survival.Factor V (F5) serves as an essential cofactor in the penultimate step of coagulation, the conversion of prothrombin to thrombin. In humans, genetic deficiency of F5 is rare and clinically diverse, with presentations ranging from neonatal intracranial hemorrhage to mild bleeding later in life. This patient variability suggests the presence of modifiers, unlinked genes inherited separately from the F5 locus. Complete loss through gene targeting of mouse F5 results in embryonic and neonatal lethality, interfering with further detailed studies. Zebrafish possess many distinct advantages for the study of coagulation and modifier genes, including high fecundity, optical clarity, external development, as well as extensive homology of the mammalian hemostatic system. Here we report the role of F5 in zebrafish development using genome editing mediated targeted mutagenesis. The f5 locus was identified through a BLAST search of zebrafish genomic sequence, identifying a protein with 48% amino acid identity and 66% similarity to human F5. In situ hybridization revealed expression of f5 mRNA localized to the developing liver at 5 days post fertilization (dpf). CRISPR RNA guided nucleases were designed to target exon 4 of F5 and injected into several hundred wild type zebrafish one cell stage embryos to produce adult fish (F0) with a panel of insertion/deletion mutations in f5. Adult fish (F1) heterozygous for a 49 base pair deletion were identified and incrossed to generate groups of offspring for analysis. This deletion was initiated at amino acid 171, within the A1 domain of F5. Genotyping was performed after phenotypic analysis so that observers were blinded during data collection. Homozygous mutants demonstrated significantly decreased survival compared to their heterozygous and wild type siblings, with die off beginning between 2 and 4 weeks post fertilization and 100% mortality by 7 months of age. Visual observation of development and circulation revealed that homozygous mutant embryos and larvae were indistinguishable from wild type siblings, with no signs of hemorrhage. Since there was no observable bleeding, we used induced occlusive thrombus formation by laser mediated endothelial ablation of the posterior cardinal vein at 3 dpf to assess hemostasis. We found that the ability to produce occlusive thrombi was absent in f5-/- mutants, a bleeding phenotype, while wild type and heterozygous siblings were phenotypically normal. This bleeding phenotype was rescued in 73% of embryos (p=0.0006) at 3 dpf after injection of zebrafish f5 cDNA at the one cell stage. In summary, we have demonstrated strong conservation of zebrafish and mammalian F5, including site of synthesis and requirement for hemostasis. Surprisingly, embryos and larvae, as well as young adults, tolerate what is a severe and lethal defect in mammals, allowing accessibility not easily achieved in murine models. This suggests the possibility of species-specific factors enabling survival in fish. Identification of these factors, combined with small molecule screens, could lead to novel therapeutic modalities for patients with bleeding disorders. Disclosures No relevant conflicts of interest to declare.
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He, Wenhui, Zhiliang Cao, Fengfeng Mao, Bijie Ren, Yunfei Li, Dan Li, Huiyu Li, et al. "Modification of Three Amino Acids in Sodium Taurocholate Cotransporting Polypeptide Renders Mice Susceptible to Infection with Hepatitis D VirusIn Vivo." Journal of Virology 90, no. 19 (July 27, 2016): 8866–74. http://dx.doi.org/10.1128/jvi.00901-16.
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ABSTRACTSodium taurocholate cotransporting polypeptide (NTCP) was identified as a functional receptor for hepatitis D virus (HDV) and its helper hepatitis B virus (HBV). In cultured cell lines, HDV infection through mouse NTCP is restricted by residues 84 to 87 of the receptor. This study shows that mice with these three amino acids altered their corresponding human residues (H84R, T86K, and S87N) in endogenous mouse NTCP supportde novoHDV infectionin vivo. HDV infection was documented by the presence of replicative forms of HDV RNA and HDV proteins in liver cells at day 6 after viral inoculation. Monoclonal antibody specifically binding to the motif centered on K86 in NTCP partially inhibited HDV infection. These studies demonstrated specific interaction between the receptor and the viral envelopesin vivoand established a novel mouse model with minimal genetic manipulation for studying HDV infection. The model will also be useful for evaluating entry inhibitors against HDV and its helper HBV.IMPORTANCENTCP was identified as a functional receptor for both HDV and HBV in cell cultures. We recently showed that neonatal C57BL/6 transgenic (Tg) mice exogenously expressing human NTCP (hNTCP-Tg) in liver support transient HDV infection. In this study, we introduced alterations of three amino acids in the endogenous NTCP of FVB mice through genome editing. The mice with the humanized NTCP residues (H84R, T86K, and S87N) are susceptible to HDV infection, and the infection can be established in both neonatal and adult mice with this editing. We also developed a monoclonal antibody specifically targeting the region of NTCP centered on lysine residue 86, and it can differentiate the modified mouse NTCP from that of the wild type and partially inhibited HDV infection. These studies shed new light on NTCP-mediated HDV infectionin vivo, and the NTCP-modified mice provide a useful animal model for studying HDV infection and evaluating antivirals against the infection.
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Wang, Lili, Yang Yang, Camilo Breton, Peter Bell, Mingyao Li, Jia Zhang, Yan Che, et al. "A mutation-independent CRISPR-Cas9–mediated gene targeting approach to treat a murine model of ornithine transcarbamylase deficiency." Science Advances 6, no. 7 (February 2020): eaax5701. http://dx.doi.org/10.1126/sciadv.aax5701.
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Ornithine transcarbamylase (OTC) deficiency is an X-linked urea cycle disorder associated with high mortality. Although a promising treatment for late-onset OTC deficiency, adeno-associated virus (AAV) neonatal gene therapy would only provide short-term therapeutic effects as the non-integrated genome gets lost during hepatocyte proliferation. CRISPR-Cas9-mediated homology-directed repair can correct a G-to-A mutation in 10% of OTC alleles in the livers of newborn OTC spfash mice. However, an editing vector able to correct one mutation would not be applicable for patients carrying different OTC mutations, plus expression would not be fast enough to treat a hyperammonemia crisis. Here, we describe a dual-AAV vector system that accomplishes rapid short-term expression from a non-integrated minigene and long-term expression from the site-specific integration of this minigene without any selective growth advantage for OTC-positive cells in newborns. This CRISPR-Cas9 gene-targeting approach may be applicable to all patients with OTC deficiency, irrespective of mutation and/or clinical state.
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Fischer, Bastian, Vanessa Schmidt, Thanh-Diep Ly, Anika Kleine, Cornelius Knabbe, and Isabel Faust-Hinse. "First Characterization of Human Dermal Fibroblasts Showing a Decreased Xylosyltransferase-I Expression Induced by the CRISPR/Cas9 System." International Journal of Molecular Sciences 23, no. 9 (May 2, 2022): 5045. http://dx.doi.org/10.3390/ijms23095045.
