Enhancing SIRT1 Gene Expression Using Small Activating RNAs: A Novel Approach for Reversing Metabolic Syndrome.

Metabolic syndrome (MetS) is a pathological condition characterized by abdominal obesity, insulin resistance, hypertension, and hyperlipidemia. Sirtuin 1 (SIRT1), a highly conserved histone deacetylase, is characterized as a key metabolic regulator and protector against aging-associated pathologies, including MetS. In this study, we investigate the therapeutic potential of activating SIRT1 using small activating RNAs (saRNA), thereby reducing inflammatory-like responses and re-establishing normal lipid metabolism. SIRT1 saRNA significantly increased SIRT1 messenger RNA (mRNA) and protein levels in both lipopolysaccharide-stimulated and nonstimulated macrophages. SIRT1 saRNA significantly decreased inflammatory-like responses, by reducing mRNA levels of key inflammatory cytokines, such as Tumor Necrosis Factor alpha, Interleukin 1 beta (IL-1ß), Interleukin 6 (IL-6), and chemokines Monocyte Chemoattractant Protein-1 and keratinocyte chemoattractant. SIRT1 overexpression also significantly reduced phosphorylation of nuclear factor-κB and c-Jun N-terminal kinase, both key signaling molecules for the inflammatory pathway. To investigate the therapeutic effect of SIRT1 upregulation, we treated a high-fat diet model with SIRT1 saRNA conjugated to a transferrin receptor aptamer for delivery to the liver and cellular internalization. Animals in the SIRT1 saRNA treatment arm demonstrated significantly decreased weight gain with a significant reduction in white adipose tissue, triglycerides, fasting glucose levels, and intracellular lipid accumulation. These suggest treatment-induced changes to lipid and glucose metabolism in the animals. The results of this study demonstrate that targeted activation of SIRT1 by saRNAs is a potential strategy to reverse MetS.

Chitosan Oleate Coated PLGA Nanoparticles as siRNA Drug Delivery System.

Oligonucleotide therapeutics such as miRNAs and siRNAs represent a class of molecules developed to modulate gene expression by interfering with ribonucleic acids (RNAs) and protein synthesis. These molecules are characterized by strong instability and easy degradation due to nuclease enzymes. To avoid these drawbacks and ensure efficient delivery to target cells, viral and non-viral vectors are the two main approaches currently employed. Viral vectors are one of the major vehicles in gene therapy; however, the potent immunogenicity and the insertional mutagenesis is a potential issue for the patient. Non-viral vectors, such as polymeric nanocarriers, provide a safer and more efficient delivery of RNA-interfering molecules. The aim of this work is to employ PLGA core nanoparticles shell-coated with chitosan oleate as siRNA carriers. An siRNA targeted on HIV-1, directed against the viral Tat/Rev transcripts was employed as a model. The ionic interaction between the oligonucleotide's moieties, negatively charged, and the positive surface charges of the chitosan shell was exploited to associate siRNA and nanoparticles. Non-covalent bonds can protect siRNA from nuclease degradation and guarantee a good cell internalization and a fast release of the siRNA into the cytosolic portion, allowing its easy activation.

MTL-CEBPA Combined with Immunotherapy or RFA Enhances Immunological Anti-Tumor Response in Preclinical Models.

