Glioblastoma is the most common form of primary brain tumor in adults, characterized by rapid progression and poor prognosis—despite the standard of care treatment including maximal safe resection, radiotherapy, and chemotherapy. Cancer vaccination has emerged as a promising strategy to harness the patient's immune system against glioblastoma. Cancer vaccination strategies can broadly be divided into cell-based or tumor antigen only (TAO), depending on whether they incorporate the use of viable immune cells. Here, we reviewed data from clinical trials that tested TAO cancer vaccination strategies for glioblastoma treatment, including personalized vaccines. Clinical safety and efficacy profiles for each vaccination strategy are summarized. Insights gained from these clinical trials are reviewed to identify opportunities for future therapeutic advancement.

ONC201 is a first-in-class, blood-brain barrier penetrant imipridone that showed promise against H3K27M gliomas. Mechanistically, H3K27M mutation inhibits EZH2 in the methylating H3K27. In this context, we hypothesized that pharmacologic inhibition of EZH2 should recapitulate the physiologic effects of H3K27M mutation, with the corollary of synthetic lethal interactions between EZH2 inhibition and ONC201. Here, we investigated i) EZH2 expression level in H3K27 wild-type diffuse intrinsic pontine gliomas correlated with ONC201 sensitivity and ii) potential synergy between ONC201 and EZH2 inhibition. pre-clinical laboratory investigation. In a panel of H3K27 wild-type diffuse intrinsic pontine glioma cell lines, EZH1/EZH2 expression correlated with ONC201 sensitivity. RNA-seq showed that ONC201 and EHZ2 inhibitor tazemetostat-treated cells exhibited similar transcriptional profiles, sharing top-regulated genes. This finding suggests that ONC201 and EHZ2 inhibition converge on regular nodes within the same linear pathway to disrupt shared cellular functions. Supporting this hypothesis, ONC201 and EZH2i-treatment caused similar changes in the profile of cytokine release. In contrast, there was no overlap in the transcription or cytokine profiles obtained after ONC201 and Panobinostat (an HDAC inhibitor) treatment. Across all H3K27 wild-type diffuse intrinsic pontine glioma cell lines, the combination of ONC201 and the EZH2 inhibitor tazemetostat resulted in synergistic cytotoxic effects. Notably, the H3K27 methylation function of EZH2 was not affected by ONC201 treatment, indicating this function does not define the principal convergence point for the physiologic effects of ONC201 and EZH2 inhibition. ONC201 and EHZ2 inhibition converge on nodes within the same linear pathway and exhibit synthetic lethal interactions. These findings bear therapeutic implications and provide the foundation for drug combinations with ONC201.

Glioblastoma, the most common form of adult primary brain cancer, remains a lethal disease with therapeutic options limited by the blood-brain barrier (BBB). ONC201 (also known as TIC10) is a first-in-class, BBB penetrant imipridone that showed promise in phase I/II glioma trials as monotherapy. The drug targets key oncogenic pathways, including ERK/AKT, integrated stress response (ISR), TRAIL/DR5, and ClpP-dependent mitochondrial stress. Here, we investigated the efficacy of ONC201 in combination with temozolomide and radiation to explore the potential as a therapeutic strategy for newly diagnosed glioblastoma. pre-clinical laboratory investigation. ONC201 synergized with the cytotoxic effects of temozolomide (TMZ) and Radiotherapy (RT) in vitro. Specifically, ONC201 enhances TMZ- or RT-induced apoptosis and integrated stress response (ISR). Consistent with the specific interaction between ClpP and ONC201, the cytotoxic effect of ONC201 was abolished upon ClpP silencing. In contrast, silencing of ClpP did not affect the cytotoxic effects of RT or TMZ. These results suggest that the mechanisms underlying ONC201’s anti-tumoral activity are distinct from those associated with TMZ or RT, with potential for synergy between these agents. Supporting this hypothesis, cytokine profiling and RNAseq experiments demonstrate that ONC201 and TMZ induced distinct responses. Additionally, ONC201 reduced Methyl-guanine methyl transferase (MGMT) expression. Further supporting the synergy between ONC201 and TMZ/RT, ONC201+RT+TMZ therapy prolonged median survival to 123 days with tail on survival curve (3-of-7 mice alive beyond 200-days) in orthotopic U251 glioblastoma model versus ONC201 (44-days; p = 0.000197), RT (63-days; p = 0.0012), TMZ (78-days; p = 0.0354), ONC201+RT (55-days; p = 0.0004), ONC201+TMZ (80-days; p = 0.0041) and RT+TMZ (103-days; p > 0.05). By day 231, the only surviving mice were in the ONC201+RT+TMZ group. These results support the consideration of incorporating ONC201 into the current standard of care for newly diagnosed glioblastomas.

