RNA interference and viruses

The Undergraduate Research Symposium was founded to honor student achievement in scientific research and to further RIT's goal of combining traditional laboratory work and classroom instruction with experiential learning. The symposium now hosts over 40 presentations a year and includes representatives from industry leaders including Eastman Kodak and Xerox

The kidneys have rarely been used as a target in the systemic delivery of siRNA when compared to other tissues or organs in the body. This review article deals with various modalities adopted to deliver siRNA to the renal system under different normal and pathophysiological states. In this article, the authors have reviewed extensive clinical data that describe the use of siRNA for the treatment of renal diseases. Conventional and 3D modeling utilizes the existing genome-based RNA libraries, which facilitated the identification of molecular pathways involved in renal diseases. Keywords: siRNA, kidney disease, targeting proteins, signal pathways

The rapid identification of highly specific and potent drug candidates continues to be a substantial challenge with traditional pharmaceutical approaches. Moreover, many targets have proven to be intractable to traditional small-molecule and protein approaches. Therapeutics based on RNA interference (RNAi) offer a powerful method for rapidly identifying specific and potent inhibitors of disease targets from all molecular classes. Numerous proof-of-concept studies in animal models of human disease demonstrate the broad potential application of RNAi therapeutics. The major challenge for successful drug development is identifying delivery strategies that can be translated to the clinic. With advances in this area and the commencement of multiple clinical trials with RNAi therapeutic candidates, a transformation in modern medicine may soon be realized.

INTRODUCTION Peste des petits ruminants (PPR) and rinderpest (RP) are very important viral diseases of domestic and wild ruminants due to their highly contagious behaviour and their high rates of morbidity and mortality. Both viruses have an economic relevance in African and Asian countries and can infect large and small ruminant species. PPRV and RPV are members of the Morbillivirus genus in the Paramyxoviridae family. Although preventive vaccines are available against these diseases, efficient therapeutics for virus control under emergency situations are desirable. Inhibition of morbillivirus replication can be achieved by post-transcriptional silencing of the nucleoprotein (N) gene by RNA interference (RNAi). Sixty two siRNA sequences targeting three genes of PPRV, RPV and measles virus (another morbillivirus affecting humans) were designed, chemically synthesized and tested for efficacy in vitro. Three sequences directed against nucleoprotein (N) gene, were found to be most effe...

The rapid identification of highly specific and potent drug candidates continues to be a substantial challenge with traditional pharmaceutical approaches. Moreover, many targets have proven to be intractable to traditional small-molecule and protein approaches. Therapeutics based on RNA interference (RNAi) offer a powerful method for rapidly identifying specific and potent inhibitors of disease targets from all molecular classes. Numerous proof-of-concept studies in animal models of human disease demonstrate the broad potential application of RNAi therapeutics. The major challenge for successful drug development is identifying delivery strategies that can be translated to the clinic. With advances in this area and the commencement of multiple clinical trials with RNAi therapeutic candidates, a transformation in modern medicine may soon be realized.

The Undergraduate Research Symposium was founded to honor student achievement in scientific research and to further RIT's goal of combining traditional laboratory work and classroom instruction with experiential learning. The symposium now hosts over 40 presentations a year and includes representatives from industry leaders including Eastman Kodak and Xerox

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism preserved during evolution. This mechanism, recently described, is mediated by small double-stranded RNAs (dsRNAs) that can specifically recognize a target mRNA sequence and mediate its cleavage or translational repression. The use of RNAi as a tool for gene therapy has been extensively studied, especially in viral infections, cancer, inherited genetic disorders, cardiovascular and rheumatic diseases. Together with data from human genome, the knowledge of gene silencing mediated by RNAi could allow a functional determination of virtually any cell expressed gene and its involvement in cellular functioning and homeostasis. Several in vitro and in vivo therapeutic studies with autoimmune disease animal models have been carried out with promising results. The pathways of tolerance breakage and inflammation are potential targets for RNAi therapy in inflammatory autoimmune diseases. This review will present the basic principles of RNAi and discuss several aspects of RNAi-based therapeutic approaches, from in vitro tool design and target identification to in vivo pre-clinical drug delivery, and tests of autoimmune diseases in human cells and animal models. Finally, this review will present some recent clinical experience with RNAi-based therapy.