The transcription factor CEBPA is a master regulator of liver homeostasis, myeloid cell differentiation and is downregulated in several oncogenic diseases. MTL-CEBPA is a small activating RNA drug which upregulates gene expression of CEBPA for treatment of hepatocellular carcinoma (HCC). We investigate whether MTL-CEBPA has immune modulatory effects by combining MTL-CEBPA with an anti-PD-1 checkpoint inhibitor (CPI) and/or radiofrequency ablation (RFA) in two preclinical models. First, mice with two flanks of HCC tumors (BNL) were treated with combinations of RFA (right flank), anti-PD-1 or MTL-CEBPA. The reduction of the left flank tumors was most pronounced in the group treated with RFA+anti-PD1+MTL-CEBPA and 7/8 animals responded. This was the only group with a significant increase in CD8+ and CD49b+/CD45+ tumor infiltrating lymphocytes (TIL). Second, a combination of anti-PD-1+MTL-CEBPA was tested in a CT26 colon cancer model and this treatment significantly reduced tumor size, modulated the tumor immune microenvironment and increased TILs. These data suggest a clinical role for combination treatment with CPIs, RFA and MTL-CEBPA through synergistic priming of the immune tumor response, enabling RFA and CPIs to have a pronounced anti-tumor effect including activity in non-treated tumors in the case of RFA.

Upregulation of C/EBPα Inhibits Suppressive Activity of Myeloid Cells and Potentiates Antitumor Response in Mice and Patients with Cancer.

PURPOSE: To evaluate the mechanisms of how therapeutic upregulation of the transcription factor, CCAAT/enhancer-binding protein alpha (C/EBPα), prevents tumor progression in patients with advanced hepatocellular carcinoma (HCC) and in different mouse tumor models. EXPERIMENTAL DESIGN: We conducted a phase I trial in 36 patients with HCC (NCT02716012) who received sorafenib as part of their standard care, and were given therapeutic C/EBPα small activating RNA (saRNA; MTL-CEBPA) as either neoadjuvant or adjuvant treatment. In the preclinical setting, the effects of MTL-CEBPA were assessed in several mouse models, including BNL-1ME liver cancer, Lewis lung carcinoma (LLC), and colon adenocarcinoma (MC38). RESULTS: MTL-CEBPA treatment caused radiologic regression of tumors in 26.7% of HCC patients with an underlying viral etiology with 3 complete responders. MTL-CEBPA treatment in those patients caused a marked decrease in peripheral blood monocytic myeloid-derived suppressor cell (M-MDSC) numbers and an overall reduction in the numbers of protumoral M2 tumor-associated macrophages (TAM). Gene and protein analysis of patient leukocytes following treatment showed CEBPA activation affected regulation of factors involved in immune-suppressive activity. To corroborate this observation, treatment of all the mouse tumor models with MTL-CEBPA led to a reversal in the suppressive activity of M-MDSCs and TAMs, but not polymorphonuclear MDSCs (PMN-MDSC). The antitumor effects of MTL-CEBPA in these tumor models showed dependency on T cells. This was accentuated when MTL-CEBPA was combined with checkpoint inhibitors or with PMN-MDSC-targeted immunotherapy. CONCLUSIONS: This report demonstrates that therapeutic upregulation of the transcription factor C/EBPα causes inactivation of immune-suppressive myeloid cells with potent antitumor responses across different tumor models and in cancer patients. MTL-CEBPA is currently being investigated in combination with pembrolizumab in a phase I/Ib multicenter clinical study (NCT04105335).

CRED9: A differentially expressed elncRNA regulates expression of transcription factor CEBPA.

Enhancer RNAs (eRNA) are non-coding transcripts produced from active enhancers and have potential gene regulatory function. CCAAT enhancer-binding protein alpha (CEBPA) is a transcription factor generally involved in metabolism, cell cycle inhibition, hematopoiesis, adipogenesis, hepatogenesis, and is associated with tumorigenesis. In this study, we demonstrate that an enhancer-associated long non-coding RNA (elncRNA), transcribed from an enhancer located 9kb downstream from the transcriptional start site (TSS) of CEBPA, positively regulates the expression of CEBPA. As a result, we named this elncRNA 'CEBPA regulatory elncRNA downstream 9kb' or 'CRED9'. CRED9 expression level positively correlates with CEBPA mRNA expression across multiple cell lines as detected by RT droplet digital PCR. Knockdown of CRED9 resulted in a reduction of CEBPA mRNA expression in Hep3B cells. Additionally, CRED9 knockdown in Hep3B and HepG2 cells resulted in lower CEBPA protein expression. We also found that knockdown of CRED9 in Hep3B cells caused a 57.8% reduction in H3K27ac levels at the +9kb CEBPA enhancer. H3K27ac has previously been described as a marker of active enhancers. Taken together, the evidence presented here supports a previously proposed model whereby, in some contexts, eRNA transcripts are necessary to amplify and maintain H3K27ac levels at a given enhancer. Ultimately, this study adds to the growing body of evidence that elncRNA transcripts have important roles in enhancer function and gene regulation.