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Background: Xylosyltransferases-I and II (XT-I and XT-II) catalyze the initial and rate limiting step of the proteoglycan (PG) biosynthesis and therefore have an import impact on the homeostasis of the extracellular matrix (ECM). The reason for the occurrence of two XT-isoforms in all higher organisms remains unknown and targeted genome-editing strategies could shed light on this issue. Methods: XT-I deficient neonatal normal human dermal fibroblasts were generated by using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated proteins (Cas) 9 system. We analyzed if a reduced XT-I activity leads to abnormalities regarding ECM-composition, myofibroblast differentiation, cellular senescence and skeletal and cartilage tissue homeostasis. Results: We successfully introduced compound heterozygous deletions within exon 9 of the XYLT1 gene. Beside XYLT1, we detected altered gene-expression levels of further, inter alia ECM-related, genes. Our data further reveal a dramatically reduced XT-I protein activity. Abnormal myofibroblast-differentiation was demonstrated by elevated alpha-smooth muscle actin expression on both, mRNA- and protein level. In addition, wound-healing capability was slightly delayed. Furthermore, we observed an increased cellular-senescence of knockout cells and an altered expression of target genes knowing to be involved in skeletonization. Conclusion: Our data show the tremendous relevance of the XT-I isoform concerning myofibroblast-differentiation and ECM-homeostasis as well as the pathophysiology of skeletal disorders.
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Tebbi, Cameron K. "Sickle Cell Disease, a Review." Hemato 3, no. 2 (May 30, 2022): 341–66. http://dx.doi.org/10.3390/hemato3020024.
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Sickle cell disease and its variants constitute the most common inherited blood disorders affecting millions of individuals worldwide. Significant information regarding the nature of the genetic mutations and modifier genes that result in increased or decreased severity of the disease are available. In recent years, detailed data regarding molecular genetics, pathophysiology, mechanisms for the development of symptoms and side effects of sickle cell disease have been published. The relationship of physiological changes, cellular interactions, coexisting coagulation disorders, effects of association with other genetic disorders and a number of intervening factors have been explored. New techniques for pre-conception, prenatal, in utero, and neonatal screening are available. Means for prediction of the severity of the disease, clinical course of the disorder, and prevention of some of its major complications have been developed. The effects of psychosocial and environmental factors have been explored. Various therapeutic strategies including bone marrow and stem cell transplantation are currently employed in the treatment of patients with sickle cell disease. Recent progress in understanding the molecular pathways controlling mammalian erythropoiesis and globin switching, as well as advances in genome engineering, particularly the gene-editing techniques, have opened a venue for genetic-based treatment of the disease. Currently, sickle cell disease is often associated with a high rate of complications and mortality. The development of new pharmacological agents, methods for gene therapy, and alterations and modification of the coexisting genetic factors and modifiers for treatment of the disease are encouraging.
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Ku, Chia-Jui, Steven Grzegorski, and Jordan A. Shavit. "Loss of Protein C in Zebrafish Results in Impaired Neutrophil Migration after Tissue Injury." Blood 136, Supplement 1 (November 5, 2020): 22. http://dx.doi.org/10.1182/blood-2020-142541.
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Hemostasis is a natural protective process that developed to retain a circulating blood system, conferred by a complicated yet sophisticated balance of factors. Disturbances of this network result in thrombosis or hemorrhage. Among many well-characterized coagulation factors, protein C (PC) exhibits multifunctional roles including anticoagulant, cytoprotective, and anti-inflammatory activities. The importance of PC has been demonstrated not only by the increased risk of venous thrombosis in individuals with heterozygous deficiency, but also the observed neonatal lethality in patients. Knockout mice exhibit similar neonatal lethality, which has made it difficult to further study complete deficiency. The zebrafish is a vertebrate organism that is characterized by a powerful genetic system, prolific breeding, rapid and transparent development, and a well described and highly conserved coagulation cascade. Here we utilize genome editing to generate a null allele of the PC gene (proc) in zebrafish and discover that its loss not only impairs hemostatic balance, but also affects neutrophil recruitment to sites of tissue injury. Through examination of publicly available zebrafish genome sequence, we determined that the proc locus is duplicated in tandem, resulting in two closely adjacent copies with nearly identical sequences. We used CRISPR/Cas9 with two single guide RNAs flanking the entire locus to produce a 17.3 kilobase deletion that knocks out both copies of proc to produce a complete null mutation, verified by sequencing and quantitative PCR. proc-/- mutants survived well into adulthood, with ~50% lethality by seven months of age. The embryonic survival and accessibility enabled us to perform intravital microscopy to evaluate the hemostatic effects of PC deficiency. We used laser-induced endothelial injury on the posterior cardinal vein (PCV) at 3 days post fertilization (dpf), which typically results in rapid formation of an occlusive fibrin-rich thrombus. proc-/- mutants had an average time to occlusion of 60 seconds versus 13 seconds in controls (p < 0.0001), consistent with a consumptive coagulopathy, as previously seen in antithrombin III (at3) mutants. A transgenic background with fluorescently labeled fibrinogen showed that more than 95% of proc-/- mutants had spontaneous thrombi in the PCV, which was not present in controls. To assess the role of PC in inflammation, we used two different injury strategies, non-vascular tail transection and chemical treatment (copper sulfate), on 3 dpf zebrafish larvae. Staining for neutrophil granules revealed homing to the site of injury within 60-75 minutes. In proc-/- mutants we found an average 50% reduction in the number of neutrophils recruited to the site of injury yet counts in the caudal hematopoietic tissue (the site of larval hematopoiesis) were unchanged. Since protein S (PS) is a cofactor for PC anticoagulant function, we hypothesized that the consumptive coagulopathy, but not the neutrophil recruitment, would be PS-dependent. We used genome editing to disrupt the PS gene (pros1) and found that loss of PS also results in a mild consumptive coagulopathy, but spontaneous thrombus formation was less common in the PCV (25%) and was often in the heart instead (80%). Neutrophil recruitment was unaffected in pros1 mutants, and evaluation of double proc/pros1 mutants revealed no synergy in any of the phenotypes. In conclusion, PC and PS deficiency in zebrafish show some similarity to our previously reported model of AT3 deficiency, but the effects are less potent, allowing robust survival that enables in vivo analyses. Our data suggest that the thrombotic phenotypes of PC and PS deficiency are not identical, and display tissue-specific phenotypes. We also found evidence for PS-independent functions of PC in neutrophil migration. We speculate this is due to the role that PC plays in inflammation and signaling but cannot exclude a role in neutrophil extracellular trap (NET) formation. This model of complete proc-/- deficiency in an accessible organism will facilitate further in vivo study of PS-dependent and independent functions of PC, as well as interplay between the two factors. Disclosures Shavit: Bayer: Consultancy; Taked: Consultancy.