MicroRNAs are key players in the regulation of gene expression by posttranscriptional suppression. They are involved in physiological processes, and thus their deregulation may contribute to the development of diseases and progression of cancer. Virus-encoded microRNAs and microRNAs of host origin play an important role in controlling the virus life cycle and immunity. The aim of this study was to determine the effect of vaccinia virus (VACV) infection on the expression of host-encoded microRNAs. A marked general suppression of most microRNAs in the infected cells was observed within 24 hours after VACV infection of a number of cell types. We demonstrate that this suppression was associated with abrogation of expression of the Dicer1 enzyme, which is a key enzyme in the generation of microRNAs.

Therapeutics based on small interfering RNA (siRNA) have promising potential as antiviral and anti-inflammatory agents. To deliver siRNA across cell membranes to reach the RNAi pathway in the cytosol of target cells, non-viral nanoparticulate delivery approaches are explored. Recently, we showed that encapsulation of siRNA in lipid-polymer hybrid nanoparticles (LPNs), based on poly(DL-lactic-co-glycolic acid) (PLGA) and cationic lipid-like materials (lipidoids), remarkably enhances intracellular delivery of siRNA as compared to siRNA delivery with LPNs modified with dioleoyltrimethylammoniumpropane (DOTAP) as the lipid component. However, the potential immune modulation by these cationic lipids remains unexplored. By testing lipidoids and DOTAP for innate immune-receptor-activating properties in vitro, we found that neither lipidoids nor DOTAP activate human Toll-like receptor (TLR) 2, 3, 7, and 9. However, in contrast to DOTAP, lipidoids are strong agonists for TLR4 and activate mu...

Human adenoviruses (HAdVs) encode for multifunctional non-coding virus-associated (VA) RNAs, which function as powerful suppressors of the cellular interferon (IFN) and RNA interference (RNAi) systems. In this study we tested the ability of various plant and animal virus encoded RNAi and IFN suppressor proteins to functionally substitute for the HAdV-5 VA RNAI. Our results revealed that only the Vaccinia virus (VACV) E3L protein was able to substitute for the HAdV-5 VA RNAI functions in virus-infected cells. Interestingly, the E3L protein rescues the translational defect but does not stimulate viral capsid mRNA accumulation observed with VA RNA. We further show that the E3L C-terminal region containing the dsRNA-binding domain is needed to enhance VA RNAI mutant virus replication. Additionally, we show that the HAdV-4 and HAdV-37 VA RNAI are more effective than the HAdV-5 VA RNAI in rescuing virus replication.

Since the failure of traditional therapy, gene therapy using functional DNA sequence and small RNA/DNA molecules (oligonucleotide) has become a promising avenue for cancer treatment. The discovery of RNA molecules has impelled researchers to investigate small regulatory RNA from various natural and artificial sources and determine a cogent target for controlling tumor progression. Small regulatory RNAs are used for therapeutic silencing of oncogenes and aberrant DNA repair response genes. Despite their advantages, therapies based on small RNAs exhibit limitations in terms of stability of therapeutic drugs, precisionbased delivery in tissues, precision-based intercellular and intracellular targeting, and tumor heterogeneity-based responses. In this study, we summarize the potential and drawbacks of small RNAs in nucleic acid therapeutics for cancer.

Since the failure of traditional therapy, gene therapy using functional DNA sequence and small RNA/DNA molecules (oligonucleotide) has become a promising avenue for cancer treatment. The discovery of RNA molecules has impelled researchers to investigate small regulatory RNA from various natural and artificial sources and determine a cogent target for controlling tumor progression. Small regulatory RNAs are used for therapeutic silencing of oncogenes and aberrant DNA repair response genes. Despite their advantages, therapies based on small RNAs exhibit limitations in terms of stability of therapeutic drugs, precisionbased delivery in tissues, precision-based intercellular and intracellular targeting, and tumor heterogeneity-based responses. In this study, we summarize the potential and drawbacks of small RNAs in nucleic acid therapeutics for cancer.

Infectious bursal disease virus (IBDV) causes an important disease in young chickens. Chicken heat-shock protein 90 (cHsp90) has been shown to be a functional component of the cellular receptor complex for IBDV infection. This study demonstrates the inhibitory effect of vector-expressed anti-cHsp90α microRNA (miRNA) on IBDV infection. The reporter vectors pcHsp90α-EGFP and pcHsp90β-EGFP were constructed to facilitate effective miRNA selection. Two anti-cHsp90α and one anti-cHsp90β miRNA-expression vectors were constructed for a stable transfection study. Poly(A)-tailed RT-PCR detected sequence-specific miRNA transcription in transfected cells. Semiquantitative RT-PCR showed inhibition of cHsp90 transcription in transfected cells. A virus-titration assay showed that the anti-cHsp90α miRNA, but not the anti-cHsp90β miRNA, had inhibitory effects on IBDV infection. These results suggest that cHsp90α is a functional component of the cellular receptor complex for IBDV infection, and that ...

