Genome-editing technologies for gene correction of hemophilia

Hemophilia is caused by various mutations in blood coagulation factor genes, including factor VIII (FVIII) and factor IX (FIX), that encode key proteins in the blood clotting pathway. Although the addition of therapeutic genes or infusion of clotting factors may be used to remedy hemophilia's symptoms, no permanent cure for the disease exists. Moreover, patients often develop neutralizing antibodies or experience adverse effects that limit the therapy's benefits. However, targeted gene therapy involving the precise correction of these mutated genes at the genome level using programmable nucleases is a promising strategy. These nucleases can induce double-strand breaks (DSBs) on genomes, and repairs of such induced DSBs by the two cellular repair systems enable a targeted gene correction. Going beyond cultured cell systems, we are now entering the age of direct gene correction in vivo using various delivery tools. Here, we describe the current status of in vivo and ex vivo genome-editing technology related to potential hemophilia gene correction and the prominent issues surrounding its application in patients with monogenic diseases.ope

Generation of a human induced pluripotent stem cell line, YCMi002-A, from a Factor VII deficiency patient carrying F7 mutations

Factor VII (FVII) deficiency is the most common among the rare bleeding disorders, which is caused by mutations in coagulation factor VII. Clinical features caused by FVII deficiency vary from mild or asymptomatic to fatal cerebral hemorrhage. We generated an induced pluripotent stem cell (iPSC) line, YCMi002-A, from FVII deficiency patient-derived fibroblasts. YCMi002-A cells are characterized by novel compound heterozygous mutations. The c.345C > A; p.C115X is well known and the second one, c.1276C > T; p.Q426X, remains novel. YCMi002-A cells may help researchers to understand correlation between these mutations and the symptoms of FVII deficiency.ope

Reversion of FMR1 Methylation and Silencing by Editing the Triplet Repeats in Fragile X iPSC-Derived Neurons

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability, resulting from a CGG repeat expansion in the fragile X mental retardation 1 (FMR1) gene. Here, we report a strategy for CGG repeat correction using CRISPR/Cas9 for targeted deletion in both embryonic stem cells and induced pluripotent stem cells derived from FXS patients. Following gene correction in FXS induced pluripotent stem cells, FMR1 expression was restored and sustained in neural precursor cells and mature neurons. Strikingly, after removal of the CGG repeats, the upstream CpG island of the FMR1 promoter showed extensive demethylation, an open chromatin state, and transcription initiation. These results suggest a silencing maintenance mechanism for the FMR1 promoter that is dependent on the existence of the CGG repeat expansion. Our strategy for deletion of trinucleotide repeats provides further insights into the molecular mechanisms of FXS and future therapies of trinucleotide repeat disorders.ope

Wnt signal activation induces midbrain specification through direct binding of the beta-catenin/TCF4 complex to the EN1 promoter in human pluripotent stem cells

The canonical Wnt signal pathway plays a pivotal role in anteroposterior patterning and midbrain specification during early neurogenesis. Activating Wnt signal has been a strategy for differentiating human pluripotent stem cells (PSCs) into midbrain dopaminergic (DA) neurons; however, the underlying molecular mechanism(s) of how the Wnt signal drives posterior fate remained unclear. In this study, we found that activating the canonical Wnt signal significantly upregulated the expression of EN1, a midbrain-specific marker, in a fibroblast growth factor signal-dependent manner in human PSC-derived neural precursor cells (NPCs). The EN1 promoter region contains a putative TCF4-binding site that directly interacts with the beta-catenin/TCF complex upon Wnt signal activation. Once differentiated, NPCs treated with a Wnt signal agonist gave rise to functional midbrain neurons including glutamatergic, GABAergic, and DA neurons. Our results provide a potential molecular mechanism that underlies midbrain specification of human PSC-derived NPCs by Wnt activation, as well as a differentiation paradigm for generating human midbrain neurons that may serve as a cellular platform for studying the ontogenesis of midbrain neurons and neurological diseases relevant to the midbrain.ope

Restoration of FVIII expression by targeted gene insertion in the FVIII locus in hemophilia A patient-derived iPSCs

