Lipid nanoparticle-targeted mRNA formulation as a treatment for ornithine-transcarbamylase deficiency model mice.

Ornithine transcarbamylase (OTC) plays a significant role in the urea cycle, a metabolic pathway functioning in the liver to detoxify ammonia. OTC deficiency (OTCD) is the most prevalent urea cycle disorder. Here, we show that intravenously delivered human OTC (hOTC) mRNA by lipid nanoparticles (LNP) was an effective treatment for OTCD by restoring the urea cycle. We observed a homotrimer conformation of hOTC proteins produced by the mRNA-LNP in cells by cryo-electron microscopy. The immunohistochemistry revealed the mitochondria localization of produced hOTC proteins in hepatocytes in mice. In livers of mice intravenously injected with hOTC-mRNA/LNP at 1.0 mg/kg, the delivered hOTC mRNA levels steeply decreased with a half-life (t1/2) of 7.1 h, whereas the produced hOTC protein levels retained for 5 days and then declined with a t1/2 of 2.2 days. In OTCD model mice (high-protein diet-fed Otcspf-ash hemizygous males), a single dose of hOTC-mRNA/LNP at 3.0 mg/kg ameliorated hyperammonemia and weight loss with prolonged survival rate (22 days) compared with that of untreated mice (11 days). Weekly repeated doses at 0.3 and 1.0 mg/kg were well tolerated in wild-type mice and showed a dose-dependent amelioration of survival rate in OTCD mice, thus, showing the therapeutic potential of LNP-formulated hOTC mRNA for OTCD.

In vitro and in vivo functions of T cells produced in complemented thymi of chimeric mice generated by blastocyst complementation.

Blastocyst complementation is an intriguing way of generating humanized animals for organ preparation in regenerative medicine and establishing novel models for drug development. Confirming that complemented organs and cells work normally in chimeric animals is critical to demonstrating the feasibility of blastocyst complementation. Here, we generated thymus-complemented chimeric mice, assessed the efficacy of anti-PD-L1 antibody in tumor-bearing chimeric mice, and then investigated T-cell function. Thymus-complemented chimeric mice were generated by injecting C57BL/6 (B6) embryonic stem cells into Foxn1nu/nu morulae or blastocysts. Flow cytometry data showed that the chimeric mouse thymic epithelial cells (TECs) were derived from the B6 cells. T cells appeared outside the thymi. Single-cell RNA-sequencing analysis revealed that the TEC gene-expression profile was comparable to that in B6 mice. Splenic T cells of chimeric mice responded very well to anti-CD3 stimulation in vitro; CD4+ and CD8+ T cells proliferated and produced IFNγ, IL-2, and granzyme B, as in B6 mice. Anti-PD-L1 antibody treatment inhibited MC38 tumor growth in chimeric mice. Moreover, in the chimeras, anti-PD-L1 antibody restored T-cell activation by significantly decreasing PD-1 expression on T cells and increasing IFNγ-producing T cells in the draining lymph nodes and tumors. T cells produced by complemented thymi thus functioned normally in vitro and in vivo. To successfully generate humanized animals by blastocyst complementation, both verification of the function and gene expression profiling of complemented organs/cells in interspecific chimeras will be important in the near future.

Selective degradation of splicing factor CAPERα by anticancer sulfonamides.

Target-protein degradation is an emerging field in drug discovery and development. In particular, the substrate-receptor proteins of the cullin-ubiquitin ligase system play a key role in selective protein degradation, which is an essential component of the anti-myeloma activity of immunomodulatory drugs (IMiDs), such as lenalidomide. Here, we demonstrate that a series of anticancer sulfonamides NSC 719239 (E7820), indisulam, and NSC 339004 (chloroquinoxaline sulfonamide, CQS) induce proteasomal degradation of the U2AF-related splicing factor coactivator of activating protein-1 and estrogen receptors (CAPERα) via CRL4DCAF15 mediated ubiquitination in human cancer cell lines. Both CRISPR-Cas9-based knockout of DCAF15 and a single amino acid substitution of CAPERα conferred resistance against sulfonamide-induced CAPERα degradation and cell-growth inhibition. Thus, these sulfonamides represent selective chemical probes for disrupting CAPERα function and designate DCAFs as promising drug targets for promoting selective protein degradation in cancer therapy.

Targeting of tumor growth and angiogenesis underlies the enhanced antitumor activity of lenvatinib in combination with everolimus.

The combination of lenvatinib, a multiple receptor tyrosine kinase inhibitor, plus everolimus, a mammalian target of rapamycin (mTOR) inhibitor, significantly improved clinical outcomes versus everolimus monotherapy in a phase II clinical study of metastatic renal cell carcinoma (RCC). We investigated potential mechanisms underlying the antitumor activity of the combination treatment in preclinical RCC models. Lenvatinib plus everolimus showed greater antitumor activity than either monotherapy in three human RCC xenograft mouse models (A-498, Caki-1, and Caki-2). In particular, the combination led to tumor regression in the A-498 and Caki-1 models. In the A-498 model, everolimus showed antiproliferative activity, whereas lenvatinib showed anti-angiogenic effects. The anti-angiogenic activity was potentiated by the lenvatinib plus everolimus combination in Caki-1 xenografts, in which fibroblast growth factor (FGF)-driven angiogenesis may contribute to tumor growth. The combination showed mostly additive activity in vascular endothelial growth factor (VEGF)-activated, and synergistic activity against FGF-activated endothelial cells, in cell proliferation and tube formation assays, as well as strongly suppressed mTOR-S6K-S6 signaling. Enhanced antitumor activities of the combination versus each monotherapy were also observed in mice bearing human pancreatic KP-1 xenografts overexpressing VEGF or FGF. Our results indicated that simultaneous targeting of tumor cell growth and angiogenesis by lenvatinib plus everolimus resulted in enhanced antitumor activity. The enhanced inhibition of both VEGF and FGF signaling pathways by the combination underlies its superior anti-angiogenic activity in human RCC xenograft models.