Objective: Morbilliviruses are responsible of important diseases in human beings and animals with economical impact in affected countries. “Peste des petits ruminants” is one of these diseases affecting goats and sheep with high mortality and morbidity. Efficient vaccines exist but they are often used in emergency situation in animals and ten days are necessary to induce a sufficient immune protection. Co-administration of an antiviral treatment with the vaccine could limit the disease impact while conferring a long-lasting protection. CIRAD has explored a biological antiviral therapy based on RNA interference. The identification of siRNA against morbilliviruses has been previously published and patented. The objective of this work was to validate the in vivo efficiency of these siRNAs. Methods: The proof of concept for an efficient in vivo delivery of anti-PPRV siRNAs was developed in a mouse model. Briefly, it is based on the siRNA dynamic extinction of a luciferase reporter gene ...

INTRODUCTION Peste des petits ruminants (PPR) and rinderpest (RP) are very important viral diseases of domestic and wild ruminants due to their highly contagious behaviour and their high rates of morbidity and mortality. Both viruses have an economic relevance in African and Asian countries and can infect large and small ruminant species. PPRV and RPV are members of the Morbillivirus genus in the Paramyxoviridae family. Although preventive vaccines are available against these diseases, efficient therapeutics for virus control under emergency situations are desirable. Inhibition of morbillivirus replication can be achieved by post-transcriptional silencing of the nucleoprotein (N) gene by RNA interference (RNAi). Sixty two siRNA sequences targeting three genes of PPRV, RPV and measles virus (another morbillivirus affecting humans) were designed, chemically synthesized and tested for efficacy in vitro. Three sequences directed against nucleoprotein (N) gene, were found to be most effe...

Peste des petits ruminants (PPR) is a highly contagious and infectious disease of domestic and wild small ruminants. It is caused by an enveloped non-segmented negative single-stranded RNA virus (PPRV). The virus is classified in the genus Morbillivirus within the family Paramyxoviridae. The disease is widely spread in Africa, the Middle-East and SouthWest Asia. In spite of the existence of efficient vaccines against this disease, no effective or specific treatments exist for infected animals. The development of a curative tool could consequently be interesting to help in the control of the disease. A promising approach is the possibility to block the expression of virus genes by RNA interference (RNAi). RNAi is a mechanism of post-transcriptional gene silencing triggered by double-stranded RNA in a sequence-specific manner. However, a major problem of all antiviral therapies is the emergence of resistant variants. Many RNA viruses escape RNAi-mediated suppression by counteracting t...

Soon aft er discovery of RNA interference (RNAi), its potential as eff ective antiviral therapy was recognized. Since then RNAi has been variously exploited for antiviral purposes which could eff ectively block viral replication in vitro. For in vivo use, however, delivery issue, toxicity, RNAi suppression and viral escape are still major hurdles. Here, we provide an overview of the RNAi strategy and review the approaches that have been developed to surpass the obstacles and to achieve targeted gene silencing for antiviral and other therapies.

Influenza A virus is unique as an RNA virus in that it replicates in the nucleus and undergoes splicing. With only ten major proteins, the virus must gain nuclear access, replicate, assemble progeny virions in the cytoplasm, and then egress. In an effort to elucidate the coordination of these events, we manipulated the transcript levels from the bicistronic nonstructural segment that encodes the spliced virus product responsible for genomic nuclear export. We find that utilization of an erroneous splice site ensures the slow accumulation of the viral nuclear export protein (NEP) while generating excessive levels of an antagonist that inhibits the cellular response to infection. Modulation of this simple transcriptional event results in improperly timed export and loss of virus infection. Together, these data demonstrate that coordination of the influenza A virus life cycle is set by a ''molecular timer'' that operates on the inefficient splicing of a virus transcript.