The androgen receptor (AR) signaling pathway plays a primary role in prostate cancer progression. Various types of second generation, non-steroidal anti-androgens (NSAA) including enzalutamide and apalutamide, have been widely used as single agents to treat patients with advanced disease. However, despite initial improvements, patients with metastatic castration-resistant prostate cancer (mCPRC) frequently develop resistance, resulting in limited overall survival benefit. Darolutamide is a novel next-generation androgen receptor-signaling inhibitor that is FDA approved for non-metastatic castration resistant prostate cancer (nmCRPC) currently in phase III clinical trials and has shown efficacy and tolerability in treating nmCRPC. ONC201/TIC10 is a first-in-class small molecule that activates the integrated stress response (ISR) and upregulates TNF-related apoptosis-inducing ligand (TRAIL). Our study investigates the ISR and AR signaling as mechanisms for antitumor efficacy with ONC201 and enzalutamide or darolutamide as single agents or in combination against mCRPC in vitro and in vivo. We previously reported that ONC201 synergizes with daroluatmide in inhibiting cell viability, upregulating ISR, inducing apoptosis, reducing prostate specific antigen (PSA) and AR signaling in CRPC cell lines 22RV1, LNCaP, DU145 and PC3. To test the novel combinatorial treatment in vivo, we established a subcutaneous CRPC mouse xenograft model with luciferase expressing 22RV1 cells, 22RV1-LUC. Tumors were routinely measured with a digital caliper and bioluminescence imaging. After tumor size reached 150mm3, mice were randomly assigned to control and treatment groups with ONC201 (50mg/kg or 100mg/kg, p.o., t.i.w or b.i.w.) and/or darolutamide (50mg/kg, p.o., b.i.d) and/or enzalutamide (20mg/kg, p.o., q.d.) for 36 days. Preliminary results demonstrated significant antitumor efficacy in the high-dose ONC201 group (100mg/kg, t.i.w.) and partial tumor regression in the combination groups. Flow cytometric analysis indicated preliminary trends of increased intratumor NK cells in mice treated with ONC201 and combination of ONC201 and darolutamide. Trends of increased TRAIL activation within NK cells were also observed in treatment groups. We are exploring the antitumor immune-modulatory effects of the combinatorial therapy that may have contributed to the partial reduction of tumor growth of 22RV1-LUC in an immunodeficient mouse model and biomarkers from the in vivo tissue samples. Our data provide insights to improved therapeutic benefits of combination treatment that can be further studied and developed for more efficient anticancer strategies. Citation Format: Jinxuan Laura Wu, Lanlan Zhou, Leiqing Zhang, Kelsey E. Huntington, Ryan Malpass, Attila Seyhan, Benedito Carneiro, Wafik S. El-Deiry. Synergistic combination therapy with ONC201/TIC10, Enzalutamide and Darolutamide in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1672.