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Gorbunova, Victoria N., Natalia V. Buchinskaia, Grigorii A. Janus, and Mikhail M. Kostik. "Lysosomal storage diseases. Sphingolipidoses — Fabry, Gaucher and Farber diseases." Pediatrician (St. Petersburg) 13, no. 2 (July 9, 2022): 61–88. http://dx.doi.org/10.17816/ped13261-88.
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Sphingolipidoses are genetically heterogeneous group of rare monogenic metabolic diseasesб caused by inherreted deficiency of enzymes involved in the degradation of sphingolipids. Sphingolipids are catabolized in lysosomes, where glycohydrolases degrade them by separation of terminal sugars to core ceramide. All sphingolipidoses are characterized by abnormal deposition of a large amount of sphingolipids and other unsplit products of lipid metabolism, mainly in parenchymal organs, bone marrow and brain. Among sphingolipidoses, such groups of diseases as glycosphingolipidoses, gangliosidoses and leukodystrophies are distinguished. This review presents the epidemiology, clinical, biochemical and molecular characteristics of the three main types of glycosphingolipidoses Fabry disease, Gaucher disease and Farber disease, caused by the mutations in the genes of -galactosidase A (GLA), glucocerebrosidase (GBA) and acid ceramidase (ASAH1), respectively. Currently, there is an increased interest in glycosphingolipidoses due to the identification of the spectrum and frequencies of mutations in the GLA, GBA and ASAH1 genes in various populations, including Russia, and the practical availability of individual molecular diagnostic methods. A description of the existing experimental models, their role in the study of the biochemical basis of the pathogenesis of these severe hereditary diseases and the development of various therapeutic approaches are given. We discuss, firstly, the possibility of early diagnosis of Fabry disease, Gaucher and Farber based on neonatal screening and examination of high risk groups of patients in order to improve the effectiveness of their prevention and treatment, as well as (secondly) the advantages and disadvantages of the main approaches to the treatment of these serious diseases, such as bone marrow and hematopoietic stem cell transplantation, enzyme replacement therapy, substrate reduction therapy, gene therapy and genome editing.
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Delhove, Juliette M., Rajvinder Karda, Lorna M. FitzPatrick, Suzanne M. K. Buckley, Simon N. Waddington, and Tristan R. McKay. "Non-invasive somatotransgenic bioimaging in living animals." F1000Research 9 (October 9, 2020): 1216. http://dx.doi.org/10.12688/f1000research.25274.1.
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Bioluminescence imaging enables noninvasive quantification of luciferase reporter gene expression in transgenic tissues of living rodents. Luciferase transgene expression can be regulated by endogenous gene promoters after targeted knock-in of the reporter gene, usually within the first intron of the gene. Even using CRISPR/Cas9 mediated genome editing this can be a time consuming and costly process. The generation of germline transgenic (GLT) rodents by targeted genomic integration of a gene expression cassette in embryonic stem (ES) cells is commonplace but results in the wastage of large numbers of animals during colony generation, back-crossing and maintenance. Using a synthetic/truncated promoter-driven luciferase gene to study promoter activity in a given tissue or organ of a GLT also often results in unwanted background luciferase activity during whole-body bioluminescent imaging as every cell contains the reporter. We have developed somatotransgenic bioimaging; a method to generate tissue-restricted transcription factor activated luciferase reporter (TFAR) cassettes in rodents that substantially reduces the number of animals required for experimentation. Bespoke designed TFARs are delivered to newborn pups using viral vectors targeted to specific organs by tissue-tropic pseudotypes. Retention and proliferation of TFARs is facilitated by stem/progenitor cell transduction and immune tolerance to luciferase due to the naïve neonatal immune system. We have successfully applied both lentiviral and adeno-associated virus (AAV) vectors in longitudinal rodent studies, targeting TFARs to the liver and brain during normal development and in well-established disease models. Development of somatotransgenic animals has broad applicability to non-invasively determine mechanistic insights into homeostatic and disease states and assess toxicology and efficacy testing. Somatotransgenic bioimaging technology is superior to current whole-body, light-emitting transgenic models as it reduces the numbers of animals used by generating only the required number of animals. It is also a refinement over current technologies given the ability to use conscious, unrestrained animals.
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Suyatno, Y. Kitamura, N. Minami, M. Yamada, and H. Imai. "192 Culture Conditions Supporting Long-Term Expansion of Bovine Spermatogonial Stem Cells Isolated from Adult and Immature Testes." Reproduction, Fertility and Development 30, no. 1 (2018): 236. http://dx.doi.org/10.1071/rdv30n1ab192.
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Spermatogonial stem cells (SSC) self-renew and differentiate into spermatocytes to produce haploid sperm. Because SSC are a small population of adult stem cells in the testis, numerous studies have been reported to derive cell lines from cultured SSC. It has been reported that neonatal and adult mouse SSC can be cultured in vitro over the long term. Male germline stem (GS) cells, embryonic stem (ES)-like cells, and multipotent male germline stem (MGS) cells were derivated from mouse SSC. However, in domestic species including cattle, information about in vitro culture of SSC is mainly available in the neonatal and immature animal. To our knowledge, there are no reports about long-term culture of SSC isolated from adult bovine testis. In this report, we established culture conditions to maintain SSC isolated from adult and immature testes. The SSC were isolated by 3-step enzymatic digestion and enriched by Percoll gradient centrifugation. For adult testicular cell suspensions, SSC were further enriched by differential plating on precoated gelatin dish. After Percoll gradient centrifugation, we found differential expression of SSC markers (GFRα-1 and UCHL-1) in the isolated cells from immature and adult testis. The RT-PCR results also confirmed the expression of differentiated spermatogonia markers (SYCP3 and STRA-8) in adult testicular cell suspensions. It suggests that isolated testicular germ cell population from adult testis are more heterogeneous than those of immature testis. The SSC isolated from adult testes were cultured in low-serum media containing 6-bromoindirubin-3′-oxime (BIO), an inhibitor of glycogen synthase kinase-3α (GSK3), and subsequently the cultures were maintained in the medium containing glial cell line-derived neurotropic factor (GDNF). The cell lines have characteristics resembling mouse GS cell lines as confirmed by their grape-like shape morphology, the expression of SSC markers (UCHL-1, DBA, and GFRa-1), and pluripotent stem cell markers (POU5F1, SOX2, KLF4). The SSC from immature testes were proliferated for more than 3 months in serum-free culture conditions in the presence of GDNF and bovine leukemia inhibitory factor (LIF). The cell lines had ES-like cell morphology, expressed pluripotent stem cell markers and SSC-specific markers. They differentiated in vitro into 3 germ layers confirmed by the expression of ectoderm (NESTIN), mesoderm (BMP4), and endoderm (GATA-6) markers by RT-PCR and neuron like-cells confirmed by the expression of glial fibrillary acidic protein (GFAP) by immunofluorescence analysis. In conclusion, these findings indicate an efficient method to enrich SSC without cell sorting method and different long-term culture systems subsequently established to maintain SSC from adult and immature testes. Furthermore, our data would be useful for further studies that aim to preserve endangered species and improve livestock production through genome editing technology.