A Multifunctional LNA Oligonucleotide-Based Strategy Blocks AR Expression and Transactivation Activity in PCa Cells.

The androgen receptor (AR) plays a critical role in the development of prostate cancer (PCa) through the activation of androgen-induced cellular proliferation genes. Thus, blocking AR-mediated transcriptional activation is expected to inhibit the growth and spread of PCa. Using tailor-made splice-switching locked nucleic acid (LNA) oligonucleotides (SSOs), we successfully redirected splicing of the AR precursor (pre-)mRNA and destabilized the transcripts via the introduction of premature stop codons. Furthermore, the SSOs simultaneously favored production of the AR45 mRNA in lieu of the full-length AR. AR45 is an AR isoform that can attenuate the activity of both full-length and oncogenic forms of AR by binding to their common N-terminal domain (NTD), thereby blocking their transactivation potential. A large screen was subsequently used to identify individual SSOs that could best perform this dual function. The selected SSOs powerfully silence AR expression and modulate the expression of AR-responsive cellular genes. This bi-functional strategy that uses a single therapeutic molecule can be the basis for novel PCa treatments. It might also be customized to other types of therapies that require the silencing of one gene and the simultaneous expression of a different isoform.

MTL-CEBPA, a Small Activating RNA Therapeutic Upregulating C/EBP-α, in Patients with Advanced Liver Cancer: A First-in-Human, Multicenter, Open-Label, Phase I Trial.

PURPOSE: Transcription factor C/EBP-α (CCAAT/enhancer-binding protein alpha) acts as a master regulator of hepatic and myeloid functions and multiple oncogenic processes. MTL-CEBPA is a first-in-class small activating RNA oligonucleotide drug that upregulates C/EBP-α. PATIENTS AND METHODS: We conducted a phase I, open-label, dose-escalation trial of MTL-CEBPA in adults with advanced hepatocellular carcinoma (HCC) with cirrhosis, or resulting from nonalcoholic steatohepatitis or with liver metastases. Patients received intravenous MTL-CEBPA once a week for 3 weeks followed by a rest period of 1 week per treatment cycle in the dose-escalation phase (3+3 design). RESULTS: Thirty-eight participants have been treated across six dose levels (28-160 mg/m2) and three dosing schedules. Thirty-four patients were evaluable for safety endpoints at 28 days. MTL-CEBPA treatment-related adverse events were not associated with dose, and no maximum dose was reached across the three schedules evaluated. Grade 3 treatment-related adverse events occurred in nine (24%) patients. In 24 patients with HCC evaluable for efficacy, an objective tumor response was achieved in one patient [4%; partial response (PR) for over 2 years] and stable disease (SD) in 12 (50%). After discontinuation of MTL-CEBPA, seven patients were treated with tyrosine kinase inhibitors (TKIs); three patients had a complete response with one further PR and two with SD. CONCLUSIONS: MTL-CEBPA is the first saRNA in clinical trials and demonstrates an acceptable safety profile and potential synergistic efficacy with TKIs in HCC. These encouraging phase I data validate targeting of C/EBP-α and have prompted MTL-CEBPA + sorafenib combination studies in HCC.

Uncovering Differently Expressed Markers and Heterogeneity on Human Pancreatic Cancer.