Target-specific genome editing, using engineered nucleases zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), is considered a promising approach to correct disease-causing mutations in various human diseases. In particular, hemophilia A can be considered an ideal target for gene modification via engineered nucleases because it is a monogenic disease caused by a mutation in coagulation factor VIII (FVIII), and a mild restoration of FVIII levels in plasma can prevent disease symptoms in patients with severe hemophilia A. In this study, we describe a universal genome correction strategy to restore FVIII expression in induced pluripotent stem cells (iPSCs) derived from a patient with hemophilia A by the human elongation factor 1 alpha (EF1α)-mediated normal FVIII gene expression in the FVIII locus of the patient. We used the CRISPR/Cas9-mediated homology-directed repair (HDR) system to insert the B-domain deleted from the FVIII gene with the human EF1α promoter. After gene targeting, the FVIIIgene was correctly inserted into iPSC lines at a high frequency (81.81%), and these cell lines retained pluripotency after knock-in and neomycin resistance cassette removal. More importantly, we confirmed that endothelial cells from the gene-corrected iPSCs could generate functionally active FVIII protein from the inserted FVIII gene. This is the first demonstration that the FVIII locus is a suitable site for integration of the normal FVIII gene and can restore FVIII expression by the EF1α promoter in endothelial cells differentiated from the hemophilia A patient-derived gene-corrected iPSCs.ope

Targeted inversion and reversion of the blood coagulation factor 8 gene in human iPS cells using TALENs

Hemophilia A, one of the most common genetic bleeding disorders, is caused by various mutations in the blood coagulation factor VIII (F8) gene. Among the genotypes that result in hemophilia A, two different types of chromosomal inversions that involve a portion of the F8 gene are most frequent, accounting for almost half of all severe hemophilia A cases. In this study, we used a transcription activator-like effector nuclease (TALEN) pair to invert a 140-kbp chromosomal segment that spans the portion of the F8 gene in human induced pluripotent stem cells (iPSCs) to create a hemophilia A model cell line. In addition, we reverted the inverted segment back to its normal orientation in the hemophilia model iPSCs using the same TALEN pair. Importantly, we detected the F8 mRNA in cells derived from the reverted iPSCs lines, but not in those derived from the clones with the inverted segment. Thus, we showed that TALENs can be used both for creating disease models associated with chromosomal rearrangements in iPSCs and for correcting genetic defects caused by chromosomal inversions. This strategy provides an iPSC-based novel therapeutic option for the treatment of hemophilia A and other genetic diseases caused by chromosomal inversions.ope

ABCD2 Is a Direct Target of β-Catenin and TCF-4: Implications for X-Linked Adrenoleukodystrophy Therapy

X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder caused by mutations in the ABCD1 gene that encodes the peroxisomal ATP-binding cassette (ABC) transporter subfamily D member 1 protein (ABCD1), which is referred to as the adrenoleukodystrophy protein (ALDP). Induction of the ABCD2 gene, the closest homolog of ABCD1, has been mentioned as a possible therapeutic option for the defective ABCD1 protein in X-ALD. However, little is known about the transcriptional regulation of ABCD2 gene expression. Here, through in silico analysis, we found two putative TCF-4 binding elements between nucleotide positions −360 and −260 of the promoter region of the ABCD2 gene. The transcriptional activity of the ABCD2 promoter was strongly increased by ectopic expression of β-catenin and TCF-4. In addition, mutation of either or both TCF-4 binding elements by site-directed mutagenesis decreased promoter activity. This was further validated by the finding that β-catenin and the promoter of the ABCD2 gene were pulled down with a β-catenin antibody in a chromatin immunoprecipitation assay. Moreover, real-time PCR analysis revealed that β-catenin and TCF-4 increased mRNA levels of ABCD2 in both a hepatocellular carcinoma cell line and primary fibroblasts from an X-ALD patient. Interestingly, we found that the levels of very long chain fatty acids were decreased by ectopic expression of ABCD2-GFP as well as β-catenin and TCF-4. Taken together, our results demonstrate for the first time the direct regulation of ABCD2 by β-catenin and TCF-4.ope

안구 내 유전자 전달을 위한 아데노바이러스 벡터와 부신 피질 호르몬제의 병용

Thesis(master`s)--서울대학교 대학원 :의학과 미생물학전공,2007.Maste

(The) effect of strategic changes in medical care aid system on usage of outpatient services