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Zhang, Isadora, Zoe Hsiao, and Fenyong Liu. "Development of Genome Editing Approaches against Herpes Simplex Virus Infections." Viruses 13, no. 2 (February 22, 2021): 338. http://dx.doi.org/10.3390/v13020338.
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Herpes simplex virus 1 (HSV-1) is a herpesvirus that may cause cold sores or keratitis in healthy or immunocompetent individuals, but can lead to severe and potentially life-threatening complications in immune-immature individuals, such as neonates or immune-compromised patients. Like all other herpesviruses, HSV-1 can engage in lytic infection as well as establish latent infection. Current anti-HSV-1 therapies effectively block viral replication and infection. However, they have little effect on viral latency and cannot completely eliminate viral infection. These issues, along with the emergence of drug-resistant viral strains, pose a need to develop new compounds and novel strategies for the treatment of HSV-1 infection. Genome editing methods represent a promising approach against viral infection by modifying or destroying the genetic material of human viruses. These editing methods include homing endonucleases (HE) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein (Cas) RNA-guided nuclease system. Recent studies have showed that both HE and CRISPR/Cas systems are effective in inhibiting HSV-1 infection in cultured cells in vitro and in mice in vivo. This review, which focuses on recently published progress, suggests that genome editing approaches could be used for eliminating HSV-1 latent and lytic infection and for treating HSV-1 associated diseases.
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Hu, Zhilian, Yang Liu, Michael Huarng, Deepak Reyon, Catherine Richter, Alexandra Stapleton, J. Keith Joung, and Jordan A. Shavit. "Factor X Mutant Zebrafish Tolerate a Severe Hemostatic Defect in Early Development Yet Develop Lethal Hemorrhage in Adulthood." Blood 126, no. 23 (December 3, 2015): 426. http://dx.doi.org/10.1182/blood.v126.23.426.426.
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Abstract Factor X (F10) deficiency is a rare inherited bleeding disorder with a heterogeneous phenotype and limited therapeutic options. Targeted knockout of F10 and other common pathway factors in mice results in embryonic/neonatal lethality with rapid resorption of homozygous mutants, hampering further studies. Several of these mutants also display yolk sac vascular defects, suggesting a role for thrombin signaling in development. The zebrafish model is characterized by external development, optical transparency, ability to generate thousands of offspring at low cost, and a highly characterized vasculature. We have used these advantages for more in depth study of the role of the coagulation cascade in developmental regulation of hemostasis and vasculogenesis. We generated a 17 base pair deletion in the zebrafish f10 locus by genome editing with TALENs. Although indistinguishable morphologically from f10+/+ and f10+/- siblings at early stages, f10-/- mutants demonstrated progressive lethality between 1 and 5 months of age. Extensive hemorrhage was identified in multiple tissues starting at 3-4 weeks of age, particularly the brain. Notably, intracranial hemorrhage is a common feature in multiple zebrafish mutants with various vascular defects, including anomalies of endothelial differentiation and specification, apoptosis, and vessel integrity/permeability. Gross inspection of f10 mutant embryos and larvae in the first week of life revealed no apparent defects in circulation or vascular development. Expression of arterial and venous endothelial markers were examined by in situ hybridization at 24 and 72 hours post fertilization, the time period during which axial and intersegmental vessels form, along with early establishment of the vascular network. Markers included ephb2a, cdh5, flk1, flt4, and ephb4, and expression patterns were indistinguishable between mutants and wild type siblings. Acridine orange staining at 5 days post fertilization (dpf) did not detect any dysregulation of apoptosis. Hemoglobin staining found no specific hemorrhage in vehicle or warfarin treated mutants. However, 3 dpf f10-/- mutants did not develop occlusive thrombi in response to laser-mediated venous endothelial injury, indicating that F10 is required for hemostasis. We used quantitative PCR to measure transcription of f10 and downstream coagulation factors in 3 dpf larvae. f10 mRNA was undetectable in homozygous mutants, presumably due to nonsense-mediated decay as a consequence of the TALEN-induced frameshift mutation. fga (fibrinogen alpha) and at3 (antithrombin III) mRNAs were increased by 1.8 and 2.3-fold, respectively (p<0.05), when compared to wild type siblings. Prothrombin (f2) mRNA transcription was slightly decreased, although the effect was not statistically significant. In summary, we have produced a zebrafish model of human F10 deficiency that exhibits a spontaneous adult lethal bleeding phenotype, although early embryonic/larval survival is unaffected despite an underlying severe hemostatic defect. Further study of this mutant may identify species specific factors enabling this early survival. qPCR analysis in mutant larvae suggests that the level of F10 mRNA or protein indirectly regulates other coagulation factors during development. We have also used this model to evaluate the longstanding hypothesis that coagulation cascade mediated thrombin signaling is required for embryonic vasculogenesis. Thus far we have not found evidence for defects in vessel development or integrity, apoptosis, or endothelial differentiation/specification. Taken together, these data suggest that the effects of F10 loss are restricted to hemostasis, and thus therapies should continue to be focused on this in deficient patients. Further study as to why mutant embryos and larvae are able to tolerate what would likely cause lethal hemorrhage in mammals, as well as small molecule screens using this model, could potentially lead to innovative therapeutic modalities for patients with bleeding disorders. Disclosures Joung: Horizon Discovery: Consultancy; Editas Medicine: Other: Financial interest; Poseida Therapeutics: Other: Financial interest; Hera Testing Laboratories: Other: Financial interest; Transposagen Biopharmaceuticals: Other: Financial interest. Shavit:CSL Behring: Consultancy; Octapharma: Consultancy; Bayer: Consultancy; Baxter: Consultancy.
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Wagenblast, Elvin, Olga I. Gan, Maria Azkanaz, Sabrina A. Smith, Joana Araújo, Lorien Shakib, Jessica L. McLeod, et al. "Understanding Pre-Leukemia in Trisomy 21 Human HSC and Modeling Progression Towards Down Syndrome Associated Leukemia Using CRISPR/Cas9 at Single Cell Resolution." Blood 134, Supplement_1 (November 13, 2019): 2531. http://dx.doi.org/10.1182/blood-2019-129259.