Discovery of biomarkers is critical to understand tumor heterogeneity and microenvironment. To determine differently expressed makers on cancer tissue for comprehensive profiling, the multiplexed tissue imaging mass cytometer (IMC) which uniquely combines time-of-flight mass spectrometry with metal-labeling technology to enable breakthrough discovery on single cell level was employed to investigate the expression of seven markers related to the epithelial-to-mesenchymal transition [α-smooth muscle actin (α-SMA), vimentin, collagen I, cytokeratin 7, pan-keratin], tumor proliferation (Ki-67), and human leucocyte antigen (HLA-DR) on human pancreatic cancer tissue. The difference was analyzed using bioinformatic tools. We observed the high expression of α-SMA, vimentin, collagen I, and Ki-67 on grade I but not on grade III. HLA-DR was highly expressed on grade I/III but not on grade II. Overall, the expression of markers has elucidated the heterogeneity intratumors. Additionally, to identify biomarkers on pancreatic cancer cells by blind systematic evolution of ligands by exponential enrichment (SELEX), aptamer pull-down assay and liquid chromatography-tandem mass spectrometry were used. Mortalin was identified as a potential a prognostic marker of pancreatic cancer. Our studies demonstrate that the IMC and blind SELEX might be implemented to discover biomarkers which can be used to better understand tumor biology and biomedical research applications.

Targeted Delivery of C/EBPα-saRNA by RNA Aptamers Shows Anti-tumor Effects in a Mouse Model of Advanced PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies; it preferentially metastasizes to the liver and is the main cause of death from this disease. In previous studies, small activating RNA against CCAAT/enhancer-binding protein-α (C/EBPα-saRNA) demonstrated efficacy of PDAC in a local subcutaneous tumor model. In this study, we focused on the efficacy of C/EBPα-saRNA in advanced stage PDAC. For targeted delivery, we selected a new anti-transferrin receptor aptamer (TR14), which demonstrated a high binding affinity to target proteins. The TR14 aptamer was internalized with clathrin-mediated endocytosis, distributed in early endosome, late endosome, and lysosome subcellularly. To investigate its anti-tumor effects to advanced PDAC, we conjugated C/EBPα-saRNA to TR14. Treatment of pancreatic cancer cells with the conjugates upregulated expression of C/EBPα and its downstream target p21, and inhibited cell proliferation. For in vivo assays, we established an advanced PDAC mouse model by engrafting luciferase reporter-PANC-1 cells directly into the livers of non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. After treatment of aptamer-C/EBPα conjugates, we observed significant reduction of tumor growth in this advanced PDAC mouse model. Combinational treatment of the conjugates with gemcitabine also demonstrated enhanced anti-tumor effects in advanced PDAC. This suggests that aptamer-C/EBPα conjugates could be used as an adjuvant, along with other conventional anti-cancer drugs in advanced PDAC. In conclusion, targeted delivery of C/EBPα-saRNAs by aptamers might have potential therapeutic effects in advanced PDAC.

Triplex Hybridization of siRNA with Bifacial Glycopolymer Nucleic Acid Enables Hepatocyte-Targeted Silencing.

Herein, we describe a versatile non-covalent strategy for packaging nucleic acid cargo with targeting modalities, based on triplex hybridization of oligo-uridylate RNA with bifacial polymer nucleic acid (bPoNA). Polyacrylate bPoNA was prepared and side chain-functionalized with N-acetylgalactosamine (GalNAc), which is known to enable delivery to hepatocytes and liver via binding to the asialoglycoprotein receptor (ASGPR). Polymer binding resulted in successful delivery of both native and synthetically modified siRNAs to HepG2 cells in culture, yielding in low nanomolar IC50 silencing of the endogenous ApoB target, in line with observations of expected Dicer processing of the polymer-siRNA targeting complex. Indeed, in vitro Dicer treatment of the polymer complex indicated that triplex hybridization does not impede RNA processing and release from the polymer. The complex itself elicited a quiescent immunostimulation profile relative to free RNA in a cytokine screen, setting the stage for a preliminary in vivo study in a high-calorie-diet mouse model. Gratifyingly, we observed significant ApoB silencing in a preliminary animal study, validating bPoNA as an in vivo carrier platform for systemic siRNA delivery. Thus, this new siRNA carrier platform exhibits generally useful function and is accessible through scalable synthesis. In addition to its utility as a carrier, the triplex-hybridizing synthetic platform could be useful for optimization screens of siRNA sequences using the identical polymer carriers, thus alleviating the need for covalent ligand modification of each RNA substrate.