Our Laboratory was established in 1994 at Univ. of Pennsylvania. Lab members demonstrated initial competencies by performing cell culture, western blots, immunofluorescence, and flow cytometry showing induction of p53/p21(WAF1) in cells treated with chemotherapy. Years later, our Laboratory of Translational Oncology & Experimental Cancer Therapeutics moved to Penn State Univ., Fox Chase Cancer Center/Temple Univ. and then Brown Univ. By 2020, with desire for inclusiveness (everyone succeeds), scientific rigor/reproducibility mandated by NIH, and as a training and mentoring activity (lab scientists/trainees/students mentoring others at High School level and beyond), we established a process for onboarding and training new cancer researchers. By Fall of 2022, there were 17 current Brown University undergraduate students (10 receiving research credit and 7 not receiving credit), HS students, 7 graduate students (PhD, masters, MD/PhD), and 6 medical students working with collaborating faculty at our laboratory at Brown’s Legorreta Cancer Center. After completion of biosafety training, and required trainings such as by IACUC, new lab members complete basic competencies in cell culture, cell viability, and western blot analysis that include technical, presentation quality output, and quantitative/statistical rigor to satisfy current standards for journal publication. For cell culture this includes pathogen free conditions, authentication, attention to details of routine procedures, documentation of morphology, freezing, thawing, passaging, seeding density, and managing cell populations to not run out of cells. Cell viability assessment includes attention to culture conditions, synergy analysis, data robustness, and presentation, and for western blots attention to quality of blots, protein quantification, loading, labeling, antibody specificity and sensitivity controls, presentation at 2022 standards, conventions for splicing, and issues with reproducibility including biological replicates, and generalizability. Additional and advanced competencies include RT-PCR, long-term colony assays, 3-D cultures (spheroids, organoids), transfection (overexpression, knockdown, CRISPR), co-culture and triculture with immune cells and fibroblasts, cytokine profiling, in vivo studies, in vivo imaging, immunohistochemistry, flow cytometric analysis, single cell techniques, viral infection, circulating tumor cell isolation, blood immune and cytokine analysis, and work with transgenic organoids and inducible cancer predisposing alleles. Modeling the tumor microenvironment, relevance to human cancer and translational directions are emphasized. Shared online lab resources, protocols, practices, videos, and manuscripts are available for lab members. The framework herein may be of interest to others involved in similar training programs. Citation Format: Wafik S. El-Deiry, Andrew George, Francesca Di Cristofano, Praveen Srinivasan, Lindsey Carlsen, Kelsey E. Huntington, Arielle De La Cruz, Leiqing Zhang, Marina Hahn, Shuai Zhao, Attila Seyhan, Bradley D. DeNardo, Aaron W. Maxwell, Dae Hee Kim, Alex Raufi, Hina Khan, Stephanie L. Graff, Don S. Dizon, Christopher Azzoli, Abbas E. Abbas, Roxanne Wood, Rishi R. Lulla, Howard P. Safran, Benedito A. Carneiro, Arunasalam Navaraj, Xiaobing Tian, Shengliang Zhang, Lanlan Zhou. Inclusive basic and advanced translational laboratory research competencies for research in cancer biology and therapeutics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4185.