Full textAbstract:
Leukemia is the most common cancer in children. Sequencing data from identical twins suggests that the first genetic alterations in childhood leukemia occur in utero. Children with Down syndrome (Trisomy 21, T21) have an increased risk of childhood leukemia. In 30% of newborns with Down syndrome, a transient pre-leukemia disease occurs, which is characterized by a clonal proliferation of immature megakaryocytes carrying somatic mutations in the GATA1 transcription factor. These acquired GATA1 mutations lead to the expression of an N-terminal truncated protein (GATA1-Short). In 20% of the cases, acute megakaryoblastic leukemia (AMKL) evolves from the pre-leukemia by acquisition of additional genetic mutations in the transient leukemia clone, predominantly in genes of the cohesin complex. It is hypothesized that this represents a multi-step process of leukemogenesis with three distinct genetic events: T21, GATA1-Short and additional cohesin mutations. Yet, it remains unclear how an extra copy of chromosome 21 predisposes towards leukemia, the mechanisms of leukemic transformation and the interplay between each genetic component. Therefore, we wanted to establish a tractable human model system to investigate the initiation and evolution of transient leukemia and AMKL using CRISPR/Cas9 genome editing in primary human hematopoietic stem cells (HSCs). To model the initiation of Down syndrome associated pre-leukemia, we utilized both neonatal cord blood and fetal liver derived LT-HSCs and other progenitor populations to express either the short or long isoform of GATA1 (GATA1-Short or GATA1-Long). This was carried out using an improved methodology that permits the in vitro and in vivo functional interrogation of CRISPR/Cas9 edited human LT-HSCs at the single cell level (Wagenblast et al., bioRxiv 609040). Importantly, in this case, expression of either GATA1 isoform remained under the regulatory control of the endogenous promoter. Culture of single LT-HSC, short-term (ST-HSC) and myelo-erythroid progenitors (MEP) revealed a drastic shift towards megakaryocytic lineage output upon exclusive expression of GATA1-Short compared to control or GATA1-Long, regardless of the developmental source of the derived cells. To investigate the functional consequences of exclusive GATA1-Short expression in LT-HSCs in vivo, we performed near-clonal xenotransplantation assays in NSG and NSGW41 mice. Strikingly, GATA1-Short edited LT-HSCs injected mice displayed a higher percentage of human CD41+CD45- megakaryocytic lineage derived cells and a decrease in human GlyA+CD45- erythroid cells compared to control. Morphological analysis revealed more immature forms of erythroid cells and fewer enucleated erythrocytes in GATA1-Short edited LT-HSCs injected mice. In order to add an additional genetic determinant to our model, we utilized T21 fetal liver derived LT-HSCs. Un-manipulated T21 LT-HSCs and other progenitor populations showed a bias towards erythroid, myeloid and megakaryocytic lineages at the expense of lymphoid fates. In vitro, the combination of T21 and CRISPR/Cas9-mediated GATA1-Short in LT-HSCs led to an increase in megakaryocytic lineage output, while decreasing erythroid output. This phenotype was similar to what was observed in normal karyotype fetal liver derived LT-HSCs. However, near clonal transplantation of GATA1-Short edited T21 LT-HSCs in NSG mice generated exclusive CD33+ myeloid grafts with disproportionate high levels of CD41+CD45- megakaryocytic lineage derived cells compared to T21 control. In addition a distinct CD34+CD41+CD71+CD45+ population was present. Thus, this phenotype is reminiscent of Down Syndrome associated transient leukemia. In summary, by using an improved CRISPR/Cas9 single cell methodology we show how GATA1 regulates lineage fate in normal and T21 LT-HSCs and other progenitor populations. Importantly, we show for the first time a humanized mouse model of Down syndrome associated transient leukemia, which was induced from T21 human fetal liver derived LT-HSCs engineered to express GATA1-Short. Current studies focus on adding additional mutations of the cohesin complex to progress transient leukemia to AMKL. Disclosures No relevant conflicts of interest to declare.
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Vogel, Georg F., Katharina M. C. Klee, Andreas R. Janecke, Thomas Müller, Michael W. Hess, and Lukas A. Huber. "Cargo-selective apical exocytosis in epithelial cells is conducted by Myo5B, Slp4a, Vamp7, and Syntaxin 3." Journal of Cell Biology 211, no. 3 (November 9, 2015): 587–604. http://dx.doi.org/10.1083/jcb.201506112.
Full textAbstract:
Mutations in the motor protein Myosin Vb (Myo5B) or the soluble NSF attachment protein receptor Syntaxin 3 (Stx3) disturb epithelial polarity and cause microvillus inclusion disease (MVID), a lethal hereditary enteropathy affecting neonates. To understand the molecular mechanism of Myo5B and Stx3 interplay, we used genome editing to introduce a defined Myo5B patient mutation in a human epithelial cell line. Our results demonstrate a selective role of Myo5B and Stx3 for apical cargo exocytosis in polarized epithelial cells. Apical exocytosis of NHE3, CFTR (cystic fibrosis transmembrane conductance regulator), and GLUT5 required an interaction cascade of Rab11, Myo5B, Slp4a, Munc18-2, and Vamp7 with Stx3, which cooperate in the final steps of this selective apical traffic pathway. The brush border enzymes DPPIV and sucrase-isomaltase still correctly localize at the apical plasma membrane independent of this pathway. Hence, our work demonstrates how Myo5B, Stx3, Slp4a, Vamp7, Munc18-2, and Rab8/11 cooperate during selective apical cargo trafficking and exocytosis in epithelial cells and thereby provides further insight into MVID pathophysiology.
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Whitley, Richard, and Joel Baines. "Clinical management of herpes simplex virus infections: past, present, and future." F1000Research 7 (October 31, 2018): 1726. http://dx.doi.org/10.12688/f1000research.16157.1.
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Infection with herpes simplex virus (HSV) types 1 and 2 is ubiquitous in the human population. Most commonly, virus replication is limited to the epithelia and establishes latency in enervating sensory neurons, reactivating periodically to produce localized recurrent lesions. However, these viruses can also cause severe disease such as recurrent keratitis leading potentially to blindness, as well as encephalitis, and systemic disease in neonates and immunocompromised patients. Although antiviral therapy has allowed continual and substantial improvement in the management of both primary and recurrent infections, resistance to currently available drugs and long-term toxicity pose a current and future threat that should be addressed through the development of new antiviral compounds directed against new targets. The development of several promising HSV vaccines has been terminated recently because of modest or controversial therapeutic effects in humans. Nevertheless, several exciting vaccine candidates remain in the pipeline and are effective in animal models; these must also be tested in humans for sufficient therapeutic effects to warrant continued development. Approaches using compounds that modulate the chromatin state of the viral genome to suppress infection and reactivation or induce enhanced antiviral immunity have potential. In addition, technologies such as CRISPR/Cas9 have the potential to edit latent viral DNA in sensory neurons, potentially curing the neuron and patient of latent infection. It is hoped that development on all three fronts—antivirals, vaccines, and gene editing—will lead to substantially less HSV morbidity in the future.