Anti-inflammatory Activity of MTL-CEBPA, a Small Activating RNA Drug, in LPS-Stimulated Monocytes and Humanized Mice.

Excessive or inappropriate inflammatory responses can cause serious and even fatal diseases. The CCAAT/enhancer-binding protein alpha (CEBPA) gene encodes C/EBPα, a transcription factor that plays a fundamental role in controlling maturation of the myeloid lineage and is also expressed during the late phase of inflammatory responses when signs of inflammation are decreasing. MTL-CEBPA, a small activating RNA targeting for upregulation of C/EBPα, is currently being evaluated in a phase 1b trial for treatment of hepatocellular carcinoma. After dosing, subjects had reduced levels of pro-inflammatory cytokines, and we therefore hypothesized that MTL-CEBPA has anti-inflammatory potential. The current study was conducted to determine the effects of C/EBPα saRNA - CEBPA-51 - on inflammation in vitro and in vivo after endotoxin challenge. CEBPA-51 led to increased expression of the C/EBPα gene and inhibition of pro-inflammatory cytokines in THP-1 monocytes previously stimulated by E. coli-derived lipopolysaccharide (LPS). Treatment with MTL-CEBPA in an LPS-challenged humanized mouse model upregulated C/EBPα mRNA, increased neutrophils, and attenuated production of several key pro-inflammatory cytokines, including TNF-α, IL-6, IL-1ß, and IFN-γ. In addition, a Luminex analysis of mouse serum revealed that MTL-CEBPA reduced pro-inflammatory cytokines and increased the anti-inflammatory cytokine IL-10. Collectively, the data support further investigation of MTL-CEBPA in acute and chronic inflammatory diseases where this mechanism has pathogenic importance.

Humanized NOD/SCID/IL2rγnull (hu-NSG) Mouse Model for HIV Replication and Latency Studies.

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in high demand. With a close genetic and behavioral resemblance to humans, small animals such as the mouse are good candidates for human disease models, through which human-like symptoms and responses can be recapitulated. Further, the mouse genetic background can be altered to accommodate diverse demands. The NOD/SCID/IL2rγnull (NSG) mouse is one of the most widely used immunocompromised mouse strains; it allows engraftment with human hematopoietic stem cells and/or human tissues and the subsequent development of a functional human immune system. This is a critical milestone in understanding the prognosis and pathophysiology of human-specific diseases such as HIV/AIDS and aiding the search for a cure. Herein, we report a detailed protocol for generating a humanized NSG mouse model (hu-NSG) by hematopoietic stem cell transplantation into a radiation-conditioned neonatal NSG mouse. The hu-NSG mouse model shows multi-lineage development of transplanted human stem cells and susceptibility to HIV-1 viral infection. It also recapitulates key biological characteristics in response to combinatorial antiretroviral therapy (cART).

An RNA Aptamer Targeting the Receptor Tyrosine Kinase PDGFRα Induces Anti-tumor Effects through STAT3 and p53 in Glioblastoma.

