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.

Non-invasive characterization of human bone marrow stimulation and reconstitution by cell-free messenger RNA sequencing.

Circulating cell-free mRNA (cf-mRNA) holds great promise as a non-invasive diagnostic biomarker. However, cf-mRNA composition and its potential clinical applications remain largely unexplored. Here we show, using Next Generation Sequencing-based profiling, that cf-mRNA is enriched in transcripts derived from the bone marrow compared to circulating cells. Further, longitudinal studies involving bone marrow ablation followed by hematopoietic stem cell transplantation in multiple myeloma and acute myeloid leukemia patients indicate that cf-mRNA levels reflect the transcriptional activity of bone marrow-resident hematopoietic lineages during bone marrow reconstitution. Mechanistically, stimulation of specific bone marrow cell populations in vivo using growth factor pharmacotherapy show that cf-mRNA reflects dynamic functional changes over time associated with cellular activity. Our results shed light on the biology of the circulating transcriptome and highlight the potential utility of cf-mRNA to non-invasively monitor bone marrow involved pathologies.

Resource utilization and toxicities after single versus tandem autologous stem cell rescue in high-risk neuroblastoma using a national administrative database.

BACKGROUND: High-dose chemotherapy with autologous stem cell rescue (HDC-ASCR) has improved event-free survival for children with high-risk neuroblastoma. Common regimens include carboplatin/etoposide/melphalan (CEM), busulfan/melphalan (BuMel), and tandem HDC-ASCR [thiotepa/cyclophosphamide (TC) followed by CEM]. To complement clinical trial data and to evaluate the clinical burden associated with these regimens, resource ultilization (RU) was evaluated. An administrative database was used to evaluate RU in a previously developed high-risk neuroblastoma cohort. Single CEM and BuMel patients were followed for 60 days from the first day of the HDC-ASCR preparative regimen or until death, whichever came first. Tandem patients were followed from the first day of the first HDC-ASCR preparative regimen through day 60 from the first day of the second HDC-ASCR. Resources compared included inpatient days, ICU-level care, and medications administered. RESULTS: A cohort of 578 patients was evaluated; 422 patients underwent single HDC-ASCR with CEM, 67 received BuMel, 72 underwent TC/CEM, and 17 received only the first portion of tandem HDC-ASCR. The median number of inpatient days and days of exposure to antibiotics, opioids, and total parenteral nutrition were higher in the tandem group than in the CEM and BuMel groups. However, the rate of use of several ICU-level resources per 1000 hospital days was lower for the tandem group. CONCLUSIONS: These data suggest that while patients undergoing tandem HDC-ASCR were hospitalized longer, the severity of illness during hospitalization was not greater in tandem patients.

Structure-based Design of Pyridone-Aminal eFT508 Targeting Dysregulated Translation by Selective Mitogen-activated Protein Kinase Interacting Kinases 1 and 2 (MNK1/2) Inhibition.

Dysregulated translation of mRNA plays a major role in tumorigenesis. Mitogen-activated protein kinase interacting kinases (MNK)1/2 are key regulators of mRNA translation integrating signals from oncogenic and immune signaling pathways through phosphorylation of eIF4E and other mRNA binding proteins. Modulation of these key effector proteins regulates mRNA, which controls tumor/stromal cell signaling. Compound 23 (eFT508), an exquisitely selective, potent dual MNK1/2 inhibitor, was designed to assess the potential for control of oncogene signaling at the level of mRNA translation. The crystal structure-guided design leverages stereoelectronic interactions unique to MNK culminating in a novel pyridone-aminal structure described for the first time in the kinase literature. Compound 23 has potent in vivo antitumor activity in models of diffuse large cell B-cell lymphoma and solid tumors, suggesting that controlling dysregulated translation has real therapeutic potential. Compound 23 is currently being evaluated in Phase 2 clinical trials in solid tumors and lymphoma. Compound 23 is the first highly selective dual MNK inhibitor targeting dysregulated translation being assessed clinically.

Endogenous miRNAa: miRNA-Mediated Gene Upregulation.

The phenomenon of RNA activation (RNAa) was initially discovered by Li and colleagues about a decade ago. Subsequently, gene activation by exogenously expressed small activating RNA has been demonstrated in different cellular contexts by a number of laboratories. Conceivably, endogenously expressed microRNAs may also utilize RNA activation as a cellular mechanism for gene regulation, which may be dysregulated in disease states such as cancer. RNA activation can be applied to gain-of-function studies and holds great promise for disease intervention. This chapter will discuss examples of promoter-targeting microRNAs discovered in recent years and their pathophysiological relevance. I will also briefly touch upon other novel classes of microRNAs with positive gene regulatory roles, including TATA-box-activating microRNAs and enhancer-associated microRNAs.