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Mehta, Suresh L., Anil K. Chokkalla, TaeHee Kim, Saivenkateshkomal Bathula, Bharath Chelluboina, Kahlilia C. Morris-Blanco, Aleah Holmes, et al. "Long Noncoding RNA Fos Downstream Transcript Is Developmentally Dispensable but Vital for Shaping the Poststroke Functional Outcome." Stroke 52, no. 7 (July 2021): 2381–92. http://dx.doi.org/10.1161/strokeaha.120.033547.
Full textAbstract:
Background and Purpose: Stroke induces the expression of several long noncoding RNAs in the brain. However, their functional significance in poststroke outcome is poorly understood. We recently observed that a brain-specific long noncoding RNA called Fos downstream transcript (FosDT) is induced rapidly in the rodent brain following focal ischemia. Using FosDT knockout rats, we presently evaluated the role of FosDT in poststroke brain damage. Methods: FosDT knockout rats were generated using CRISPR-Cas9 genome editing on a Sprague-Dawley background. Male and female FosDT −/− and FosDT +/+ cohorts were subjected to transient middle cerebral artery occlusion. Postischemic sensorimotor deficits were evaluated between days 1 and 7 and lesion volume on day 7 of reperfusion. The developmental expression profile of FosDT was determined with real-time polymerase chain reaction and mechanistic implications of FosDT in the ischemic brain were conducted with RNA-sequencing analysis and immunostaining of pathological markers. Results: FosDT expression is developmentally regulated, with the adult cerebral cortex showing significantly higher FosDT expression than neonates. FosDT −/− rats did not show any anomalies in growth and development, fertility, brain cytoarchitecture, and cerebral vasculature. However, when subjected to transient focal ischemia, FosDT −/− rats of both sexes showed enhanced sensorimotor recovery and reduced brain damage. RNA-sequencing analysis showed that improved poststroke functional outcome in FosDT −/− rats is partially associated with curtailed induction of inflammatory genes, reduced apoptosis, mitochondrial dysfunction, and oxidative stress. Conclusions: Our study shows that FosDT is developmentally dispensable, mechanistically important, and a functionally promising target to reduce ischemic brain damage and facilitate neurological recovery.
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Walter, Roland B., Olivier Humbert, George S. Laszlo, Sophie R. Sichel, Christina M. Ironside, Kevin G. Haworth, Olivia M. Bates, Mary E. Beddoe, Ray R. Carillo, and Hans-Peter Kiem. "Engineering Resistance to CD33-Targeted Immunotherapy in Normal Hematopoiesis By CRISPR/Cas9-Deletion of CD33 Exon 2." Blood 132, Supplement 1 (November 29, 2018): 2200. http://dx.doi.org/10.1182/blood-2018-99-117856.
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Abstract Background:Improved survival with gemtuzumab ozogamicin (GO) in some people with acute myeloid leukemia has validated CD33 as immunotherapeutic target and sparked interested in developing new, highly potent CD33-directed therapeutics. As a limitation of this treatment strategy, CD33 expression on maturing and mature myeloid cells causes significant on-target, off-leukemia effects. Toxicity of CD33-targeted immunotherapy should be minimal in the presence of normal hematopoietic stem and progenitor cells (HSPCs) engineered to lack CD33 variants recognized by therapeutic antibodies. Indeed, very recent studies have shown that CRISPR/Cas9 with a single guide RNA (gRNA) designed to target the CD33coding region reduces display of CD33 and protects engineered cells from CD33 CAR T-cells. However, this approach resulted in low levels of CD33disruption in vivoand off-target activity. We therefore developed an approach in which the Cas9 protein is complexed with two synthetic gRNAs for precise excision of the intervening sequence (i.e. more controlled genome editing than what can be accomplished with a single gRNA). We directed these two gRNAs to intronic sequences for precise excision of exon 2, which encodes the V-set domain of CD33 that is recognized by all current CD33 therapeutics including GO. This approach eliminates exonic indels and protects engineered cells from CD33-targeted immunotherapy while maintaining expression of an exon 2-free variant of CD33 (CD33∆E2), which has previously been identified as natural isoform in human HSPCs. Methods: We used human myeloid ML-1 cells and human fetal liver CD34+ HSPCs for CRISPR/Cas9 editing, which was carried out by electroporation of purified Cas9 protein complexed with synthetic gRNAs. In vitro cytotoxicity assays were performed to test sensitivity to GO and the CD33/CD3 bispecific antibody AMG 330.For in vivoassessment of engineered HSPCs, NSG neonate mice were infused with 6.0x105human CD34+ cells, and peripheral blood analyzed biweekly for 14 weeks. Results:Delivery of Cas9/sgRNA ribonucleoproteins (RNPs) in ML-1 cells resulted in exon 2 deletion and time-dependent reduction in cell surface display of full-length CD33 (CD33FL). Pure populations of CD33∆E2cells were generated via single-cell cloning. These sublines lacked surface display of CD33FLbut, instead, expressed increased levels of the CD33∆E2transcript. CD33∆E2ML-1 cells were completely resistant to GO and AMG 330, whereas wild-type ML-1 cells were highly sensitive to these two drugs. Consistent with the findings in ML-1 cells, delivery of CRISPR/Cas9 RNPs to human fetal liver CD34+ HSPCs resulted in the expected CD33exon 2 deletion and decreased CD33FLsurface expression, while it did not impact the distribution of colony-forming cellsand only minimally reduced colony-forming potential. In NSG mouse xenotransplantation experiments, we found these CD33∆E2human CD34+ HSPCs to have comparable engraftment and multilineage differentiation potential relative to HSPCs expressing CD33FL(Fig. 1A). As expected, CD33FLexpression was substantially reduced in peripheral blood monocytes from CD33∆E2 HSPC-engrafted animals (Fig. 1B). In addition, robust engraftment of CD33∆E2 -engineered HSPCs in bone marrow was demonstrated by over 50% biallelic CD33exon 2 deletion at time of necropsy from colony-forming cells analysis (Fig. 1C). To determine if in vivodifferentiated myeloid CD33∆E2cells derived from infused human CD34+ HSPCs are indeed resistant to CD33-directed therapies, we treated mice with GO intravenously. GO administration reduced the number of circulating CD33FLCD14+ myeloid cells while leaving the number of CD33∆E2cells unaffected. Conclusion:These findings support a novel strategy in which CD33∆E2-engineeredHSPCs are used to widen the therapeutic window of CD33-directed immunotherapies. Such cells could be envisioned for people at risk for the need of CD33-immunotherapy, e.g. AML patients in remission (where CD33-immunotherapy could be used to prevent/treat relapse) or, pre-emptively, for people with genetic AML-predisposition syndromes (such that CD33-targeted immunotherapy could be given safely if AML occurred). With this potential, further development of CD33∆E2-engineered HSPCs toward clinical application is warranted. Disclosures Walter: Amphivena Therapeutics, Inc: Consultancy, Other: Clinical Trial Support, Research Funding; Aptevo Therapeutics, Inc: Consultancy, Other: Clinical Trial Support, Research Funding; Covagen AG: Consultancy, Other: Clinical Trial Support, Research Funding; Actinium Pharmaceuticals, Inc: Other: Clinical Trial support , Research Funding; Amgen Inc: Other: Clinical Trial Support, Research Funding; Boehringer Ingelheim Pharma GmbH & Co. KG: Consultancy; Pfizer, Inc: Consultancy; Seattle Genetics, Inc: Consultancy, Other: Clinical Trial Support, Research Funding. Kiem:Magenta: Consultancy; Homology Medicine: Consultancy; Rocket Pharmaceuticals: Consultancy.