Abstract Novel therapies are much needed in treating pediatric sarcomas, such as Ewing sarcoma. In the setting of metastatic Ewing sarcoma, survival expectancy ranges around 10%. A novel class of small molecules, imipridones, target G protein-coupled receptors and mitochondrial protease ClpP. Imipridones inactivate cell proliferation kinases Akt/ERK and induce cell death through the pro-apoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor DR5. In pre-clinical and clinical trials, imipridone ONC201 shows increased activity against tumors with mutations in histones, specifically histone H3 at the location of lysine 27 (H3K27). Changes in histone acetylation and the epigenetic effects of imipridones remain under investigation. Most commonly caused by a fusion of the EWS-FLI1 genes, Ewing sarcoma exhibits epigenetic modifications with the EWS-FLI1 protein acting as a transcription factor that causes covalent modifications of histone H3, including acetylation and activation of H3K27. Ewing sarcomas have also shown in vitro response to ERK pathway inhibition, leading to apoptosis and inhibition of metastasis formation. We hypothesized that Ewing sarcoma cell lines, with increased H3K27 acetylation due to EWS-FLI1 acting as an activating transcription factor, would respond to the combination of imipridones with histone deacetylase inhibitors. We performed combinatorial drug treatment on Ewing sarcoma cell lines SK-N-MC and RD-ES with 3 imipridones (ONC201, ONC206, and ONC212) and 3 HDAC inhibitors (vorinostat, entinostat, and panobinostat). Cell viability was measured after drug treatment in vitro. We investigated markers of cell death in established pediatric sarcoma cell lines via protein analysis and flow cytometry analysis. Using protein quantification studies and downstream target analysis on combination drug-treated cells, we investigated the mechanisms of how these two targeted therapies interact synergistically to cause cell death. We show that imipridones inactivate cellular proliferation through the Akt/ERK pathway and induce cell death through the TRAIL pathway, with ONC212 exhibiting the highest cell killing potency. Protein quantification by Western blotting revealed treatment with imipridone ONC201 affected the mitochondrial Clp protease complex by decreasing the chaperone subunit ClpX. Cell viability studies and analysis with Compusyn demonstrate potent synergistic effects causing tumor cell death when imipridones are combined with HDAC inhibitors. Our work presents a novel therapeutic combination for the treatment of Ewing sarcoma. Citation Format: Wen-i Chang, Lanlan Zhou, Attila Seyhan, Varun V. Prabhu, Wafik S. El-Deiry. Combinatorial therapy of imipridones and histone deacetylase inhibitors in Ewing sarcoma cell lines demonstrates synergistic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1060.

ONC201 is a promising anti-cancer agent that kills tumor cells by triggering an integrated stress response (ISR) dependent on ATF4. ONC201 demonstrated tumor regression and prolonged disease stability in patients with histone H3K27M-mutated midline glioma. The Enhancer of Zeste Homolog 2 (EZH2), a subunit of the polycomb repressive complex 2 (PRC2), is a histone methyltransferase that tri-methylates H3K27 (H3K27me3) and silences target genes. EZH2 inhibitors (EZH2i) reduce global H3K27 methylation. Based on the fact that the H3K27 mutation reduces H3K27 dimethylation (H3K27me2) and trimethylation (H3K27me3), we hypothesized that ONC201 sensitivity and tumor cell death may be enhanced by reducing H3K27 methylation with EZH2i as a mimic of H3K27M-mutation and by increasing H3K27 acetylation with histone deacetylase inhibitors (HDACi). We evaluated synergy of EZH2i EPZ-6438 or HDACi vorinostat with ONC201 against GBM cell lines, U251 and T98G-1 and DMG cell line, SF8638. Cell viability was determined with the Cell Titer Glo assay. Apoptosis was evaluated through immunoblotting of cleaved PARP and flow cytometry analysis of cell distribution. ISR activity was evaluated using immunoblotting of ATF4. Our result demonstrate that ONC201 synergistically reduced cell viability with vorinostat in U251, T98G-1 and SF8628 cell lines, induced apoptosis in combination with vorinostat in U251 and SF8628. ONC201 synergistically reduced cell viability and induced apoptosis with EPZ-6438 in U251. The immunoblotting detected no enhancement of ATF4 by addition of EPZ-6438 to ONC201. Immunoblotting analysis showed that EPZ-6438 reduced H3K27me3 in U251. Our results unravel potent synergy between ONC201 and EZH2i or HDACi in GBM and DMG cell lines, and provide further insights into the role of H3K27me3 in ONC201 drug sensitivity.

