It is anticipated that gamma-secretase inhibitors (gamma-Sec-I) that modulate Notch processing will alter differentiation in tissues whose architecture is governed by Notch signaling. To explore this hypothesis, Han Wistar rats were dosed for up to 5 days with 10-100 micromol/kg b.i.d. gamma-Sec-I from three chemical series that inhibit Notch processing in vitro at various potencies (Notch IC(50)). These included an arylsulfonamide (AS) (142 nM), a dibenzazepine (DBZ) (1.7 nM), and a benzodiazepine (BZ) (2.2 nM). The DBZ and BZ caused dose-dependent intestinal goblet cell metaplasia. In contrast, the AS produced no detectable in vivo toxicity, despite higher exposure to free drug. In a time course using BZ, small intestinal crypt cell and large intestinal glandular cell epithelial apoptosis was observed on days 1-5, followed by goblet cell metaplasia on days 2-5 and crypt epithelial and glandular epithelial regenerative hyperplasia on days 4-5. Gene expression profiling of duodenal samples from BZ-dosed animals revealed significant time-dependent deregulation of mRNAs for various panendocrine, hormonal, and transcription factor genes. Somatostatin, secretin, mucin, CCK, and gastrin mRNAs were elevated twofold or more by day 2, and a number of candidate "early-predictive" genes were altered on days 1-2, remaining changed for 4-5 days; these included Delta1, NeuroD, Hes1-regulated adipsin, and the Hes-regulated transcriptional activator of gut secretory lineage differentiation, the rat homolog of Drosophila atonal, Rath1. Western blotting of fecal protein from BZ-and DBZ-dosed animals exhibited increased levels of both anti-Rath1 reactive protein and anti-adipsin reactive proteins, confirming their potential value as noninvasive biomarkers of intestinal goblet metaplasia.

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Background Isolated methylmalonic acidemia/aciduria (MMA) is an ultra-rare, serious, inherited metabolic disorder with significant morbidity and mortality. Exogenously delivered mRNA encoding human methylmalonyl-CoA mutase (hMUT), the enzyme most frequently mutated in MMA, is a potential therapy to produce functional MUT enzyme in liver. Methods Two 12-week repeat-dose studies were conducted to evaluate the efficacy and safety of intravenously-administered hMUT mRNA encapsulated in lipid nanoparticles in two murine models of MMA. Findings In MMA hypomorphic mice, hMUT mRNA treatment resulted in dose-dependent and reproducible biomarker responses after each dose. Enzymatically-active MUT protein was produced in liver in a dose-dependent manner. hMUT mRNA was well-tolerated with no adverse effects, as indicated by the lack of clinical observations, minimal changes in clinical chemistry parameters, and histopathology examination across all tissues. In severe MMA mice, hMUT mRNA led to substantially improved survival and growth and ameliorated biochemical abnormalities, all of which are cardinal clinical manifestations in severely affected patients. Interpretation These data demonstrate durable functional benefit of hMUT mRNA and support development of this new class of therapy for a devastating, pediatric disorder. Fund This work was funded by Moderna, Inc.

Stable, efficient gene transfer to normal human peripheral viruses, and unselected cultured cells were assayed 24 to blood mononuclear cells (PBMC) is a prerequisite for ther-48 h later for expression of transduced genes by immunoapy of a number of diseases, both hereditary and acquired, chemistry and Northern blot analysis. Expression of both affecting these cells. Current approaches to gene transfer luciferase and HBSAg was detected using both to PBMC entail ex vivo mitogenic stimulation and multiple approaches. Levels of expression of luciferase were transduction steps followed by selection, usually of pro-slightly higher in PBMC which were stimulated with congenitor populations. Thus, the ability to transfer gene canavalin A (con A). Conversely, expression of HBSAg expression to normal, resting PBMC could complement was less in con A-stimulated PBMC, compared with gene transfer strategies that target dividing precursor cells. unstimulated cells. Con A stimulation did not alter infectivity We report successful short-term transduction of human of PBMC by SV40-derivative virus. While longevity and PBMC using two different SV40-derived viral vectors. stability of expression in vitro are as yet unknown, this SV40-derivative viruses were constructed by cloning demonstration of successful gene transfer to resting, nor-cDNAs for firefly luciferase (luc), or hepatitis B surface anti-mal human PBMC, assayed on unselected cells, suggests gen (HBSAg), into shuttle plasmids, to create the SV40 that SV40-based transduction systems may be potential derivative viruses SVluc and SV(HBS) respectively. Both candidates for use in transient gene transfer to monogenes were cloned downstream from SV40 early promoter. nuclear blood cells. Normal, resting, human PBMC were exposed to these

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