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Lin, Steven, Brett T. Staahl, Ravi K. Alla, and Jennifer A. Doudna. "Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery." eLife 3 (December 15, 2014). http://dx.doi.org/10.7554/elife.04766.
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The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (<xref ref-type="bibr" rid="bib13">Jinek et al., 2013</xref>), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.
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Silva-Pinheiro, Pedro, Pavel A. Nash, Lindsey Van Haute, Christian D. Mutti, Keira Turner, and Michal Minczuk. "In vivo mitochondrial base editing via adeno-associated viral delivery to mouse post-mitotic tissue." Nature Communications 13, no. 1 (February 8, 2022). http://dx.doi.org/10.1038/s41467-022-28358-w.
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AbstractMitochondria host key metabolic processes vital for cellular energy provision and are central to cell fate decisions. They are subjected to unique genetic control by both nuclear DNA and their own multi-copy genome - mitochondrial DNA (mtDNA). Mutations in mtDNA often lead to clinically heterogeneous, maternally inherited diseases that display different organ-specific presentation at any stage of life. For a long time, genetic manipulation of mammalian mtDNA has posed a major challenge, impeding our ability to understand the basic mitochondrial biology and mechanisms underpinning mitochondrial disease. However, an important new tool for mtDNA mutagenesis has emerged recently, namely double-stranded DNA deaminase (DddA)-derived cytosine base editor (DdCBE). Here, we test this emerging tool for in vivo use, by delivering DdCBEs into mouse heart using adeno-associated virus (AAV) vectors and show that it can install desired mtDNA edits in adult and neonatal mice. This work provides proof-of-concept for use of DdCBEs to mutagenize mtDNA in vivo in post-mitotic tissues and provides crucial insights into potential translation to human somatic gene correction therapies to treat primary mitochondrial disease phenotypes.
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YAMAMOTO, Hiromi, Keisuke YAMAMURA, Haruka NAGASAKI, Takamasa SUZUKI, Fumiko NINOMIYA, Kenji MATSUBARA, Naomoto HARADA, and Shuichi OHKUBO. "Genome editing of Nf1, Pten, and Trp53 in neonatal mice induces glioblastomas positive for oligodendrocyte lineage transcription factor 2." Journal of Toxicologic Pathology, 2021. http://dx.doi.org/10.1293/tox.2021-0029.
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Yang, Qian, Yan Hong, Ting Zhao, Hongjun Song, and Guo-li Ming. "What Makes Organoids Good Models of Human Neurogenesis?" Frontiers in Neuroscience 16 (April 14, 2022). http://dx.doi.org/10.3389/fnins.2022.872794.
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Human neurogenesis occurs mainly in embryonic, fetal, and neonatal stages and generates tremendously diverse neural cell types that constitute the human nervous system. Studies on human neurogenesis have been limited due to a lack of access to human embryonic and fetal tissues. Brain organoids derived from human pluripotent stem cells not only recapitulate major developmental processes during neurogenesis, but also exhibit human-specific features, thus providing an unprecedented opportunity to study human neurodevelopment. First, three-dimensional brain organoids resemble early human neurogenesis with diverse stem cell pools, including the presence of primate-enriched outer radial glia cells. Second, brain organoids recapitulate human neurogenesis at the cellular level, generating diverse neuronal cell types and forming stratified cortical layers. Third, brain organoids also capture gliogenesis with the presence of human-specific astrocytes. Fourth, combined with genome-editing technologies, brain organoids are promising models for investigating functions of human-specific genes at different stages of human neurogenesis. Finally, human organoids derived from patient iPSCs can recapitulate specific disease phenotypes, providing unique models for studying developmental brain disorders of genetic and environmental causes, and for mechanistic studies and drug screening. The aim of this review is to illustrate why brain organoids are good models to study various steps of human neurogenesis, with a focus on corticogenesis. We also discuss limitations of current brain organoid models and future improvements.
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VanDusen, Nathan J., Yanjiang Zheng, Catalina E. Butler, Qing Ma, Justin S. King, and William T. Pu. "Abstract 106: Efficient In Vivo Homology-Directed Repair Within Cardiomyocytes." Circulation Research 129, Suppl_1 (September 3, 2021). http://dx.doi.org/10.1161/res.129.suppl_1.106.