cotransfected with a luciferase expression plasmid into mouse liver by hydrodynamic transfection, this construct induced ∼90% silencing relative to a random-hairpin control. The construct was next adapted to target hepatitis B virus (HBV) s-antigen (sAg), and packaged as a double-strand in pseudotyped adeno-associated virus serotype 8 (dsAAV8). The hairpin sequence used was for the expression of a 25mer shRNA that had proven to be toxic when expressed from a U6 promoter. Previous work demonstrated that the toxic effect observed for this U6 construct was such that the ultimate result was either loss of the RNAiinduced silencing effect or the death of recipient mice due to liver failure less than a month following injection of expression cassetteharboring AAV. We tested the ApoE/hAAT expression construct in transgenic mice harboring an integrated HBV genome. Over the course of three months, the ApoE/hAAT construct was well tolerated and induced a stable level of sAg silencing of ∼80% of starting levels. Healthy FVB mice were used in a separate toxicity study for direct comparison of U6-and ApoE/hAAT-driven 25mers. Serum ALT levels were followed over three months as an indicator of liver toxicity. Mice that received the ApoE/hAAT-driven construct had stable, normal ALT levels, while those of mice that received the U6driven construct spiked to peak around three weeks before gradually returning to normal. While we continue to optimize its efficacy, we conclude that this pol II system is an effective and safer HBV therapeutic in a mouse model of human disease.

The Undergraduate Research Symposium was founded to honor student achievement in scientific research and to further RIT's goal of combining traditional laboratory work and classroom instruction with experiential learning. The symposium now hosts over 40 presentations a year and includes representatives from industry leaders including Eastman Kodak and Xerox

The discovery and characterization of the RNA interference (RNAi) pathway has been one of the most important scientific developments of the last 12 years. RNAi is a cellular pathway wherein small RNAs control the expression of genes by either degrading homologous RNAs or preventing the translation of RNAs with partial homology. It has impacted basic biology on two major fronts. The first is the discovery of microRNAs (miRNAs), which regulate almost every cellular process and are required for some viral infections, including hepatitis C virus (HCV). The second front is the use of small interfering RNAs (siRNAs) as the first robust tool for mammalian cellular genetics. This has led to the identification of hundreds of cellular genes that are important for HCV infection. There is now a major push to adapt RNAi technology to the clinic. In this review, we explore the impact of RNAi in understanding HCV biology, the progress in design of RNAi-based therapeutics for HCV, and remaining obs...

siRNA interference, commonly referred to as gene silence, is a biological mechanism that inhibits gene expression in disorders such as cancer. It may enhance the precision, efficacy, and stability of medicines, especially genetic therapies to some extent. However, obstacles such as the delivery of oligonucleotide drugs to inaccessible areas of the body and the prevalence of severe side effects must be overcome. To maximize their potential, it is thus essential to optimize their distribution to target locations and limit their toxicity to healthy cells. The action of siRNA may be harnessed to delete a similar segment of mRNA that encodes a protein that causes sickness. The absence of an efficient delivery mechanism that shields siRNA from nuclease degradation, delivers it to cancer cells and releases it into the cytoplasm of specific cancer cells without causing side effects is currently the greatest obstacle to the practical implementation of siRNA therapy. This article focuses on c...

Gene silencing induced by RNAi represents a promising antiviral development strategy. This review will summarise the current state of RNAi therapeutics for treating acute and chronic human virus infections. The gene silencing pathways exploited by RNAi therapeutics will be described and include both classic RNAi, inducing cytoplasmic mRNA degradation post-transcription and novel RNAi, mediating epigenetic modifications at the transcription level in the nucleus. Finally, the challenge of delivering gene modifications via RNAi will be discussed, along with the unique characteristics of respiratory versus systemic administration routes to highlight recent advances and future potential of RNAi antiviral treatment strategies.

Highlights d Individual roles for the RNAi/miRNA effector proteins AGO1, AGO3, and AGO4 are elusive d AGO4 is uniquely antiviral in mammalian immune cells d Mammalian AGO4 has evolved to both elicit antiviral RNAi and boost IFN d AGO4 is indispensable for control of influenza in vivo

RNA interference (RNAi) is an exciting new tool to effect acute in vivo knockdown of genes for pharmacological target validation. Testing the application of this technology to metabolic disease targets, three RNAi delivery methods were compared in two frequently utilized preclinical models of obesity and diabetes, the diet-induced obese (DIO) and B6.V-Lep<ob>/J (ob/ob) mouse. Intraperitoneal (i.p.) and high pressure hydrodynamic intravenous (i.v.) administration of naked siRNA, and low pressure i.v. administration of shRNA-expressing adenovirus were assessed for both safety and gene knockdown efficacy using constructs targeting cJun N-terminal kinase 1 (JNK1). Hydrodynamic delivery of siRNA lowered liver JNK1 protein levels 40% in DIO mice, but was accompanied by iatrogenic liver damage. The ob/ob model proved even more intolerant of this technique, with hydrodynamic delivery resulting in severe liver damage and death of most animals. While well-tolerated, i.p. injections of s...