보건정보관리학과/석사이 연구는 의료급여 수급권자의 과다 의료이용 등 도덕적 해이를 줄이고 적정의료이용 유도 및 재정안정을 도모하기 위하여 시행된 여러 유형의 의료급여제도 정책변화가 환자의 의료이용에 어떠한 변화를 주었는지 도입 전•후 외래 진료일수와 외래 진료비 등에 미치는 영향을 분석하였다.요양기관에서 청구한 의료급여수급권자의 진료비 지급자료로 국민건강보험 공단이 보유하고 있는D/B의 의료급여 진료정보를 이용하였으며, 연구대상자 는 371,811명(남자 147,364명, 여자 224,447명)이며, 의료급여제도가 도입되기 이전인 2005년 7월 1일부터 2007년 6월 30일까지 2년간 및 제도도입 후 2007년7월 1일부터 2009년 6월 30일 까지 2년간 외래로 요양기관을 방문한 진료내역을 연구자료로 이용하였다. 종속변수는 외래 진료일수와 외래 진료비이며, 설명변수는 제도도입 전후 이다. 혼란변수는 성, 연령, 거주지역, 요양기관 종별, 다빈도 질환명을 변수로 사용하여 각 변수와 진료일수 및 진료비의 차이는 카이제곱 검정(chi-square test)을 이용하여 분석하였고, 각 변수마다 진료일수 및 진료비에 미친 영향을 분석하기 위하여 일반화 추정방정식(GEE : Generalized Estimating Equation)을 시행하였다.연구결과는 제도도입 전후 외래 진료일수는 월 평균 1.95일에서 1.13일로 약 0.9일이 줄었고, 외래 진료비는 월 평균 50,859원에서 34,352원으로 16,507원이 줄어 들었으며, 통계적으로도 유의하였다(p<0.0001).외래 의료이용의 빈도가 높은 순으로 7개를 선정하여 질환별 의료이용을 분석해 본 결과, 모든 질환에서 유의하게 감소한 것으로 나타났으며, 특히 추간판장애나 무릎관절증 등 도덕적 해이가 심한 질환에서 더 크게 감소한 것으로 나타났고, 이외에 만성질환으로 평소 진료가 필요한 질환인 고혈압성 질환이나 갱년기 장애 등에 있어서는 소폭 줄어든 것으로 나타났다.입원의 경우는 대부분의 질환에서 입원일수의 소폭 증가를 보였으나, 악성신생물의 경우 22.1일에서 3.3일로 18.8일이 대폭 감소하였다. 요양기관 종별로는 의원급에서 진료일수는 2.3일(54.2%)와 진료비는 28,013원(52.9%) 각각 큰 폭으로 줄어들었으며, 통계적으로도 유의하였다(P<0.0001). 병원급에서도 약간의 감소를 보였지만 통계적으로 유의하지 않았다(P<0.1034).의료급여제도 정책변화 이후 진료일수가 감소하는 것에 대해서는 효과가 입증되었으며, 제도도입 후 2년간의 종합병원 이용률의 감소는 외래 진료일수의 감소로 이어 졌다고 볼 때, 정책의 변화가 병원 이용에 대해서는 단기적 효과만 있었으나, 내원일수에 대해서는 장기적 효과도 있었다고 판단된다.이와 같은 결과들은 의료급여제도 정책변화의 근본 취지인 불필요한 과다 의료이용 줄이고 적정 의료이용 유도 및 재정안정 도모 등 긍정적인 결과를 가져왔다는 것에 의의가 있다 할 것이며, 계속될 의료급여 정책 개선 및 보완에 참고가 되기를 기대한다. 향후 다른 연구에서는 의료급여제도 정책변화가 환자들의 의료이용 뿐만 아니라 건강의 유지관리에 어떤 영향을 미치는지와 경제적 부담 완화에 얼마나 기여를 했는지, 스스로의 건강관리 책임성이 얼마나 높아졌는지 등에 대한 검토와 제도적 측면에서 보완하고 개선해야 할 부분은 무엇인지에 대한 심도 있는 연구가 이루어져야 할 것이다.ope

Modeling and correction of structural variations in patient-derived iPSCs using CRISPR/Cas9

Genome engineering technology using engineered nucleases has been rapidly developing, enabling the efficient correction of simple mutations. However, the precise correction of structural variations (SVs) such as large inversions remains limited. Here we describe a detailed procedure for the modeling or correction of large chromosomal rearrangements and short nucleotide repeat expansions using engineered nucleases in human induced pluripotent stem cells (hiPSCs) from a healthy donor and patients with SVs. This protocol includes the delivery of engineered nucleases with no donor template to hiPSCs, and genotyping and derivation/characterization of gene-manipulated hiPSC clones. With engineered nucleases, genomic inversions, reversions, and deletions of short nucleotide expansions can be identified in 2 weeks, and desired clones can be generated in as little as 3-4 weeks. This protocol enables the correction of large inverted segments and short nucleotide repeat expansions in diseases such as hemophilia A, fragile X syndrome, Hunter syndrome, and Friedreich's ataxia.restrictio