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CRISPR/Cas9-based genome editing technologies provide powerful tools for genetic manipulation. Delivery of Cas9 and a homology directed repair (HDR) template using adeno-associated virus (AAV; CASAAV-HDR), was recently shown to enable creation of precise genomic edits, even within postmitotic cells. Here we studied CASAAV-HDR in cardiomyocytes. We constructed an AAV9 vector containing a gRNA targeting the ventricle specific Myl2 gene, and a promoterless HDR template that replaces the native Myl2 stop codon with a self-cleaving 2A peptide followed by mScarlet, a red fluorescent protein. When this vector was injected into Cas9 expressing newborn mice, we observed mScarlet expression within a remarkably high fraction of cardiomyocytes, approximately 45%. Expression was ventricle specific, consistent with the Myl2 expression profile. Similarly, when we targeted the atrial specific Myl7 gene, we observed mScarlet expression in ~20% of atrial cardiomyocytes. Amplicon sequencing of Myl2 and Myl7 transcripts showed that the vast majority of transcripts with an insertion were mutation-free, indicating that CASAAV-HDR is precise. Furthermore, CASAAV-HDR efficiency was comparable when AAV was delivered to fetal, neonatal, or mature mice. Next we targeted seven additional loci: Yap1, Tmem43, Nfatc3, Bdh1, Mkl1, Ttn, and Pln, fusing either an HA tag or mScarlet to each. Insertion efficiency varied dramatically between loci, with HDR efficiency generally correlating with target gene expression. TTN-mScarlet and mScarlet-PLN fusion proteins localized to the sarcomere and sarcoplasmic reticulum, respectively, consistent with the localization of the endogenous proteins. Collectively these data indicate that systemic delivery of CASAAV-HDR vectors can achieve efficient, precise, in vivo somatic genome modification that does not require cardiomyocyte proliferation. We successfully used this technology to monitor protein localization and anticipate it will be useful for many other applications, such as precise introduction of mutations to model disease or probe gene function. CASAAV-HDR may also enable efficient, permanent, and precisely targeted delivery of therapeutic transgenes to validated loci.
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Shakya, Manita, Surbhi Gahlot, Anoop Arunagiri, Anis Hassan, Peter Arvan, Malcolm James Low, and Iris Lindberg. "SUN-669 The G209R Mutant Mouse as a Model for Human PCSK1 Polyendocrinopathy." Journal of the Endocrine Society 4, Supplement_1 (April 2020). http://dx.doi.org/10.1210/jendso/bvaa046.913.
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Abstract A striking number of SNPs and rare mutations have been identified in PCSK1, the gene that codes for the enzyme proprotein convertase 1/3 (PC1/3) which proteolytically activates prohormones within the secretory pathway. All infants bearing two copies of catalytically inactivating mutations, including G209R, exhibit severe neonatal malabsorption requiring parenteral nutrition for months and subsequently develop additional endocrinopathies, often including diabetes and obesity. In order to create a mouse model to explore the underlying mechanism of the malabsorption phenomenon and the endocrinopathies, a G209R point mutation was introduced into exon 6 of mouse Pcsk1 using CRISPR-Cas9 genome editing. Fifty-six live pups were collected at postnatal days one or two; however, most homozygous G209R mutant pups succumbed by day 2, and surviving pups were severely dwarfed. In homozygous, but not heterozygous pups, blood glucose levels were significantly lower with elevated plasma insulin-like immunoreactivity and accumulation of unprocessed proinsulin in G209R pancreas compared to the wild type pups from the same litters. The POMC product α-MSH (produced by PC2 from PC1/3-generated ACTH) has been strongly implicated in obesity mechanisms. We found pituitary POMC processing to ACTH was also affected by the G209R mutation in combined anterior and intermediate pituitary lobes. ACTH was markedly reduced in homozygote pituitary, with significant accumulation of POMC. Using Western blotting, we observed a significant reduction in PC1/3 protein in homozygote brains, while PC2 protein levels remained unaffected. Most likely due to the continued presence of PC2, pituitary and brain levels of α-MSH were not impaired, suggesting that α-MSH itself is not involved in the phenotype. Prior studies have shown that G209R PC1/3 is not efficiently trafficked out of the ER; further studies will examine the contribution of misfolded G209R PC1/3 to possible cellular ER stress, as well as determine peptide hormone levels in brain and peripheral tissues.
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Böck, Desirée, Tanja Rothgangl, Lukas Villiger, Lukas Schmidheini, Mai Matsushita, Nicolas Mathis, Eleonora Ioannidi, et al. "In vivo prime editing of a metabolic liver disease in mice." Science Translational Medicine 14, no. 636 (March 16, 2022). http://dx.doi.org/10.1126/scitranslmed.abl9238.
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Prime editing is a highly versatile CRISPR-based genome editing technology that works without DNA double-strand break formation. Despite rapid technological advances, in vivo application for the treatment of genetic diseases remains challenging. Here, we developed a size-reduced Sp Cas9 prime editor (PE) lacking the RNaseH domain (PE2 Δ RnH ) and an intein-split construct (PE2 p.1153) for adeno-associated virus–mediated delivery into the liver. Editing efficiencies reached 15% at the Dnmt1 locus and were further elevated to 58% by delivering unsplit PE2 Δ RnH via human adenoviral vector 5 (AdV). To provide proof of concept for correcting a genetic liver disease, we used the AdV approach for repairing the disease-causing Pah enu2 mutation in a mouse model of phenylketonuria (PKU) via prime editing. Average correction efficiencies of 11.1% (up to 17.4%) in neonates led to therapeutic reduction of blood phenylalanine, without inducing detectable off-target mutations or prolonged liver inflammation. Although the current in vivo prime editing approach for PKU has limitations for clinical application due to the requirement of high vector doses (7 × 10 14 vg/kg) and the induction of immune responses to the vector and the PE, further development of the technology may lead to curative therapies for PKU and other genetic liver diseases.
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Mehta, Suresh L., Kahlilia Morris-Blanco, Anil Kiran Chokkalla, Saivenkateshkomal Bathula, Taehee Kim, Bharath Chelluboina, Aleah Holmes, et al. "Abstract P835: Long Noncoding RNA FosDT is Developmentally Dispensable but Critical for Shaping the Therapeutic Post-Stroke Functional Outcome." Stroke 52, Suppl_1 (March 2021). http://dx.doi.org/10.1161/str.52.suppl_1.p835.
Full textAbstract:
Stroke induces the expression of several long noncoding RNAs (lncRNAs) in the brain. However, their functional significance in post-stroke outcome is poorly understood. We recently observed that a brain-specific lncRNA called Fos downstream transcript (FosDT) is induced rapidly in rodent brain following focal ischemia. We here show that FosDT expression is developmentally regulated with adults specifically display higher expression in the cerebral cortex than neonates. To understand its significance in ischemic brain damage, we developed FosDT knockout rats using CRISPR-Cas9 genome editing. We found that FosDT knockout rats did not show any anomalies in growth and development, fertility, brain cytoarchitecture and cerebral vasculature. However, when subjected to transient focal ischemia, FosDT knockout rats of both sexes showed enhanced sensorimotor recovery and reduced brain damage. To further understand the mechanistic implications of FosDT in the ischemic brain, we conducted RNA-seq analysis. The result showed that improved post-stroke functional outcome in FosDT knockout rats is partially associated with curtailed post-ischemic induction of inflammatory genes. When rats subjected to transient focal ischemia were treated with FosDT siRNA, there was significant neuroprotection and better functional outcome irrespective of sex and age. FosDT siRNA was efficacious when administered peripherally and also in a delayed manner. Thus, preventing FosDT activation is beneficial for the post-stroke outcome.