Small hairpin RNAs (shRNAs) are widely used as gene silencing tools and typically consist of a duplex stem of 19–29 bp, a loop, and often a dinucleotide overhang at the 3′ end. Like siRNAs, shRNAs show promise as potential therapeutic agents due to their high level of specificity and potency, although effective delivery to target tissues remains a challenge. Algorithms used to predict siRNA performance are frequently used to design shRNAs as well. However, the differences between these two kinds of RNAi mediators indicate that the factors affecting target gene silencing will not be the same for siRNAs and shRNAs. Stem and loop lengths, structures of the termini, the identity of nucleotides adjacent to and near the loop, and the position of the guide (antisense) strand all affect the efficacy of shRNAs. In addition, shRNAs with 19-bp or shorter stem lengths are processed and function differently than those with longer stems. In this review, we describe studies of targeting the hepati...

The discovery and characterization of the RNA interference (RNAi) pathway has been one of the most important scientific developments of the last 12 years. RNAi is a cellular pathway wherein small RNAs control the expression of genes by either degrading homologous RNAs or preventing the translation of RNAs with partial homology. It has impacted basic biology on two major fronts. The first is the discovery of microRNAs (miRNAs), which regulate almost every cellular process and are required for some viral infections, including hepatitis C virus (HCV). The second front is the use of small interfering RNAs (siRNAs) as the first robust tool for mammalian cellular genetics. This has led to the identification of hundreds of cellular genes that are important for HCV infection. There is now a major push to adapt RNAi technology to the clinic. In this review, we explore the impact of RNAi in understanding HCV biology, the progress in design of RNAi-based therapeutics for HCV, and remaining obs...

Highlights d Individual roles for the RNAi/miRNA effector proteins AGO1, AGO3, and AGO4 are elusive d AGO4 is uniquely antiviral in mammalian immune cells d Mammalian AGO4 has evolved to both elicit antiviral RNAi and boost IFN d AGO4 is indispensable for control of influenza in vivo

siRNA interference, commonly referred to as gene silence, is a biological mechanism that inhibits gene expression in disorders such as cancer. It may enhance the precision, efficacy, and stability of medicines, especially genetic therapies to some extent. However, obstacles such as the delivery of oligonucleotide drugs to inaccessible areas of the body and the prevalence of severe side effects must be overcome. To maximize their potential, it is thus essential to optimize their distribution to target locations and limit their toxicity to healthy cells. The action of siRNA may be harnessed to delete a similar segment of mRNA that encodes a protein that causes sickness. The absence of an efficient delivery mechanism that shields siRNA from nuclease degradation, delivers it to cancer cells and releases it into the cytoplasm of specific cancer cells without causing side effects is currently the greatest obstacle to the practical implementation of siRNA therapy. This article focuses on combinations of siRNA with chemotherapeutic drug delivery systems for the treatment of cancer and gives an overview of several nanocarrier formulations in both research and clinical applications.

Since the failure of traditional therapy, gene therapy using functional DNA sequence and small RNA/DNA molecules (oligonucleotide) has become a promising avenue for cancer treatment. The discovery of RNA molecules has impelled researchers to investigate small regulatory RNA from various natural and artificial sources and determine a cogent target for controlling tumor progression. Small regulatory RNAs are used for therapeutic silencing of oncogenes and aberrant DNA repair response genes. Despite their advantages, therapies based on small RNAs exhibit limitations in terms of stability of therapeutic drugs, precisionbased delivery in tissues, precision-based intercellular and intracellular targeting, and tumor heterogeneity-based responses. In this study, we summarize the potential and drawbacks of small RNAs in nucleic acid therapeutics for cancer.

MicroRNAs are key players in the regulation of gene expression by posttranscriptional suppression. They are involved in physiological processes, and thus their deregulation may contribute to the development of diseases and progression of cancer. Virus-encoded microRNAs and microRNAs of host origin play an important role in controlling the virus life cycle and immunity. The aim of this study was to determine the effect of vaccinia virus (VACV) infection on the expression of host-encoded microRNAs. A marked general suppression of most microRNAs in the infected cells was observed within 24 hours after VACV infection of a number of cell types. We demonstrate that this suppression was associated with abrogation of expression of the Dicer1 enzyme, which is a key enzyme in the generation of microRNAs.