Leiodermatolide, a novel marine natural product, has potent cytotoxic and antimitotic activity against cancer cells, appears to affect microtubule dynamics, and exhibits antitumor activity.

Pancreatic cancer, the fourth leading cause of cancer death in the United States, has a negative prognosis because metastasis occurs before symptoms manifest. Leiodermatolide, a polyketide macrolide with antimitotic activity isolated from a deep water sponge of the genus Leiodermatium, exhibits potent and selective cytotoxicity toward the pancreatic cancer cell lines AsPC-1, PANC-1, BxPC-3, and MIA PaCa-2, and potent cytotoxicity against skin, breast and colon cancer cell lines. Induction of apoptosis by leiodermatolide was confirmed in the AsPC-1, BxPC-3 and MIA PaCa-2 cells. Leiodermatolide induces cell cycle arrest but has no effects on in vitro polymerization or depolymerization of tubulin alone, while it enhances polymerization of tubulin containing microtubule associated proteins (MAPs). Observations through confocal microscopy show that leiodermatolide, at low concentrations, causes minimal effects on polymerization or depolymerization of the microtubule network in interphase cells, but disruption of spindle formation in mitotic cells. At higher concentrations, depolymerization of the microtubule network is observed. Visualization of the growing microtubule in HeLa cells expressing GFP-tagged plus end binding protein EB-1 showed that leiodermatolide stopped the polymerization of tubulin. These results suggest that leiodermatolide may affect tubulin dynamics without directly interacting with tubulin and hint at a unique mechanism of action. In a mouse model of metastatic pancreatic cancer, leiodermatolide exhibited significant tumor reduction when compared to gemcitabine and controls. The antitumor activities of leiodermatolide, as well as the proven utility of antimitotic compounds against cancer, make leiodermatolide an interesting compound with potential chemotherapeutic effects that may merit further research.

Analysis of membrane topology and identification of essential residues for the yeast endoplasmic reticulum inositol acyltransferase Gwt1p.

Glycosylphosphatidylinositol (GPI) is a post-translational modification that anchors cell surface proteins to the plasma membrane, and GPI modifications occur in all eukaryotes. Biosynthesis of GPI starts on the cytoplasmic face of the endoplasmic reticulum (ER) membrane, and GPI precursors flip from the cytoplasmic side to the luminal side of the ER, where biosynthesis of GPI precursors is completed. Gwt1p and PIG-W are inositol acyltransferases that transfer fatty acyl chains to the inositol moiety of GPI precursors in yeast and mammalian cells, respectively. To ascertain whether flipping across the ER membrane occurs before or after inositol acylation of GPI precursors, we identified essential residues of PIG-W and Gwt1p and determined the membrane topology of Gwt1p. Guided by algorithm-based predictions of membrane topology, we experimentally identified 13 transmembrane domains in Gwt1p. We found that Gwt1p, PIG-W, and their orthologs shared four conserved regions and that these four regions in Gwt1p faced the luminal side of the ER membrane. Moreover, essential residues of Gwt1p and PIG-W faced the ER lumen or were near the luminal edge of transmembrane domains. The membrane topology of Gwt1p suggested that inositol acylation occurred on the luminal side of the ER membrane. Rather than stimulate flipping of the GPI precursor across the ER membrane, inositol acylation of GPI precursors may anchor the precursors to the luminal side of the ER membrane, preventing flip-flops.

MPR1 as a novel selection marker in Saccharomyces cerevisiae.

L-Azetidine-2-carboxylic acid (AZC) is a toxic four-membered ring analogue of L-proline that is transported into cells by proline transporters. AZC and L-proline in the cells are competitively incorporated into nascent proteins. When AZC is present in a minimum medium, misfolded proteins are synthesized in the cells, thereby inhibiting cell growth. The MPR1 gene has been isolated from the budding yeast Saccharomyces cerevisiae Sigma1278b as a multicopy suppressor of AZC-induced growth inhibition. MPR1 encodes a novel acetyltransferase that detoxifies AZC via N-acetylation. Since MPR1 is absent in the laboratory strain of S. cerevisiae S288C, it could be a positive selection marker that confers AZC resistance in the S288C background strains. To examine the usefulness of MPR1, we constructed some plasmid vectors that harboured MPR1 under the control of various promoters and introduced them into the S288C-derived strains. The expression of MPR1 conferred AZC resistance that was largely dependent on the expression level of MPR1. In an additional experiment, the galactose-inducible MPR1 and ppr1(+), the fission yeast Schizosaccharomyces pombe homologue of MPR1, were used for gene disruption by homologous recombination, and here AZC-resistant colonies were also successfully selected. We concluded that our MPR1-AZC system provides a powerful tool for yeast transformation.