Human glioblastoma (GBM) is the most aggressive malignancy of the CNS, with less than 5% survival. Despite great efforts to find effective therapeutics, current options remain very limited. To develop a targeted cancer therapeutic, we selected RNA aptamers against platelet-derived growth factor receptor α (PDGFRα), which is a receptor tyrosine kinase. One RNA aptamer (PDR3) with high affinity (0.25 nM) showed PDGFRα specificity and was internalized in U251-MG cells. Following treatment with the PDR3 aptamer, expression of the transcription factor STAT3 (signal transducer and activator of transcription 3) was inhibited, whereas the expression of the histone demethylase JMJD3 and the tumor suppressor p53 were upregulated. PDR3 also upregulated serine phosphorylation of p53, which subsequently mediated apoptosis through the death receptors: tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptors 1/2 (TRAIL-R1/R2), Fas-associated via death domain (FADD), and Fas. PDR3 significantly decreased cell viability in a dose-dependent manner. Furthermore, translocation of PDR3 into the nucleus induced hypomethylation at the promoters of cyclin D2. To assess the feasibility of targeted delivery, we conjugated PDR3 aptamer with STAT3-siRNA for a chimera. The PDR3-siSTAT3 chimera successfully inhibited the expression of target genes and showed significant inhibition of cell viability. In summary, our results show that well-tailored RNA aptamers targeting the PDGFRα-STAT3 axis have the potential to act as anti-cancer therapeutics in GBM.

Targeted Molecular Imaging Using Aptamers in Cancer.

Imaging is not only seeing, but also believing. For targeted imaging modalities, nucleic acid aptamers have features such as superior recognition of structural epitopes and quick uptake in target cells. This explains the emergence of an evolved new class of aptamers into a wide spectrum of imaging applications over the last decade. Genetically encoded biosensors tagged with fluorescent RNA aptamers have been developed as intracellular imaging tools to understand cellular signaling and physiology in live cells. Cancer-specific aptamers labeled with fluorescence have been used for assessment of clinical tissue specimens. Aptamers conjugated with gold nanoparticles have been employed to develop innovative mass spectrometry tissue imaging. Also, use of chemically conjugated cancer-specific aptamers as probes for non-invasive and high-resolution imaging has been transformative for in vivo imaging in multiple cancers.

Multi-center phase II trial of bortezomib and rituximab maintenance combination therapy in patients with mantle cell lymphoma after consolidative autologous stem cell transplantation.

BACKGROUND: Mantle cell lymphoma (MCL) is an aggressive and incurable lymphoma. Standard of care for younger patients with MCL is induction chemotherapy followed by autologous stem cell transplantation (auto-HCT). Rituximab maintenance after auto-HCT has been shown to improve progression-free survival (PFS) and overall survival (OS) in MCL. Bortezomib maintenance therapy has also been shown to be tolerable and feasible in this setting. However, the combination of bortezomib and rituximab as maintenance therapy post-auto-HCT has not been studied. METHODS: We conducted a multicenter, phase II trial of bortezomib given in combination with rituximab as maintenance in MCL patients after consolidative auto-HCT. Enrolled patients (n = 23) received bortezomib 1.3 mg/m2 subcutaneously weekly for 4 weeks every 3 months (up to 24 months) and rituximab 375 mg/m2 intravenously weekly for 4 weeks every 6 months (up to 24 months) for a total duration of 2 years. The primary study endpoint was disease-free survival (DFS). RESULTS: With a median follow-up of 35.9 months, the 2-year DFS probability was 90.2% (95% CI 66-97), and 2-year OS was 94.7% (95% CI 68-99). The most frequent grade 3/4 toxic events were neutropenia (in 74% of patients) and lymphopenia (in 35%). The incidence of peripheral neuropathy was 48% for grade 1, 9% for grade 2, and 0% for grade 3/4. We also examined the role of quantitative cyclin D1 (CCND1) mRNA in monitoring minimal residual disease. CONCLUSION: Combined bortezomib and rituximab as maintenance therapy in MCL patients following auto-HCT is an active and well-tolerated regimen. TRIAL REGISTRATION: ClinicalTrials.gov NCT01267812 , registered Dec 29, 2010.

Development of MTL-CEBPA: Small Activating RNA Drug for Hepatocellular Carcinoma.