The influenza A non-structural protein 1 (NS1) is known for its ability to hinder the synthesis of type I interferon (IFN) during viral infection. Influenza viruses lacking NS1 (ΔNS1) are under clinical development as live attenuated human influenza virus vaccines and induce potent influenza virus-specific humoral and cellular adaptive immune responses. Attenuation of ΔNS1 influenza viruses is due to their high IFN inducing properties, that limit their replication in vivo. This study demonstrates that pre-treatment with a ΔNS1 virus results in an antiviral state which prevents subsequent replication of homologous and heterologous viruses, preventing disease from virus respiratory pathogens, including SARS-CoV-2. Our studies suggest that ΔNS1 influenza viruses could be used for the prophylaxis of influenza, SARS-CoV-2 and other human respiratory viral infections, and that an influenza virus vaccine based on ΔNS1 live attenuated viruses would confer broad protection against influenza ...

MicroRNAs (miRNAs) are powerful regulators of gene expression and fine-tuning genes in all tissues. Cellular miRNAs can control 100s of biologic processes (e.g., morphogenesis of embryonic structures, differentiation of tissue-specific cells, and metabolic control in specific cell types) and have been involved in the regulation of nearly all cellular pathways. Inherently to their involvement in different physiologic processes, miRNAs deregulation has been associated with several diseases. Moreover, several viruses have been described as either, avoid and block cellular miRNAs or synthesize their own miRNA to facilitate infection and pathogenesis. Adenoviruses genome encodes two non-coding RNAs, known as viral-associated (VA) RNA I and VA RNA II , which seem to play an important role either by blocking important proteins from miRNA pathway, such as Exportin-5 and Dicer, or by targeting relevant cellular factors. Drastic changes in cellular miRNA expression profile are also noticeable and several cellular functions are affected by these changes. This review focuses on the mechanisms underlying the biogenesis and molecular interactions of miRNAs providing basic concepts of their functions as well as in the interplay between miRNAs and human adenoviruses.

Background: Polio eradication is a global health priority and Pakistan is among last three endemic countries. Supplementary immunization activities (SIAs) with oral poliovirus vaccines are usually separated by four week intervals; however, shorter intervals have been used in security compromised areas and for rapid outbreak response. We assessed immunogenicity of monovalent type 1 oral poliovirus vaccines (mOPV1) administered at shorter than usual intervals in a cohort of infants in Karachi, Pakistan. Methods & Materials: A randomized controlled trial was conducted to compare immunogenicity of two doses of mOPV1 given at 7 or 14 day-intervals with standard 30 day interval. In addition, bivalent OPV1&3 (bOPV) was administered at 30-day interval. Birth trivalent OPV and two study OPV doses were administered and blood samples obtained at birth, six weeks of age and one month after the last OPV dose. Blood was tested for poliovirus neutralizing antibodies. Results: A total of 1009 newborns were enrolled, and 829 (82%) met eligibility criteria for randomization at 6 weeks; 554 (55%) were included in the per protocol analysis. Seroprevalence of poliovirus neutralizing antibodies for poliovirus type 1 after three doses of OPV was > 95% for all arms. Among those who did not seroconvert after birth dose, no significant differences in seroconversion to poliovirus type 1 after two study OPV doses were found between study arms (75.0% [CI95% = 65-83%] for mOPV1 given at 7 day interval, 75.0% [CI95% = 65-83%] for mOPV1 at 14 day interval, 78.1% [CI95% = 69-86%] for mOPV1 at 30 day interval and 72.6% [CI95% = 63-81%] for bOPV at 30 day interval). Conclusion: We found no differences in immunogenicity of mOPV1 administered in shorter than standard intervals. These results provide the scientific justification for the expanded use of the short-interval strategy to rapidly increase population immunity, to control outbreaks, prevent importations, and in areas of conflict where limited window of access can be created.

