Upregulation of LHPP by saRNA inhibited hepatocellular cancer cell proliferation and xenograft tumor growth.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer worldwide and no pharmacological treatment is available that can achieve complete remission of HCC. Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) is a recently identified HCC tumor suppressor gene which plays an important role in the development of HCC and its inactivation and reactivation has been shown to result in respectively HCC tumorigenesis and suppression. Small activating RNAs (saRNAs) have been used to achieve targeted activation of therapeutic genes for the restoration of their encoded protein through the RNAa mechanism. Here we designed and validated saRNAs that could activate LHPP expression at both the mRNA and protein levels in HCC cells. Activation of LHPP by its saRNAs led to the suppression of HCC proliferation, migration and the inhibition of Akt phosphorylation. When combined with targeted anticancer drugs (e.g., regorafenib), LHPP saRNA exhibited synergistic effect in inhibiting in vitro HCC proliferation and in vivo antitumor growth in a xenograft HCC model. Findings from this study provides further evidence for a tumor suppressor role of LHPP and potential therapeutic value of restoring the expression of LHPP by saRNA for the treatment of HCC.

Intrathecal administration of a novel siRNA modality extends survival and improves motor function in the SOD1G93A ALS mouse model.

Antisense oligonucleotides (ASOs) were the first modality to pioneer targeted gene knockdown in the treatment of amyotrophic lateral sclerosis (ALS) caused by mutant superoxide dismutase 1 (SOD1). RNA interference (RNAi) is another mechanism of gene silencing in which short interfering RNAs (siRNAs) effectively degrade complementary transcripts. However, delivery to extrahepatic tissues like the CNS has been a bottleneck in the clinical development of RNAi. Herein, we identify potent siRNA duplexes for the knockdown of human SOD1 in which medicinal chemistry and conjugation to an accessory oligonucleotide (ACO) enable activity in CNS tissues. Local delivery via intracerebroventricular or intrathecal injection into SOD1G93A mice delayed disease progression and extended animal survival with superior efficacy compared with an ASO resembling tofersen in sequence and chemistry. Treatment also prevented disease-related declines in motor function, including improvements in animal mobility, muscle strength, and coordination. The ACO itself does not target any specific complementary nucleic acid sequence; rather, it imparts benefits conducive to bioavailability and delivery through its chemistry. The complete conjugate (i.e., siRNA-ACO) represents a novel modality for delivery of duplex RNA (e.g., siRNA) to the CNS that is currently being tested in the clinic for treatment of ALS.

p21CIP/WAF1 saRNA inhibits proliferative vitreoretinopathy in a rabbit model.

PURPOSE: Proliferative vitreoretinopathy (PVR) is a disease process resulting from proliferation of retinal pigment epithelial (RPE) cells in the vitreous and periretinal area, leading to periretinal membrane formation and traction and eventually to postoperative failure after vitreo-retinal surgery for primary rhegmatogenous retinal detachment (RRD). The present study was designed to test the therapeutic potential of a p21CIP/WAF1 (p21) inducing saRNA for PVR. METHODS: A chemically modified p21 saRNA (RAG1-40-53) was tested in cultured human RPE cells for p21 induction and for the inhibition of cell proliferation, migration and cell cycle progression. RAG1-40-53 was further conjugated to a cholesterol moiety and tested for pharmacokinetics and pharmacodynamics in rabbit eyes and for therapeutic effects after intravitreal administration in a rabbit PVR model established by injecting human RPE cells. RESULTS: RAG1-40-53 (0.3 mg, 1 mg) significantly induced p21 expression in RPE cells and inhibited cell proliferation, the progression of cell cycle at the G0/G1 phase and TGF-ß1 induced migration. After a single intravitreal injection into rabbit eyes, cholesterol-conjugated RAG1-40-53 exhibited sustained concentration in the vitreal humor beyond at least 8 days and prevented the progression of established PVR. CONCLUSION: p21 saRNA could represent a novel therapeutics for PVR by exerting a antiproliferation and antimigration effect on RPE cells.

Small activating RNA activation of ATOH1 promotes regeneration of human inner ear hair cells.

The loss of inner ear hair cells leads to irreversible acoustic injury in mammals, and regeneration of inner ear hair cells to restore hearing loss is challenging. ATOH1 is a key gene in the development and regeneration of hair cells. Small activating RNAs (saRNAs) can target a gene to specifically upregulate its expression. This study aimed to explore whether small activating RNAs could induce the differentiation of human adipose-derived mesenchymal stem cells into hair cell-like cells with a combination of growth factors in vitro and thus provide a new strategy for hair cell regeneration and the treatment of sensorineural hearing loss. Fifteen small activating RNAs targeting the human ATOH1 gene were designed and screened in 293 T and human adipose-derived mesenchymal stem cells, and 3 of these candidates were found to be capable of effectively and stably activating ATOH1 gene expression. The selected small activating RNAs were then transfected into hair cell progenitor cells, and hair cell markers were examined 10 days after transfection. After transfection of the selected small activating RNAs, the expression of the characteristic markers of inner ear hair cells, POU class 4 homeobox 3 (POU4F3) and myosin VIIA (MYO7A), was detected. Human adipose-derived mesenchymal stem cells have the potential to differentiate into human hair cell progenitor cells. In vitro, small activating RNAs were able to induce the differentiation of hair cell progenitor cells into hair cell-like cells. Therefore, RNA activation technology has the potential to provide a new strategy for the regeneration of hair cells.

