Bachelor of Arts, St. John Fisher College (2006)
Master of Science, University of Rochester (2011)
Doctor of Philosophy, University of Rochester, Toxicology (2014)
CRISPR/Cas9 ß-globin gene targeting in human haematopoietic stem cells.
The β-haemoglobinopathies, such as sickle cell disease and β-thalassaemia, are caused by mutations in the β-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure β-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult β-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for β-haemoglobinopathies.
View details for DOI 10.1038/nature20134
View details for PubMedID 27820943
Aryl hydrocarbon receptor deletion in cerebellar granule neuron precursors impairs neurogenesis
2016; 76 (5): 533-550
The aryl hydrocarbon receptor (AhR) is a ligand-activated member of the basic-helix-loop-helix/PER-ARNT-SIM(PAS) transcription factor superfamily that also mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increasing evidence suggests that AhR influences the development of many tissues, including the central nervous system. Our previous studies suggest that sustained AhR activation by TCDD and/or AhR deletion disrupts cerebellar granule neuron precursor (GNP) development. In the current study, to determine whether endogenous AhR controls GNP development in a cell-autonomous manner, we created a GNP-specific AhR deletion mouse, AhR(fx/fx) /Math1(CRE/+) (AhR CKO). Selective AhR deletion in GNPs produced abnormalities in proliferation and differentiation. Specifically, fewer GNPs were engaged in S-phase, as demonstrated by ∼25% reductions in thymidine (in vitro) and Bromodeoxyuridine (in vivo) incorporation. Furthermore, total granule neuron numbers in the internal granule layer at PND21 and PND60 were diminished in AhR conditional knockout (CKO) mice compared with controls. Conversely, differentiation was enhanced, including ∼40% increase in neurite outgrowth and 50% increase in GABARα6 receptor expression in deletion mutants. Our results suggest that AhR activity plays a role in regulating granule neuron number and differentiation, possibly by coordinating this GNP developmental transition. These studies provide novel insights for understanding the normal roles of AhR signaling during cerebellar granule cell neurogenesis and may have important implications for the effects of environmental factors in cerebellar dysgenesis.
View details for DOI 10.1002/dneu.22330
View details for Web of Science ID 000373930600005
View details for PubMedID 26243376
The Aryl Hydrocarbon Receptor Contributes to the Proliferation of Human Medulloblastoma Cells
2012; 81 (5): 669-678
The aryl hydrocarbon receptor (AhR), a ligand-activated member of the basic helix-loop-helix (bHLH)/PER-ARNT-SIM (PAS) transcription superfamily, is known to regulate the toxicity of polyaromatic halogenated hydrocarbon environmental chemicals, most notably dioxin. However, the AhR has also been implicated in multiple stages of tumorigenesis. Medulloblastoma (MB), a primary cerebellar brain tumor arising in infants and children, is thought to originate from abnormally proliferating cerebellar granule neuron precursors (GNPs). GNPs express high levels of the AhR in the external germinal layer of the developing cerebellum. Moreover, our laboratory has previously reported that either abnormal activation or deletion of the AhR leads to dysregulation of GNP cell cycle activity and maturation. These observations led to the hypothesis that the AhR promotes the growth of MB. Therefore, this study evaluated whether the AhR serves a pro-proliferative role in an immortalized MB tumor cell line (DAOY). We produced a stable AhR knockdown DAOY cell line [AhR short hairpin RNA (shRNA)], which exhibited a 70% reduction in AhR protein levels. Compared with wild-type DAOY cells, AhR shRNA DAOY cells displayed an impaired G(1)-to-S cell cycle transition, decreased DNA synthesis, and reduced proliferation. Furthermore, these cell cycle perturbations were correlated with decreased levels of the pro-proliferative gene Hes1 and increased levels of the cell cycle inhibitor p27(kip1). Supplementation experiments with human AhR restored the proliferative activity in AhR shRNA DAOY cells. Taken together, our data show that the AhR promotes proliferation of MB cells, suggesting that this pathway should be considered as a potential therapeutic target for MB treatment.