BACKGROUND: Oligonucleotide drug development has revolutionised the drug discovery field. Within this field, 'small' or 'short' activating RNAs (saRNA) are a more recently discovered category of short double-stranded RNA with clinical potential. saRNAs promote transcription from target loci, a phenomenon widely observed in mammals known as RNA activation (RNAa). OBJECTIVE: The ability to target a particular gene is dependent on the sequence of the saRNA. Hence, the potential clinical application of saRNAs is to increase target gene expression in a sequence-specific manner. saRNA-based therapeutics present opportunities for expanding the "druggable genome" with particular areas of interest including transcription factor activation and cases of haploinsufficiency. RESULTS AND CONCLUSION: In this mini-review, we describe the pre-clinical development of the first saRNA drug to enter the clinic. This saRNA, referred to as MTL-CEBPA, induces increased expression of the transcription factor CCAAT/enhancer-binding protein alpha (CEBPα), a tumour suppressor and critical regulator of hepatocyte function. MTL-CEBPA is presently in Phase I clinical trials for hepatocellular carcinoma (HCC). The clinical development of MTL-CEBPA will demonstrate "proof of concept" that saRNAs can provide the basis for drugs which enhance target gene expression and consequently improve treatment outcome in patients.

Therapeutic Potential of Small Activating RNAs (saRNAs) in Human Cancers.

BACKGROUND: RNA is increasingly recognized as a powerful molecule that can be used to control gene expression. Sophisticated, well-engineered RNA-based regulators are being developed as oligotherapeutics. METHODS: In particular, small activating RNAs (saRNAs) are promising therapeutic options for targeting human diseases. Numerous saRNAs targeting multiple cancers have been developed in preclinical models. One saRNA targeting C/EBPα is currently undergoing clinical trials in liver cancer. RESULTS AND CONCLUSION: In this review, we describe the current working model of the intracellular mechanism of saRNA, discuss the recent progress of saRNA therapeutics in preclinical and clinical trials, and current advances in targeted delivery using aptamers in detail.

Receptor-targeted aptamer-siRNA conjugate-directed transcriptional regulation of HIV-1.

Gene-based therapies represent a promising therapeutic paradigm for the treatment of HIV-1, as they have the potential to maintain sustained viral inhibition with reduced treatment interventions. Such an option may represent a long-term treatment alternative to highly active antiretroviral therapy. Methods: We previously described a therapeutic approach, referred to as transcriptional gene silencing (TGS), whereby small noncoding RNAs directly inhibit the transcriptional activity of HIV-1 by targeting sites within the viral promoter, specifically the 5' long terminal repeat (LTR). TGS differs from traditional RNA interference (RNAi) in that it is characterized by concomitant silent-state epigenetic marks on histones and DNA. To deliver TGS-inducing RNAs, we developed functional RNA conjugates based on the previously reported dual function of the gp120 (A-1) aptamer conjugated to 27-mer Dicer-substrate anti-HIV-1 siRNA (dsiRNA), LTR-362. Results: We demonstrate here that high levels of processed guide RNAs localize to the nucleus in infected T lymphoblastoid CEM cell line and primary human CD4+ T-cells. Treatment of the aptamer-siRNA conjugates induced TGS with an ~10-fold suppression of viral p24 levels as measured at day 12 post infection. To explore the silencing efficacy of aptamer-siRNA conjugates in vivo, HIV-1-infected humanized NOD/SCID/IL2 rγnull mice (hu-NSG) were treated with the aptamer-siRNA conjugates. Systemic delivery of the A-1-stick-LTR-362 27-mer siRNA conjugates suppressed HIV-1 infection and protected CD4+ T cell levels in viremia hu-NSG mice. Principle conclusions: Collectively these data suggest that the gp120 aptamer-dsiRNA conjugate design is suitable for systemic delivery of small RNAs that can be used to suppress HIV-1.

Gene activation of CEBPA using saRNA: preclinical studies of the first in human saRNA drug candidate for liver cancer.