Abstract ONC201 is a promising anti-cancer agent that upregulates cytotoxic TRAIL pathway signaling in cancer cells. It has broad spectrum anti-tumor activity and achieved tumor regression and prolonged disease stabilization in clinical trials, especially in patients with H3K27M mutant gliomas. Based on the fact that the H3K27M mutation reduces the level of H3K27 dimethylation (H3K27me2) and trimethylation (H3K27me3), we hypothesized that cancer cells could be sensitized to ONC201 by epigenetic modulators that impact on histone methylation or acetylation. We tested the synergy between epigenetic modulators and ONC201 in tumor cell lines. We treated cancer cells from different tissue origins including breast cancer, pancreatic cancer, colorectal cancer, GBM, and DIPG with single agent ONC201, EZH2 inhibitor EPZ-6438 or the combination of ONC201 plus EPZ-6438. We also treated cancer cell lines originating from colorectal cancer, breast cancer, gastric cancer, prostate cancer, GBM and DIPG with combination of histone deacetylase inhibitor (HADCi), vorinostat, and ONC201. Cell viability was determined with the Cell Titer-Glo assay. Apoptosis was evaluated through flow cytometry analysis of cell distribution. Integrated stress response (ISR) activity widely observed in ONC201-treated tumor cells was evaluated using Western blot analysis of ATF4 and apoptosis by analysis of PARP cleavage. Cytotoxic TRAIL pathway signaling was evaluated using Western blot analysis of DR5. Our results demonstrate that ONC201 synergistically reduced cell viability, induced integrated stress response, cytotoxic TRAIL pathway signaling and apoptosis in combination with epigenetic modulators. There was greater induction of ATF4 and DR5 in combination therapy-treated tumor cells versus monotherapy. Immunoblotting analysis showed that combination of ONC201 and EZH2 inhibitor EPZ-6438 reduced H3K27me3 and EZH2. ONC201 is an antagonist of dopamine receptor D2 (DRD2). Dopamine receptor D5 (DRD5) is a dopamine receptor family member that opposes DRD2 signaling. The DRD2+DRD5- biomarker signature is associated with enhanced ONC201 tumor cell sensitivity. In order to investigate the role of dopamine receptors in anti-tumor effect of ONC201, we also tested the mRNA level of dopamine receptors by RT-PCR and observed that both vorinostat and EPZ-6438 upregulated DRD5 more than DRD2. Thus, we suspect other target genes may be involved in sensitization to ONC201 due to epigenetic modulation. We are currently investigating the interplay between H3K27 methylation versus acetylation in ONC201 sensitivity with or without additional therapeutic modulation by epigenetic drugs. Our results unravel potent synergy between ONC201 and EZH2 inhibitors or HDAC inhibitors and provide further insights into the role of H3K27me3 in ONC201 drug sensitivity. Citation Format: Yiqun Zhang, Lanlan Zhou, Michael Glantz, Howard Safran, Attila Seyhan, Wafik S. El-Deiry. Potent synergistic tumor cell suppression from combination of ONC201 and epigenetic modulators EZH2 or HDAC inhibitors provides a novel treatment strategy for solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4042.

