Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce Immunogenicity without HPLC Purification.

The Cas9/guide RNA (Cas9/gRNA) system is commonly used for genome editing. mRNA expressing Cas9 can induce innate immune responses, reducing Cas9 expression. First-generation Cas9 mRNAs were modified with pseudouridine and 5-methylcytosine to reduce innate immune responses. We combined four approaches to produce more active, less immunogenic second-generation Cas9 mRNAs. First, we developed a novel co-transcriptional capping method yielding natural Cap 1. Second, we screened modified nucleotides in Cas9 mRNA to identify novel modifications that increase Cas9 activity. Third, we depleted the mRNA of uridines to improve mRNA activity. Lastly, we tested high-performance liquid chromatography (HPLC) purification to remove double-stranded RNAs. The activity of these mRNAs was tested in cell lines and primary human CD34+ cells. Cytokines were measured in whole blood and mice. These approaches yielded more active and less immunogenic mRNA. Uridine depletion (UD) most impacted insertion or deletion (indel) activity. Specifically, 5-methoxyuridine UD induced indel frequencies as high as 88% (average ± SD = 79% ± 11%) and elicited minimal immune responses without needing HPLC purification. Our work suggests that uridine-depleted Cas9 mRNA modified with 5-methoxyuridine (without HPLC purification) or pseudouridine may be optimal for the broad use of Cas9 both in vitro and in vivo.

Downregulation of TLX induces TET3 expression and inhibits glioblastoma stem cell self-renewal and tumorigenesis.

Glioblastomas have been proposed to be maintained by highly tumorigenic glioblastoma stem cells (GSCs) that are resistant to current therapy. Therefore, targeting GSCs is critical for developing effective therapies for glioblastoma. In this study, we identify the regulatory cascade of the nuclear receptor TLX and the DNA hydroxylase Ten eleven translocation 3 (TET3) as a target for human GSCs. We show that knockdown of TLX expression inhibits human GSC tumorigenicity in mice. Treatment of human GSC-grafted mice with viral vector-delivered TLX shRNA or nanovector-delivered TLX siRNA inhibits tumour development and prolongs survival. Moreover, we identify TET3 as a potent tumour suppressor downstream of TLX to regulate the growth and self-renewal in GSCs. This study identifies the TLX-TET3 axis as a potential therapeutic target for glioblastoma.

T-cadherin (Cdh13) in association with pancreatic ß-cell granules contributes to second phase insulin secretion.

Glucose homeostasis depends on adequate control of insulin secretion. We report the association of the cell-adhesion and adiponectin (APN)-binding glycoprotein T-cadherin (Cdh13) with insulin granules in mouse and human ß-cells. Immunohistochemistry and electron microscopy of islets in situ and targeting of RFP-tagged T-cadherin to GFP-labeled insulin granules in isolated ß-cells demonstrate this unusual location. Analyses of T-cadherin-deficient (Tcad-KO) mice show normal islet architecture and insulin content. However, T-cadherin is required for sufficient insulin release in vitro and in vivo. Primary islets from Tcad-KO mice were defective in glucose-induced but not KCl-mediated insulin secretion. In vivo, second phase insulin release in T-cad-KO mice during a hyperglycemic clamp was impaired while acute first phase release was unaffected. Tcad-KO mice showed progressive glucose intolerance by 5 mo of age without concomitant changes in peripheral insulin sensitivity. Our analyses detected no association of APN with T-cadherin on ß-cell granules although colocalization was observed on the pancreatic vasculature. These data identify T-cadherin as a novel component of insulin granules and suggest that T-cadherin contributes to the regulation of insulin secretion independently of direct interactions with APN.