Resource Utilization and Toxicities After Carboplatin/Etoposide/Melphalan and Busulfan/Melphalan for Autologous Stem Cell Rescue in High-Risk Neuroblastoma Using a National Administrative Database.

BACKGROUND: High-dose chemotherapy with autologous stem cell rescue (ASCR) is a key component of high-risk neuroblastoma therapy. Resources required to support patients treated with ASCR conditioning regimens [carboplatin/etoposide/melphalan (CEM) and busulfan/melphalan (BuMel)] have not been directly compared. PROCEDURE: An administrative database was used to analyze resource utilization and outcomes in a cohort of high-risk neuroblastoma patients. Patients were followed for 60 days from start of conditioning or until death. Length of hospitalization, length of intensive care unit (ICU) level of care, incidence of sepsis and sinusoidal obstruction syndrome (SOS), and duration of use of specific supportive care resources were analyzed. RESULTS: Six of 171 CEM patients and zero of 59 BuMel patients died during the study period (P = 0.34). Duration of hospitalization was longer following BuMel (median 35 vs. 31 days; P = 0.01); however, there was no difference in duration of ICU-level care. Antibiotic use was longer following CEM (median 19 vs. 15 days; P = 0.01), as was antihypertensive use (median 5 vs. 1.6 days; P = 0.0024). Duration of opiate and nonnarcotic analgesic use was longer following CEM early in the study period. Resources consistent with a diagnosis of SOS were used in a higher proportion of BuMel patients. A higher proportion of BuMel treated patients required mechanical ventilation (17% vs. 6%; P = 0.03). CONCLUSIONS: We used administrative billing data to compare resources associated with ASCR conditioning regimens. CEM patients required more extended use of analgesics, antibiotics, and antihypertensives, while duration of hospitalization was longer, and SOS and the use of mechanical ventilation were more frequent following BuMel.

Inducing gene expression by targeting promoter sequences using small activating RNAs.

Vector-based systems comprised of exogenous nucleic acid sequences remain the standard for ectopic expression of a particular gene. Such systems offer robust overexpression, but have inherent drawbacks such as the tedious process of construction, excluding sequences (e.g. introns and untranslated regions) important for gene function and potential insertional mutagenesis of host genome associated with the use of viral vectors. We and others have recently reported that short double-stranded RNAs (dsRNAs) can induce endogenous gene expression by targeting promoter sequences in a phenomenon referred to as RNA activation (RNAa) and such dsRNAs are termed small activating RNAs (saRNAs). To date, RNAa has been successfully utilized to induce the expression of different genes such as tumor suppressor genes. Here, we describe a detailed protocol for target selection and dsRNA design with associated experiments to facilitate RNAa in cultured cells. This technique may be applied to selectively activate endogenous gene expression for studying gene function, interrogating molecular pathways and reprogramming cell fate.

Identification of small activating RNAs that enhance endogenous OCT4 expression in human mesenchymal stem cells.

Ectopic overexpression of transcription factors has been used to reprogram cell fate. For example, virus-mediated overexpression of four transcription factors OCT4, SOX2, MYC, and KLF4, known as Yamanaka factors, can convert somatic cells to induced pluripotent stem (iPS) cells. However, gene-specific switch-on of endogenous gene production without the use of foreign DNA remains a challenge. The small RNA machinery that comprised small RNAs and Argonaute proteins is known to silence gene expression, but can be repurposed to activate gene expression when directed to gene promoters, a phenomenon known as RNA activation or RNAa. By screening of dsRNAs targeting OCT4 promoter, we identified a small activating RNA (saRNA) that activated OCT4 expression in several types of human mesenchymal stem cells (MSCs). We found that saRNA-induced OCT4 activation can be further enhanced by a histone deacetylase inhibitor, valproic acid. Furthermore, introducing OCT4 saRNA in combination with viruses encoding the remaining three Yamanaka factors (SOX2, MYC, and KLF4) into MSCs led to the derivation of partially reprogrammed iPS cells. Findings from this study suggest that, with further optimization, RNAa can be a powerful tool to reprogram cell fate by inducing the expression of endogenous genes.

Demystifying the nuclear function of Argonaute proteins.