Introduction: Obesity is a significant risk factor for the development of type 2 diabetes (T2D) and cardiovascular disease (CVD), but is also associated with an increased risk for multiple gastrointestinal cancers, including colon and pancreatic cancers. In addition to decreasing risk for T2D and CVD, bariatric surgery significantly reduces the risk of obesity associated cancers. MicroRNAs (miRs) are small noncoding RNAs implicated in a broad range of metabolic processes and regulatory pathways. Circulating miRs have been explored as novel biomarkers of disease risk, activity and response to treatment for a variety of indications including T2D, CVD and cancer. Methods: We profiled the effects of bariatric surgery on a panel of 94 circulating miRs implicated in T2D and CVD as part of the Physical Activity Following Surgery Induced Weight Loss study (clinicaltrials.gov identifier: NCT00692367). Here we describe a subanalysis exploring gender differences in levels of miRs responsive to bariatric surgery. Archived plasma samples were used from 22 subjects who were recruited after bariatric surgery and followed for 6 months, with (N=11) or without (N=11) exercise intervention. Mixed-effect models for repeated measures were performed in R. Results: Plasma levels of miR-221 decreased after bariatric surgery (p = 0.007). Women had significantly higher plasma levels of miR-221 than men both pre- and post- surgery (p = 0.047). miR-221 has been implicated as a pro-oncogenic regulator and has been explored as a target for modulation of cancer cell growth. In addition, miR-106b and miR-15a both increased after bariatric surgery (p = 0.009 and p = 0.007, respectively) with a marginally significant gender difference (p = 0.093 and p = 0.058, respectively), both with higher levels in men. Downregulation of miR-106b has been implicated in cellular senescence in diet-induced obese (DIO) mice, and is upregulated in DIO mice following a weight loss intervention. miR-15a is decreased in obesity in prior studies of obese men, and in patients with T2D. Conclusion: These three miRs change with bariatric surgery and demonstrate gender differences. Specifically, levels of each miR in women correspond to a higher risk profile both pre- and post- surgery. Our analysis suggests miR-221, miR-106b, and miR-15a may be useful biomarkers of the response to bariatric surgery. Gender differences in the levels of these three miRs may contribute to differential risk profiles for gastrointestinal cancers and T2D among men and women.

Background Heterogeneity of clinical manifestations and underlying biology has confounded treatment development and optimal care of systemic lupus erythematosus (SLE) patients. About half of adult SLE patients tend to have stable, high expression levels of genes in the interferon (IFN) signaling pathways. We and others have previously reported differences in expression levels between IFN groups of genes in pathways other than IFN signaling pathways1,2,3,4,5. Objectives To determine how gene expression changes with disease flare in SLE patients with high versus low expression of IFN pathway genes. Methods The BOLD study enrolls patients with active disease who are withdrawn from potentially confounding background immunosuppressive therapy and given brief intramuscular steroids (depomedrol) to induce improvement (Improving Visit), and followed until flare (Flare Visit). An interim analysis was conducted by TaqMan® RT-PCR on 23 patients who completed the study. Expression levels of 272 genes (selected based on reported associations with lupus and/or inflammation) were compared between Improving Visit and Flare Visit. Results Striking expression differences between Improving Visit and Flare Visit were observed for some genes, predominantly associated with the IFN High Group. Expression of most (76%) genes tested did not change with flare (p>0.1). IFN groups did not differ in demographic features. There was a trend towards higher disease activity (CLASI score, p=0.06, BILAG scores p=0.1) at baseline in the IFN high group. Conclusions This interim report suggests biomarkers in several pathways might be tested as sensitive indicators to guide patient selection and dosing in the development of treatments which affect the TLR/IFN pathway, and to optimize the eventual use of these agents in clinic. Given the heterogeneity of patients with SLE it could be that some of these downstream indicators could help differentiate between patients who are or are not intrinsically good candidates for targeting of IFN signaling pathways. References O9Toole et al., ACR 2011, Abstract 1407. Baechler et al., PNAS 2003. Bennett et al., J. Exp. Med 2003. Kirou et al., Arthritis and Rheumatism 2005. Petri et al, Lupus 2009. Disclosure of Interest A. Seyhan Employee of: Pfizer, M. O9Toole Employee of: Pfizer, Y. Zhang Employee of: Pfizer, F. Immermann Employee of: Pfizer, A. Hill Employee of: Pfizer, P. Reddy Employee of: Pfizer, J. Masferrer Employee of: Pfizer, T. Zhou Employee of: Pfizer, W. Mounts Employee of: Pfizer, M. Whitley Employee of: Pfizer, T. Walker Employee of: Pfizer, S. Kamp: None Declared, J. James: None Declared, S. Sridharan Employee of: Pfizer, J. Merrill: None Declared, M. Honczarenko Employee of: Pfizer

