Academic literature on the topic 'Neonatal genome editing'

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.

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.

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.

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.

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.

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.

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.

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.

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 &lt; 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.

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.

In vivo gene therapy with adeno-associated viral (AAV) vectors has been successful at treating several inherited diseases, specifically those caused by loss of function mutations which require transfer of a correct copy of a gene. This would not benefit dominant diseases due to gain of function mutations which produce toxic protein products. In addition, since AAV genomes persist as episomes in target cells, AAV mediated transgene expression might be short lived in tissues where cell proliferation occurs when newborn or after damage, like for example the liver. To overcome these challenges, I have developed AAV-based therapeutic approaches which use genome editing to introduce stable modifications at specific genomic loci. First, an allele-specific approach which targets the Rhodopsin P347S dominant mutation was developed and tested both in vitro and in vivo. I achieved allele-specific targeting of human P347S rhodopsin, which reduced mRNA levels and improved retinal electrical function in a mouse model of autosomal dominant retinitis pigmentosa. Second, I developed a mutation- and homology-independent targeted integration (HITI) approach for gene correction in photoreceptors. I demonstrated feasibility of this approach in mouse and pig photoreceptors using a reporter gene and characterized on-target precision of HITI in the murine rhodopsin locus. I then tested the therapeutic potential of this approach in a mouse model of autosomal dominant retinitis pigmentosa and observed mild and transient improvement of retinal function in treated eyes, which suggests that the levels of editing obtained need optimization. Third, I developed a HITI approach for expressing therapeutic genes from the liver by targeting the albumin locus, which is highly transcribed in hepatocytes. I demonstrated feasibility and efficiency of this approach using a reporter gene, and characterized on-target precision of HITI, as well as off-target integration due to Cas9 cleavage. I then tested the therapeutic potential of the integration of a copy of the human arylsulfatase B (ARSB) gene, which is mutated in a rare lysosomal storage disease, mucopolysaccharidosis type VI (MPS VI), in the albumin locus in the liver of newborn MPSVI mice. I demonstrated that this approach achieves stable expression of ARSB at levels that reduce glucosaminoglycan (GAG) urinary secretion, one of the main readouts of MPSVI phenotype. This stable expression of ARSB is contrary to the decrease of transgene expression observed in neonatal MPSVI mice injected with the same dose of a conventional gene therapy vector, thus overcoming the potential loss of transgene expression caused by hepatocyte proliferation. Overall, I have developed different genome editing approaches for conditions that are inherited as either dominant or recessive. I have tested these approaches in two relevant tissues for gene therapy like retina and liver and shown the potential to provide AAV with persistent transgene expression in proliferating tissues like the newborn liver.

Throughout his history, man has selected animals that have the most useful features by carrying out their intraspecific crossing leading to the fixation of these features. Survival in the neonatal period has always remained one of the most important factors in breeding animals, and with the advent of genome editing, through which mutations, often negative for health, appear in the genome, the high survival rate of genetically edited animals becomes a paramount issue.