Liver diseases are a growing epidemic worldwide. If unresolved, liver fibrosis develops and can lead to cirrhosis and clinical decompensation. Around 5% of cirrhotic liver diseased patients develop hepatocellular carcinoma (HCC), which in its advanced stages has limited therapeutic options and negative survival outcomes. CEPBA is a master regulator of hepatic function where its expression is known to be suppressed in many forms of liver disease including HCC. Injection of MTL-CEBPA, a small activating RNA oligonucleotide therapy (CEBPA-51) formulated in liposomal nanoparticles (NOV340- SMARTICLES) upregulates hepatic CEBPA expression. Here we show how MTL-CEBPA therapy promotes disease reversal in rodent models of cirrhosis, fibrosis, hepatosteatosis, and significantly reduces tumor burden in cirrhotic HCC. Restoration of liver function markers were observed in a carbon-tetrachloride-induced rat model of fibrosis following 2 weeks of MTL-CEBPA therapy. At 14 weeks, animals showed reduction in ascites and enhanced survival rates. MTL-CEBPA reversed changes associated with hepatosteatosis in non-alcoholic methionine and cholic-deficient diet-induced steaotic liver disease. In diethylnitrosamine induced cirrhotic HCC rats, MTL-CEBPA treatment led to a significant reduction in tumor burden. The data included here and the rapid adoption of MTL-CEBPA into a Phase 1 study may lead to new therapeutic oligonucleotides for undruggable diseases.

HIV Replication and Latency in a Humanized NSG Mouse Model during Suppressive Oral Combinational Antiretroviral Therapy.

Although current combinatorial antiretroviral therapy (cART) is therapeutically effective in the majority of HIV patients, interruption of therapy can cause a rapid rebound in viremia, demonstrating the existence of a stable reservoir of latently infected cells. HIV latency is therefore considered a primary barrier to HIV eradication. Identifying, quantifying, and purging the HIV reservoir is crucial to effectively curing patients and relieving them from the lifelong requirement for therapy. Latently infected transformed cell models have been used to investigate HIV latency; however, these models cannot accurately represent the quiescent cellular environment of primary latently infected cells in vivo For this reason, in vivo humanized murine models have been developed for screening antiviral agents, identifying latently infected T cells, and establishing treatment approaches for HIV research. Such models include humanized bone marrow/liver/thymus mice and SCID-hu-thy/liv mice, which are repopulated with human immune cells and implanted human tissues through laborious surgical manipulation. However, no one has utilized the human hematopoietic stem cell-engrafted NOD/SCID/IL2rγnull (NSG) model (hu-NSG) for this purpose. Therefore, in the present study, we used the HIV-infected hu-NSG mouse to recapitulate the key aspects of HIV infection and pathogenesis in vivo Moreover, we evaluated the ability of HIV-infected human cells isolated from HIV-infected hu-NSG mice on suppressive cART to act as a latent HIV reservoir. Our results demonstrate that the hu-NSG model is an effective surgery-free in vivo system in which to efficiently evaluate HIV replication, antiretroviral therapy, latency and persistence, and eradication interventions.IMPORTANCE HIV can establish a stably integrated, nonproductive state of infection at the level of individual cells, known as HIV latency, which is considered a primary barrier to curing HIV. A complete understanding of the establishment and role of HIV latency in vivo would greatly enhance attempts to develop novel HIV purging strategies. An ideal animal model for this purpose should be easy to work with, should have a shortened disease course so that efficacy testing can be completed in a reasonable time, and should have immune correlates that are easily translatable to humans. We therefore describe a novel application of the hematopoietic stem cell-transplanted humanized NSG model for dynamically testing antiretroviral treatment, supporting HIV infection, establishing HIV latency in vivo The hu-NSG model could be a facile alternative to humanized bone marrow/liver/thymus or SCID-hu-thy/liv mice in which laborious surgical manipulation and time-consuming human cell reconstitution is required.