Dominant negative genetic disorders, in which a mutant allele of a gene causes disease in the presence of a second, normal copy, have been challenging since there is no cure and treatments are only to alleviate the symptoms. Current therapies involving pharmacological and biological drugs are not suitable to target mutant genes selectively due to structural indifference of the normal variant of their targets from the disease-causing mutant ones. In instances when the target contains single nucleotide polymorphism (SNP), whether it is an enzyme or structural or receptor protein are not ideal for treatment using conventional drugs due to their lack of selectivity. Therefore, there is a need to develop new approaches to accelerate targeting these previously inaccessible targets by classical therapeutics. Although there is a cooling trend by the pharmaceutical industry for the potential of RNA interference (RNAi), RNAi and other RNA targeting drugs (antisense, ribozyme, etc.) still hold their promise as the only drugs that provide an opportunity to target genes with SNP mutations found in dominant negative disorders, genes specific to pathogenic tumor cells, and genes that are critical for mediating the pathology of various other diseases. Because of its exquisite specificity and potency, RNAi has attracted a considerable interest as a new class of therapeutic for genetic diseases including amyotrophic lateral sclerosis, Huntington’s disease (HD), Alzheimer’s disease (AD), Parkinson’s disease (PD), spinocerebellar ataxia, dominant muscular dystrophies, and cancer. In this review, progress and challenges in developing RNAi therapeutics for genetic diseases will be discussed.

Vitamin D may play an important role in modifying the risk of type 2 diabetes. Supplementation with cholecalciferol has been shown to improve β cell function and to attenuate the rise in glycated hemoglobin in people at high risk of diabetes. We examined whether circulating microRNAs (miRNAs) reflect disease progression and/or respond to vitamin D supplementation. We measured plasma levels of select miRNAs implicated in diabetes in people with prediabetes treated either with placebo (n=21) or 2000 U of cholecalciferol daily (n=21) for 4 months in the Calcium and Vitamin D for Diabetes Mellitus trial and compared the baseline-adjusted changes after correcting for age, body mass index, race, time of study entry (season) and baseline disposition index. Circulating levels of miR-7 (sixfold reduction, P=.01), miR-152 (1.5-fold increase, P=.03), and miR-192 (1.7-fold reduction, P=.026) displayed significant treatment-by-time interactions between the placebo- and the vitamin-D-treated groups. Plasma levels of miR-7 were reduced in the vitamin D and increased in the placebo group. The change in miR-152 positively correlated with the change in levels of the circulating metabolite 25-hydroxyvitamin D (r=0.33, P=.046) and negatively correlated with the change in glycated hemoglobin (r=−0.37, P=.024). The change in miR-192 positively correlated with the change in fasting glucose (r=0.41, P<.011). In conclusion, reduction of circulating miR-7 and miR-192, accompanied by elevation of miR-152, reflects a beneficial metabolic response to vitamin D treatment in people with prediabetes. These miRNAs may be useful biomarkers in diabetes prevention trials and other studies of vitamin D.

Self-processing hairpin ribozymes have been synthesized from promoterless singlestranded DNA circles (73 nt) within mammalian cells. Following lipid-mediated transient transfection, DNA circles were efficiently internalized by mouse L cells (OST7–1) that stably express T7 RNA polymerase confining it to the cytoplasm. Cellular uptake of circular DNA templates and intracellular accumulation of ribozyme RNA transcripts from these DNA circles were progressive, both peaking at 24 h after transfection. Intracellular transcription generated RNA concatemers accumulating to a level of ~100 copies per cell. Transcription appears to be independent of specific promoter sequences but depends on T7 RNA polymerase. The data presented here may support the hypothesis that single stranded bubble regions within duplex DNA can serve as de novo initiation sites for RNA transcription not only in vitro but also in the cytoplasm of mammalian cells. These results may provide a model for the rolling circle transcription of small circular nucleic acids in mammalian cells.