Biology subjects

Baik, R.

Publications and source records attributed to Baik, R..

3 recordsLinked to original sources

bioRxiv2021

Targeted replacement of full-length CFTR in human airway stem cells by CRISPR/Cas9 for pan-mutation correction in the endogenous locus

Cystic fibrosis (CF) is a monogenic disease caused by impaired production and/or function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Although we have previously shown correction of the most common pathogenic mutation, there are many other pathogenic mutations throughout the CF gene. An autologous airway stem cell therapy in which the CFTR cDNA is precisely inserted into the CFTR locus may enable the development of a durable cure for almost all CF patients, irrespective of the causal mutation. Here, we use CRISPR/Cas9 and two adeno-associated viruses (AAV) carrying the two halves of the CFTR cDNA to sequentially insert the full CFTR cDNA along with a truncated CD19 (tCD19) enrichment tag in upper airway basal stem cells (UABCs) and human bronchial basal stem cells (HBECs). The modified cells were enriched to obtain 60-80% tCD19+ UABCs and HBECs from 11 different CF donors with a variety of mutations. Differentiated epithelial monolayers cultured at air-liquid interface showed restored CFTR function that was >70% of the CFTR function in non-CF controls. Thus, our study enables the development of a therapy for almost all CF patients, including patients who cannot be treated using recently approved modulator therapies.

bioengineering↗

bioRxiv2020

Cas9-AAV6 Gene Correction of Beta-Globin in Autologous HSCs Improves Sickle Cell Disease Erythropoiesis in Mice

CRISPR/Cas9-mediated beta-globin (HBB) gene correction of Sickle Cell Disease (SCD) patient-derived hematopoietic stem cells (HSCs) in combination with autologous transplantation represents a novel paradigm in gene therapy. Although several Cas9-based HBB-correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated. Here, we used a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated HBB-correction in functional HSCs within the context of autologous transplantation. We discover that long-term multipotent HSCs can be gene corrected ex vivo and stable hemoglobin-A production can be achieved in vivo from HBB-corrected HSCs following autologous transplantation. We observed a direct correlation between increased HBB-corrected myeloid chimerism and normalized in vivo RBC features, but even low levels of chimerism resulted in robust hemoglobin-A levels. Moreover, this study offers a platform for gene editing of mouse HSCs for both basic and translational research.

developmental biology↗

bioRxiv2019

Genome editing to model and reverse a prevalent mutation associated with myeloproliferative neoplasms

Myeloproliferative neoplasms (MPNs) cause the over-production of blood cells such as erythrocytes (polycythemia vera) or platelets (essential thrombocytosis). JAK2 V617F is the most prevalent somatic mutation in many MPNs, but previous modeling of this mutation in mice relied on transgenic overexpression and resulted in diverse phenotypes that were in some cases attributed to expression level. CRISPR-Cas9 engineering offers new possibilities to model and potentially cure genetically encoded disorders via precise modification of the endogenous locus in primary cells. Here we develop "scarless" Cas9-based reagents to create and reverse the JAK2 V617F mutation in an immortalized human erythroid progenitor cell line (HUDEP-2), CD34+ adult human hematopoietic stem and progenitor cells (HSPCs), and immunophenotypic long-term hematopoietic stem cells (LT-HSCs). We find no overt in vitro increase in proliferation associated with an endogenous JAK2 V617F allele, but co-culture with wild type cells unmasks a competitive growth advantage provided by the mutation. Acquisition of the V617F allele also promotes terminal differentiation of erythroid progenitors, even in the absence of hematopoietic cytokine signaling. Taken together, these data are consistent with the gradually progressive manifestation of MPNs and reveals that endogenously acquired JAK2 V617F mutations may yield more subtle phenotypes as compared to transgenic overexpression models.

cancer biology↗