Reciprocal roles of SIRT1 and SKIP in the regulation of RAR activity: implication in the retinoic acid-induced neuronal differentiation of P19 cells.

Human sirtuin 1 (SIRT1) is a NAD(+)-dependent deacetylase that participates in cell death/survival, senescence and metabolism. Although its substrates are well characterized, no direct regulators have been defined. Here, we show that SIRT1 associates with SKI-interacting protein (SKIP) and modulates its activity as a coactivator of retinoic acid receptor (RAR). Binding assays indicated that SKIP interacts with RAR in a RA-dependent manner, through a region that overlaps the binding site for SIRT1. SKIP augmented the transcriptional activation activity of RAR by cooperating with SRC-1, and SIRT1 suppressed SKIP/SRC-1-enhanced RAR transactivation activity. The suppression was dependent on the deacetylase activity of SIRT1 and was enhanced by a SIRT1 activator, resveratrol. In contrast, the suppression was relieved by SIRT1 knockdown, overexpression of SKIP and treatment with a SIRT1 inhibitor, splitomicin. Upon SKIP overexpression, the recruitment of SIRT1 to the endogenous RARbeta2 promoter was severely impaired, and SKIP was recruited to the promoter instead. Finally, resveratrol treatment inhibited RA-induced neuronal differentiation of P19 cells, accompanied by reductions in the neuronal marker nestin and a RAR target gene, RARbeta2. This inhibition was relieved by either knockdown of SIRT1 or overexpression of SKIP. These data suggest that SIRT1 and SKIP play reciprocal roles in the regulation of RAR activity, which is implicated in the regulation of RA-induced neuronal differentiation of P19 cells.

A novel benzimidazole analogue inhibits the hypoxia-inducible factor (HIF)-1 pathway.

Hypoxia-inducible factor (HIF)-1 is a therapeutic target in solid tumors. We report the novel benzimidazole analogue AC1-004, obtained from a chemical library using an HRE-dependent cell-based assay in colorectal carcinoma HCT-116 cells. The accumulation of hypoxia-induced HIF-1alpha was inhibited by compound AC1-004 in various cancer cells, including HCT-116, MDA-MB435, SK-HEP1, and Caki-1. Further, AC1-004 down-regulated VEGF and EPO, target genes of HIF-1, and inhibited in vitro tube formation of HUVEC, suggesting its potential inhibitory activity on angiogenesis. Importantly, AC1-004 was found to regulate the stability of HIF-1alpha through the Hsp90-Akt pathway, leading to the degradation of HIF-1alpha. An in vivo antitumor study demonstrated that AC1-004 reduced tumor size significantly (i.e., by 58.6%), without severe side effects. These results suggest the benzimidazole analogue AC1-004 is a novel HIF inhibitor that targets HIF-1alpha via the Hsp90-Akt pathway, and that it can be used as a new lead in developing anticancer drugs.

Active regulator of SIRT1 cooperates with SIRT1 and facilitates suppression of p53 activity.

Human SIRT1 is an NAD+-dependent deacetylase protein that plays a role in cell death/survival, senescence, and endocrine signaling. While its substrates, including p53, have been well characterized, no direct regulators are known. We describe here a nuclear protein, active regulator of SIRT1 (AROS), which directly regulates SIRT1 function. AROS enhanced SIRT1-mediated deacetylation of p53 both in vitro and in vivo, and it inhibited p53-mediated transcriptional activity. AROS activity was abrogated by the SIRT1 inhibitors splitomicin and nicotinamide and by SIRT1 small interfering RNA (siRNA). In addition, AROS was unable to cooperate in p53 inactivation in an AROS-binding-defective SIRT1 mutant. Finally, knockdown of endogenous AROS using an antisense expression vector enhanced p21WAF1 expression and increased both the G0/G1 population and apoptosis in response to DNA damage, while AROS overexpression improved cell survival. To our knowledge, AROS is the first direct SIRT1 regulator to be identified that modulates p53-mediated growth regulation.

Structure-activity relationship studies of a series of novel delta-lactam-based histone deacetylase inhibitors.

We synthesized a series of delta-lactam-based HDAC inhibitors that were identified with various degrees of anti-inflammatory and cell growth inhibitory activities. Compounds possessing significant HDAC inhibitory activity exhibited both anti-inflammatory and cell growth inhibitory activities as well as significant tumor growth inhibition in the in vivo tumor xenograft experiments. Besides, these compounds demonstrated anti-inflammatory properties in vitro via suppression of the production of the proinflammatory cytokine TNF-alpha and nitric oxide by LPS-stimulated RAW264.7 cells.