Distinct cis-acting regions control six6 expression during eye field and optic cup stages of eye formation.

The eye field transcription factor, Six6, is essential for both the early (specification and proliferative growth) phase of eye formation, as well as for normal retinal progenitor cell differentiation. While genomic regions driving six6 optic cup expression have been described, the sequences controlling eye field and optic vesicle expression are unknown. Two evolutionary conserved regions 5' and a third 3' to the six6 coding region were identified, and together they faithfully replicate the endogenous X. laevis six6 expression pattern. Transgenic lines were generated and used to determine the onset and expression patterns controlled by the regulatory regions. The conserved 3' region was necessary and sufficient for eye field and optic vesicle expression. In contrast, the two conserved enhancer regions located 5' of the coding sequence were required together for normal optic cup and mature retinal expression. Gain-of-function experiments indicate endogenous six6 and GFP expression in F1 transgenic embryos are similarly regulated in response to candidate trans-acting factors. Importantly, CRISPR/CAS9-mediated deletion of the 3' eye field/optic vesicle enhancer in X. laevis, resulted in a reduction in optic vesicle size. These results identify the cis-acting regions, demonstrate the modular nature of the elements controlling early versus late retinal expression, and identify potential regulators of six6 expression during the early stages of eye formation.

A lipid-encapsulated mRNA encoding a potently neutralizing human monoclonal antibody protects against chikungunya infection.

Infection with chikungunya virus (CHIKV) causes an acute illness characterized by fever, rash, and arthralgia. However, CHIKV infection can sometimes progress to chronic arthritis or even lethal disease. CHIKV continues to cause substantial morbidity worldwide as its vector mosquitoes expand and spread. There are currently no approved vaccines or antiviral drugs available for the prevention or treatment of CHIKV. Although antibody therapy has shown promise in the prevention or treatment of CHIKV disease in preclinical models, challenges remain for implementing such therapies. Here, from the B cells of a survivor of natural CHIKV infection, we isolated ultrapotent neutralizing human monoclonal antibodies (mAbs) and encoded their sequences into mRNA molecules delivered by infusion. One human mAb, CHKV-24, was expressed to biologically significant levels in vivo after infusion of mRNAs in lipid nanoparticles in mice. We evaluated the protective capacity of CHKV-24 mAb immunoglobulin G protein or mRNA in mouse models of CHIKV infection. Treatment with CHKV-24 mRNA protected mice from arthritis, musculoskeletal tissue infection, and lethality and reduced viremia to undetectable levels at 2 days after inoculation. Infusion of macaques with CHKV-24 mRNA achieved a mean maximal mAb concentration of 10.1 to 35.9 micrograms per milliliter, with a half-life of 23 days, a level well above what is needed for protection in mice. Studies with CHKV-24 mRNA in macaques demonstrated a dose-response effect after the first dose of mRNA and maintained levels after second dose. These preclinical data with CHKV-24 mRNA suggest that it might be useful to prevent human disease.

ZFHX4 interacts with the NuRD core member CHD4 and regulates the glioblastoma tumor-initiating cell state.

Glioblastoma (GBM) harbors subpopulations of therapy-resistant tumor-initiating cells (TICs) that are self-renewing and multipotent. To understand the regulation of the TIC state, we performed an image-based screen for genes regulating GBM TIC maintenance and identified ZFHX4, a 397 kDa transcription factor. ZFHX4 is required to maintain TIC-associated and normal human neural precursor cell phenotypes in vitro, suggesting that ZFHX4 regulates differentiation, and its suppression increases glioma-free survival in intracranial xenografts. ZFHX4 interacts with CHD4, a core member of the nucleosome remodeling and deacetylase (NuRD) complex. ZFHX4 and CHD4 bind to overlapping sets of genomic loci and control similar gene expression programs. Using expression data derived from GBM patients, we found that ZFHX4 significantly affects CHD4-mediated gene expression perturbations, which defines ZFHX4 as a master regulator of CHD4. These observations define ZFHX4 as a regulatory factor that links the chromatin-remodeling NuRD complex and the GBM TIC state.

Coordinate activation of Shh and PI3K signaling in PTEN-deficient glioblastoma: new therapeutic opportunities.

In glioblastoma, phosphatidylinositol 3-kinase (PI3K) signaling is frequently activated by loss of the tumor suppressor phosphatase and tensin homolog (PTEN). However, it is not known whether inhibiting PI3K represents a selective and effective approach for treatment. We interrogated large databases and found that sonic hedgehog (SHH) signaling is activated in PTEN-deficient glioblastoma. We demonstrate that the SHH and PI3K pathways synergize to promote tumor growth and viability in human PTEN-deficient glioblastomas. A combination of PI3K and SHH signaling inhibitors not only suppressed the activation of both pathways but also abrogated S6 kinase (S6K) signaling. Accordingly, targeting both pathways simultaneously resulted in mitotic catastrophe and tumor apoptosis and markedly reduced the growth of PTEN-deficient glioblastomas in vitro and in vivo. The drugs tested here appear to be safe in humans; therefore, this combination may provide a new targeted treatment for glioblastoma.