The Argonaute family of proteins is highly evolutionarily conserved and plays essential roles in small RNA-mediated gene regulatory pathways and in a wide variety of cellular processes. They were initially discovered by genetics studies in plants and have been well characterized as key components of gene silencing pathways guided by small RNAs, a phenomenon known as RNA interference. Conventionally, guided by different classes of small RNAs, Argonautes bind to and silence homologous target sequences at the post-transcriptional level. Increasing lines of evidence support their multi-functional roles in the nucleus. Advances in high-throughput genome-wide methodologies have greatly facilitated our understanding of their functions in post-transcriptional gene silencing as well as in other nuclear events. In this point-of-view, we will summarize key findings from genome-wide analyses of the Ago subfamily of proteins in mammals and Drosophila, discuss their nuclear functions in the regulation of transcription and alternative splicing identified in recent years, and briefly touch upon their potential implications in cancer.

RNAa in action: from the exception to the norm.

Small RNA programmed Argonautes are sophisticated cellular effector platforms known to be involved in a diverse array of functions ranging from mRNA cleavage, translational inhibition, DNA elimination, epigenetic silencing, alternative splicing and even gene activation. First observed in human cells, small RNA-induced gene activation, also known as RNAa, involves the targeted recruitment of Argonaute proteins to specific promoter sequences followed by induction of stable epigenetic changes which promote transcription. The existence of RNAa remains contentious due to its elusive mechanism. A string of recent studies in C. elegans provides unequivocal evidence for RNAa's fundamental role in sculpting the epigenetic landscape and maintaining active transcription of endogenous genes and supports the presence of a functionally sophisticated network of small RNA-Argonaute pathways consisting of opposite yet complementary "yin and yang" regulatory elements. In this review, we summarize key findings from recent studies of endogenous RNAa in C. elegans, with an emphasis on the Argonaute protein CSR-1.

Ago1 Interacts with RNA polymerase II and binds to the promoters of actively transcribed genes in human cancer cells.

Argonaute proteins are often credited for their cytoplasmic activities in which they function as central mediators of the RNAi platform and microRNA (miRNA)-mediated processes. They also facilitate heterochromatin formation and establishment of repressive epigenetic marks in the nucleus of fission yeast and plants. However, the nuclear functions of Ago proteins in mammalian cells remain elusive. In the present study, we combine ChIP-seq (chromatin immunoprecipitation coupled with massively parallel sequencing) with biochemical assays to show that nuclear Ago1 directly interacts with RNA Polymerase II and is widely associated with chromosomal loci throughout the genome with preferential enrichment in promoters of transcriptionally active genes. Additional analyses show that nuclear Ago1 regulates the expression of Ago1-bound genes that are implicated in oncogenic pathways including cell cycle progression, growth, and survival. Our findings reveal the first landscape of human Ago1-chromosomal interactions, which may play a role in the oncogenic transcriptional program of cancer cells.

Targeted induction of endogenous NKX3-1 by small activating RNA inhibits prostate tumor growth.

BACKGROUND: RNA activation (RNAa) is a small RNA-mediated gene regulation mechanism by which expression of a particular gene can be induced by targeting its promoter using small double-stranded RNA also known as small activating RNA (saRNA). We used saRNA as a molecular tool to examine NKX3-1's role as a tumor suppressor and tested in vitro and in vivo antitumor effects of NKX3-1 induction by saRNA. MATERIALS AND METHODS: NKX3-1 saRNA was transfected into human prostate cancer cells including LNCaP, CWR22R, PC-3, CWR22RV1, DuPro, LAPC4, and DU145. The transfected cells were used for analysis of gene expression by RT-PCR and immunoblotting, proliferation, apoptosis and cell cycle distribution. PC-3 xenograft models were established in immunocompromised mice and treated with NKX3-1 saRNA. RESULTS: NKX3-1 saRNA induced NKX3-1 expression in different prostate cancer cell lines, resulting in inhibited cell proliferation and survival, cell cycle arrest and apoptotic cell death. These effects were partly mediated by NKX3-1's regulation of several downstream genes including the upregulation of p21 and p27, and the inhibition of VEGFC expression. Treatment of mouse xenograft prostate tumors with intratumoral delivery of NKX3-1 saRNA formulated in lipid nanoparticles significantly inhibited tumor growth and prolonged animal survival. CONCLUSIONS: By revealing several important target genes of NKX3-1, our findings corroborated NKX3-1's role as a tumor suppressor gene through direct regulation of the cell cycle and growth/survival pathways. This study also validated the therapeutic potential of saRNA for the treatment of prostate cancer via targeted activation of tumor suppressor genes.

miRNA goes nuclear.

microRNAs (miRNAs), defined as 21-24 nucleotide non-coding RNAs, are important regulators of gene expression. Initially, the functions of miRNAs were recognized as post-transcriptional regulators on mRNAs that result in mRNA degradation and/or translational repression. It is becoming evident that miRNAs are not only restricted to function in the cytoplasm, they can also regulate gene expression in other cellular compartments by a spectrum of targeting mechanisms via coding regions, 5' and 3'untransalated regions (UTRs), promoters, and gene termini. In this point-of-view, we will specifically focus on the nuclear functions of miRNAs and discuss examples of miRNA-directed transcriptional gene regulation identified in recent years.