View details for DOI 10.1124/mol.111.077305
View details for Web of Science ID 000302934500005
View details for PubMedID 22311706
Subchronic Polychlorinated Biphenyl (Aroclor 1254) Exposure Produces Oxidative Damage and Neuronal Death of Ventral Midbrain Dopaminergic Systems
2012; 125 (2): 496-508
Recent epidemiologic studies have demonstrated a link between organochlorine and pesticide exposure to an enhanced risk for neurodegenerative disorders such as Parkinson's disease (PD). A common biological phenomenon underlying cell injury associated with both polychlorinated biphenyl (PCB) exposure and dopaminergic neurodegeneration during aging is oxidative stress (OS). In this study, we tested the hypothesis that oral PCB exposure, via food ingestion, impairs dopamine systems in the adult murine brain. We determined whether PCB exposure was associated with OS in dopaminergic neurons, a population of cells that selectively degenerate in PD. After 4 weeks of oral exposure to the PCB mixture Aroclor 1254, several congeners, mostly ortho substituted, accumulated throughout the brain. Significant increases in locomotor activity were observed within 2 weeks, which persisted after cessation of PCB exposure. Stereologic analyses revealed a significant loss of dopaminergic neurons within the substantia nigra and ventral tegmental area. However, striatal dopamine levels were elevated, suggesting that compensatory mechanisms exist to maintain dopamine homeostasis, which could contribute to the observed increases in locomotor activity following PCB exposure. Biochemical experiments revealed alterations in OS markers, including increases in SOD and HO-1 levels and the presence of oxidatively modified lipids and proteins. These findings were accompanied by elevated iron levels within the striatal and midbrain regions, perhaps due to the observed dysregulation of transferrin receptors and ferritin levels following PCB exposure. In this study, we suggest that both OS and the uncoupling of iron regulation contribute to dopamine neuron degeneration and hyperactivity following PCB exposure.
View details for DOI 10.1093/toxsci/kfr313
View details for Web of Science ID 000299346000017
View details for PubMedID 22094459
2,3,7,8-tetracholorodibenzo-p-dioxin exposure disrupts granule neuron precursor maturation in the developing mouse cerebellum
2008; 103 (1): 125-136
The widespread environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been linked to developmental neurotoxicity associated with abnormal cerebellar maturation in both humans and rodents. TCDD mediates toxicity via binding to the aryl hydrocarbon receptor (AhR), a transcription factor that regulates the expression of xenobiotic metabolizing enzymes and growth regulatory molecules. Our previous studies demonstrated that cerebellar granule neuron precursor cells (GNPs) express transcriptionally active AhR during critical developmental periods. TCDD exposure also impaired GNP proliferation and survival in vitro. Therefore, this study tested the hypothesis that TCDD exposure disrupts cerebellar development by interfering with GNP differentiation. In vivo experiments indicated that TCDD exposure on postnatal day (PND) 6 resulted in increased expression of a mitotic marker and increased thickness of the external granule layer (EGL) on PND10. Expression of the early differentiation marker TAG-1 was also more pronounced in postmitotic, premigratory granule neurons of the EGL, and increased apoptosis of GNPs was observed. On PND21, expression of the late GNP differentiation marker GABA(A alpha 6) receptor (GABAR(A alpha 6)) and total estimated cell numbers were both reduced following exposure on PND6. Studies in unexposed adult AhR(-/-) mice revealed lower GABAR(A alpha 6) levels and DNA content. In vitro studies showed elevated expression of the early differentiation marker p27/Kip1 and the GABAR(A alpha 6) in GNPs following TCDD exposure, and the expression patterns of proteins related to granule cell neurite outgrowth, beta III-tubulin and polysialic acid neural cell adhesion molecule, were consistent with enhanced neuroblast differentiation. Together, our data suggest that TCDD disrupts a normal physiological role of AhR, resulting in compromised GNP maturation and neuroblast survival, which impacts final cell number in the cerebellum.
View details for DOI 10.1093/toxsci/kfn017
View details for Web of Science ID 000254955500014
View details for PubMedID 18227101