Browsing by Author "Lee, Ciaran"
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Item Collagen-rich airway smooth muscle cells are a metastatic niche for tumor colonization in the lung(Springer Nature, 2019) Lee, Yu-Cheng; Kurtova, Antonina V.; Xiao, Jing; Nikolos, Fotis; Hayashi, Kazukuni; Tramel, Zoe; Jain, Antrix; Chen, Fengju; Chokshi, Mithil; Lee, Ciaran; Bao, Gang; Zhang, Xiang; Shen, Jianjun; Mo, Qianxing; Jung, Sung Yun; Rowley, David; Chan, Keith SysonMetastases account for the majority of cancer deaths. While certain steps of the metastatic cascade are well characterized, identification of targets to block this process remains a challenge. Host factors determining metastatic colonization to secondary organs are particularly important for exploration, as those might be shared among different cancer types. Here, we showed that bladder tumor cells expressing the collagen receptor, CD167a, responded to collagen I stimulation at the primary tumor to promote local invasion and utilized the same receptor to preferentially colonize at airway smooth muscle cells (ASMCs)—a rich source of collagen III in lung. Morphologically, COL3-CD167a-driven metastatic foci are uniquely distinct from typical lung alveolar metastatic lesions and exhibited activation of the CD167a-HSP90-Stat3 axis. Importantly, metastatic lung colonization could be abrogated using an investigational drug that attenuates Stat3 activity, implicating this seed-and-soil interaction as a therapeutic target for eliminating lung metastasis.Item Collagen-rich airway smooth muscle cells are a metastatic niche for tumor colonization in the lung(Springer Nature, 2019) Lee, Yu-Cheng; Kurtova, Antonina V.; Xiao, Jing; Nikolos, Fotis; Hayashi, Kazukuni; Tramel, Zoe; Jain, Antrix; Chen, Fengju; Chokshi, Mithil; Lee, Ciaran; Bao, Gang; Zhang, Xiang; Shen, Jianjun; Mo, Qianxing; Jung, Sung Yun; Rowley, David; Chan, Keith SysonMetastases account for the majority of cancer deaths. While certain steps of the metastatic cascade are well characterized, identification of targets to block this process remains a challenge. Host factors determining metastatic colonization to secondary organs are particularly important for exploration, as those might be shared among different cancer types. Here, we showed that bladder tumor cells expressing the collagen receptor, CD167a, responded to collagen I stimulation at the primary tumor to promote local invasion and utilized the same receptor to preferentially colonize at airway smooth muscle cells (ASMCs)—a rich source of collagen III in lung. Morphologically, COL3-CD167a-driven metastatic foci are uniquely distinct from typical lung alveolar metastatic lesions and exhibited activation of the CD167a-HSP90-Stat3 axis. Importantly, metastatic lung colonization could be abrogated using an investigational drug that attenuates Stat3 activity, implicating this seed-and-soil interaction as a therapeutic target for eliminating lung metastasis.Item Fine-mapping within eQTL credible intervals by expression CROP-seq(Oxford University Press, 2020) Pan, Yidan; Tian, Ruoyu; Lee, Ciaran; Bao, Gang; Gibson, GregThe majority of genome-wide association study (GWAS)-identified SNPs are located in noncoding regions of genes and are likely to influence disease risk and phenotypes by affecting gene expression. Since credible intervals responsible for genome-wide associations typically consist of ≥100 variants with similar statistical support, experimental methods are needed to fine map causal variants. We report here a moderate-throughput approach to identifying regulatory GWAS variants, expression CROP-seq, which consists of multiplex CRISPR-Cas9 genome editing combined with single-cell RNAseq to measure perturbation in transcript abundance. Mutations were induced in the HL60/S4 myeloid cell line nearby 57 SNPs in three genes, two of which, rs2251039 and rs35675666, significantly altered CISD1 and PARK7 expression, respectively, with strong replication and validation in single-cell clones. The sites overlap with chromatin accessibility peaks and define causal variants for inflammatory bowel disease at the two loci. This relatively inexpensive approach should be scalable for broad surveys and is also implementable for the fine mapping of individual genes.Item Gene correction for SCID-X1 in long-term hematopoietic stem cells(Springer Nature, 2019) Pavel-Dinu, Mara; Wiebking, Volker; Dejene, Beruh T.; Srifa, Waracharee; Mantri, Sruthi; Nicolas, Carmencita E.; Lee, Ciaran; Bao, Gang; Kildebeck, Eric J.; Punjya, Niraj; Sindhu, Camille; Inlay, Matthew A.; Saxena, Nivedita; DeRavin, Suk See; Malech, Harry; Roncarolo, Maria Grazia; Weinberg, Kenneth I.; Porteus, Matthew H.Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45%) in CD34+ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl.Item Human genome-edited hematopoietic stem cells phenotypically correct Mucopolysaccharidosis type I(Springer Nature, 2019) Gomez-Ospina, Natalia; Scharenberg, Samantha G.; Mostrel, Nathalie; Bak, Rasmus O.; Mantri, Sruthi; Quadros, Rolen M.; Gurumurthy, Channabasavaiah B.; Lee, Ciaran; Bao, Gang; Suarez, Carlos J.; Khan, Shaukat; Sawamoto, Kazuki; Tomatsu, Shunji; Raj, Nitin; Attardi, Laura D.; Aurelian, Laure; Porteus, Matthew H.Lysosomal enzyme deficiencies comprise a large group of genetic disorders that generally lack effective treatments. A potential treatment approach is to engineer the patient’s own hematopoietic system to express high levels of the deficient enzyme, thereby correcting the biochemical defect and halting disease progression. Here, we present an efficient ex vivo genome editing approach using CRISPR-Cas9 that targets the lysosomal enzyme iduronidase to the CCR5 safe harbor locus in human CD34+ hematopoietic stem and progenitor cells. The modified cells secrete supra-endogenous enzyme levels, maintain long-term repopulation and multi-lineage differentiation potential, and can improve biochemical and phenotypic abnormalities in an immunocompromised mouse model of Mucopolysaccharidosis type I. These studies provide support for the development of genome-edited CD34+ hematopoietic stem and progenitor cells as a potential treatment for Mucopolysaccharidosis type I. The safe harbor approach constitutes a flexible platform for the expression of lysosomal enzymes making it applicable to other lysosomal storage disorders.Item In Vivo Ryr2 Editing Corrects Catecholaminergic Polymorphic Ventricular Tachycardia(American Heart Association, 2018) Pan, Xiaolu; Philippen, Leonne; Lahiri, Satadru K.; Lee, Ciaran; Park, So Hyun; Word, Tarah A.; Li, Na; Jarrett, Kelsey E.; Gupta, Rajat; Reynolds, Julia O.; Lin, Jean; Bao, Gang; Lagor, William R.; Wehrens, Xander H.T.Rationale:Autosomal-dominant mutations in ryanodine receptor type 2 (RYR2) are responsible for ≈60% of all catecholaminergic polymorphic ventricular tachycardia. Dysfunctional RyR2 subunits trigger inappropriate calcium leak from the tetrameric channel resulting in potentially lethal ventricular tachycardia. In vivo CRISPR/Cas9-mediated gene editing is a promising strategy that could be used to eliminate the disease-causing Ryr2 allele and hence rescue catecholaminergic polymorphic ventricular tachycardia.Objective:To determine if somatic in vivo genome editing using the CRISPR/Cas9 system delivered by adeno-associated viral (AAV) vectors could correct catecholaminergic polymorphic ventricular tachycardia arrhythmias in mice heterozygous for RyR2 mutation R176Q (R176Q/+).Methods and Results:Guide RNAs were designed to specifically disrupt the R176Q allele in the R176Q/+ mice using the SaCas9 (Staphylococcus aureus Cas9) genome editing system. AAV serotype 9 was used to deliver Cas9 and guide RNA to neonatal mice by single subcutaneous injection at postnatal day 10. Strikingly, none of the R176Q/+ mice treated with AAV-CRISPR developed arrhythmias, compared with 71% of R176Q/+ mice receiving control AAV serotype 9. Total Ryr2 mRNA and protein levels were significantly reduced in R176Q/+ mice, but not in wild-type littermates. Targeted deep sequencing confirmed successful and highly specific editing of the disease-causing R176Q allele. No detectable off-target mutagenesis was observed in the wild-type Ryr2 allele or the predicted putative off-target site, confirming high specificity for SaCas9 in vivo. In addition, confocal imaging revealed that gene editing normalized the enhanced Ca2+ spark frequency observed in untreated R176Q/+ mice without affecting systolic Ca2+ transients.Conclusions:AAV serotype 9–based delivery of the SaCas9 system can efficiently disrupt a disease-causing allele in cardiomyocytes in vivo. This work highlights the potential of somatic genome editing approaches for the treatment of lethal autosomal-dominant inherited cardiac disorders, such as catecholaminergic polymorphic ventricular tachycardia.