Induction of NANOG expression by targeting promoter sequence with small activating RNA antagonizes retinoic acid-induced differentiation.

RNAa (RNA activation) is a mechanism by which small dsRNA (double-stranded RNA), termed saRNA (small activating RNA), target promoter sequences to induce gene expression. This technique represents a novel approach to gene overexpression without the use of exogenous DNA. In the present study, we investigated whether RNAa can modulate expression of the development-related gene NANOG and manipulate cell fate. Using a lentivirus-based reporter system as a screening tool, we identified synthetic saRNAs that stimulate NANOG expression in human NCCIT embryonic carcinoma cells. Mismatch mutations to saRNA duplexes define sequence requirement for gene activation. Functional analysis of NANOG induction reveals saRNA treatment predictably modulates the expression of several known downstream target genes, including FOXH1 (forkhead box H1), REST (RE1-silencing transcription factor), OCT4 (octamer-binding protein 4) and REX1 (reduced expression protein 1). Treatment with RA (retinoic acid) triggers NCCIT cell differentiation, reducing NANOG and OCT4 expression and up-regulating several neural markers [i.e. ASCL1 (achaete-scute complex homologue 1), NEUROD1 (neuronal differentiation 1) and PAX6 (paired box 6)]. However, co-treatment with saRNA antagonizes NANOG down-regulation and RA-induced differentiation. Ectopic overexpression of NANOG via lentiviral transduction further recapitulates saRNA results, providing proof-of-concept that RNAa may be utilized to activate development-related genes and manipulate cell fate.

Formulation of Small Activating RNA Into Lipidoid Nanoparticles Inhibits Xenograft Prostate Tumor Growth by Inducing p21 Expression.

Application of RNA interference (RNAi) in the clinic has improved with the development of novel delivery reagents (e.g., lipidoids). Although RNAi promises a therapeutic approach at silencing gene expression, practical methods for enhancing gene production still remain a challenge. Previously, we reported that double-stranded RNA (dsRNA) can activate gene expression by targeting promoter sequence in a phenomenon termed RNA activation (RNAa). In the present study, we investigate the therapeutic potential of RNAa in prostate cancer xenografts by using lipidoid-based formulation to facilitate in vivo delivery. We identify a strong activator of gene expression by screening several dsRNAs targeting the promoter of tumor suppressor p21(WAF1/ Cip1) (p21). Chemical modification is subsequently implemented to improve the medicinal properties of the candidate duplex. Lipidoid-encapsulated nanoparticle (LNP) formulation is validated as a delivery vehicle to mediate p21 induction and inhibit growth of prostate tumor xenografts grown in nude mice following intratumoral injection. We provide insight into the stepwise creation and analysis of a putative RNAa-based therapeutic with antitumor activity. Our results provide proof-of-principle that RNAa in conjunction with lipidioids may represent a novel approach for stimulating gene expression in vivo to treat disease.

Upregulation of Cyclin B1 by miRNA and its implications in cancer.

It is largely recognized that microRNAs (miRNAs) function to silence gene expression by targeting 3'UTR regions. However, miRNAs have also been implicated to positively-regulate gene expression by targeting promoter elements, a phenomenon known as RNA activation (RNAa). In the present study, we show that expression of mouse Cyclin B1 (Ccnb1) is dependent on key factors involved in miRNA biogenesis and function (i.e. Dicer, Drosha, Ago1 and Ago2). In silico analysis identifies highly-complementary sites for 21 miRNAs in the Ccnb1 promoter. Experimental validation identified three miRNAs (miR-744, miR-1186 and miR-466d-3p) that induce Ccnb1 expression in mouse cell lines. Conversely, knockdown of endogenous miR-744 led to decreased Ccnb1 levels. Chromatin immunoprecipitation (ChIP) analysis revealed that Ago1 was selectively associated with the Ccnb1 promoter and miR-744 increased enrichment of RNA polymerase II (RNAP II) and trimethylation of histone 3 at lysine 4 (H3K4me3) at the Ccnb1 transcription start site. Functionally, short-term overexpression of miR-744 and miR-1186 resulted in enhanced cell proliferation, while prolonged expression caused chromosomal instability and in vivo tumor suppression. Such phenotypes were recapitulated by overexpression of Ccnb1. Our findings reveal an endogenous system by which miRNA functions to activate Ccnb1 expression in mouse cells and manipulate in vivo tumor development/growth.

Small RNA and transcriptional upregulation.

Small RNA molecules, such as microRNA and small interfering RNA, have emerged as master regulators of gene expression through their ability to suppress target genes in a phenomenon collectively called RNA interference (RNAi). There is growing evidence that small RNAs can also serve as activators of gene expression by targeting gene regulatory sequences. This novel mechanism, known as RNA activation (RNAa), appears to be conserved in at least mammalian cells and triggered by both endogenous and artificially designed small RNAs. RNAa depends on Argonaute proteins, but possesses kinetics distinct from that of RNAi. Epigenetic changes are associated with RNAa and may contribute to transcriptional activation of target genes, but the underlying mechanism remains elusive. Given the potential of RNAa as a molecular tool for studying gene function and as a therapeutic for disease, further research is needed to completely elucidate its molecular mechanism in order to refine the rules for target selection and improve strategies for exploiting it therapeutically. WIREs RNA 2011 2 748-760 DOI: 10.1002/wrna.90 For further resources related to this article, please visit the WIREs website.

Prognostic value and function of KLF4 in prostate cancer: RNAa and vector-mediated overexpression identify KLF4 as an inhibitor of tumor cell growth and migration.

KLF4/GLKF4 is a transcription factor that can have divergent functions in different malignancies. The role of KLF4 in prostate cancer etiology remains unclear. We have recently reported that small double-stranded RNA can induce gene expression by targeting promoter sequence in a phenomenon referred to as RNA activation (RNAa). In this study, we examine KLF4 levels in prostate cancer tissue and utilize RNAa as a tool for gene overexpression to investigate its function. Expression analysis indicated that KLF4 is significantly downregulated in prostate cancer cell lines compared with nontumorigenic prostate cells. Meta-analysis of existing cDNA microarray data also revealed that KLF4 is frequently depleted in prostate cancer tissue with more pronounced reduction in metastases. In support, tissue microarray analysis of tumors and patient-matched controls indicated downregulation of KLF4 in metastatic tumor samples. Logistic regression analysis found that tumors with a KLF4 staining score less than 5 had a 15-fold higher risk for developing metastatic prostate cancer (P = 0.001; 95% confidence interval, 3.0-79.0). In vitro analysis indicated that RNAa-mediated overexpression of KLF4 inhibited prostate cancer cell proliferation and survival and altered the expression of several downstream cell-cycle-related genes. Ectopic expression of KLF4 via viral transduction recapitulated the RNAa results, validating its inhibitory effects on cancer growth. Reactivation of KLF4 also suppressed migration and invasion of prostate cancer cells. These results suggest that KLF4 functions as an inhibitor of tumor cell growth and migration in prostate cancer and decreased expression has prognostic value for predicting prostate cancer metastasis.

RNAa is conserved in mammalian cells.

BACKGROUND: RNA activation (RNAa) is a newly discovered mechanism of gene activation triggered by small double-stranded RNAs termed 'small activating RNAs' (saRNAs). Thus far, RNAa has only been demonstrated in human cells and is unclear whether it is conserved in other mammals. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we evaluated RNAa in cells derived from four mammalian species including nonhuman primates (African green monkey and chimpanzee), mouse, and rat. Previously, we identified saRNAs leading to the activation of E-cadherin, p21, and VEGF in human cells. As the targeted sequences are highly conserved in primates, transfection of each human saRNA into African green monkey (COS1) and chimpanzee (WES) cells also resulted in induction of the intended gene. Additional saRNAs targeting clinically relevant genes including p53, PAR4, WT1, RB1, p27, NKX3-1, VDR, IL2, and pS2 were also designed and transfected into COS1 and WES cells. Of the nine genes, p53, PAR4, WT1, and NKX3-1 were induced by their corresponding saRNAs. We further extended our analysis of RNAa into rodent cell types. We identified two saRNAs that induced the expression of mouse Cyclin B1 in NIH/3T3 and TRAMP C1 cells, which led to increased phosphorylation of histone H3, a downstream marker for chromosome condensation and entry into mitosis. We also identified two saRNAs that activated the expression of CXCR4 in primary rat adipose-derived stem cells. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that RNAa exists in mammalian species other than human. Our findings also suggest that nonhuman primate disease models may have clinical applicability for validating RNAa-based drugs.