The discovery of host-microRNA (miRNA) targets in the genomes of many vertebrate viruses indicates that the corresponding miRNAs are a part of the host's innate antiviral defense. However, given that viruses evolve much faster than host miRNAs, it is surprising that viral variants lacking these "antiviral" miRNA target sequences have not become established. We present an alternate view that miRNAs are among the host molecules that viruses co-opt to suppress their own replication to evade immune elimination and establish a persistent infection. The presence of host-miRNA targets in the genomes of rapidly evolving viruses probably reflects the adaptation of these viruses to the cellular miRNA milieu. RNA silencing and immunity RNA silencing (Figure 1) is a widely conserved mechanism of post-transcriptional gene regulation. RNA interference (RNAi) is also an integral part of the antiviral defense in plants and invertebrates, which can process virus-derived long double stranded RNAs (dsRNA) into small interfering RNAs (siRNAs), amplify siRNAs by RNA dependent RNA polymerases (RDRP) and induce systemic RNAi [1]. The presence of a viral counter-defense in the form of viral suppressors of RNA silencing (VSRs) in numerous plant and invertebrate viruses is strong supporting evidence for the antiviral nature of RNAi in plants and invertebrates (Box 1). However, in vertebrates, the interferon (IFN) response is the primary form of innate antiviral defense. It is triggered by innate immune recognition of various viral intermediates and products, such as 5′-triphosphate on viral RNA, cytosolic DNA and dsRNA. Binding of IFNs to their receptors initiates signaling that leads to a global shutdown in protein translation, cellular RNA degradation and deamination and often the death of virus-infected cells [2]. As with RNAi in plants and invertebrates, numerous vertebrate viruses have evolved mechanisms to evade the IFN response [3]. It is not yet established whether small RNAs encoded in the vertebrate genome have a direct role in innate antiviral responses. Synthetic siRNAs have been shown to be able to inhibit the replication of a wide range of pathogenic viruses when introduced into mammalian cells and animal models [4,5]. In addition, several endogenous host microRNAs (miRNAs) have been

Background RNA polymerase III (pol III)-dependent transcripts are involved in many fundamental activities in a cell, such as splicing and protein synthesis. They also regulate cell growth and influence tumor formation. During recent years vector-based systems for expression of short hairpin (sh) RNA under the control of a pol III promoter have been developed as gene-based medicines. Therefore, there is an increasing interest in means to regulate pol III-dependent transcription. Recently, we have developed a novel anti-gene molecule 'Zorro LNA (Locked Nucleic Acid)', which simultaneously hybridizes to both strands of super-coiled DNA and potently inhibits RNA polymerase II-derived transcription. We have now applied Zorro LNA in an attempt to also control U6 promoter-driven expression of shRNA. Methods In this study, we constructed pshluc and pshluc2BS plasmids, in which U6 promoter-driven small hairpin RNA specific for luciferase gene (shluc) was without or with Zorro LNA binding sites, respectively. After hybridization of Zorro LNA to pshluc2BS, the LNA-bound plasmid was cotransfected with pEGFPluc into mammalian cells and into a mouse model. In cellular experiments, cotransfection of unhybridized pshluc2BS, Zorro LNA and pEGFPluc was also performed. Results The results showed that the Zorro LNA construct efficiently inhibited pol III-dependent transcription as an anti-gene reagent in a cellular context, including in vivo in a mouse model. Conclusions Thus, this new form of gene silencer 'Zorro LNA' could potentially serve as a versatile regulator of pol III-dependent transcription, including various forms of shRNAs.

siRNAs confer sequence specific and robust silencing of mRNA. By virtue of these properties, siRNAs have become therapeutic candidates for disease intervention. However, their use as therapeutic agents can be hampered by unintended off-target effects by either or both strands of the siRNA duplex. We report here that unlocked nucleobase analogs (UNAs) confer desirable properties to siRNAs. Addition of a single UNA at the 5 0-terminus of the passenger strand blocks participation of the passenger strand in RISC-mediated target down-regulation with a concomitant increase in guide strand activity. Placement of a UNA in the seed region of the guide strand prevents miRNA-like off-target silencing without compromising siRNA activity. Most significantly, combined substitution of UNA at the 3 0-termini of both strands, the addition of a UNA at the 5 0-terminus of the passenger strand, and a single UNA in the seed region of the guide strand, reduced the global off-target events by more than 10-fold compared to unmodified siRNA. The reduction in off-target events was specific to UNA placement in the siRNA, with no apparent new off-target events. Taken together, these results indicate that when strategically placed, UNA substitutions have important implications for the design of safe and effective siRNAbased therapeutics.

The Undergraduate Research Symposium was founded to honor student achievement in scientific research and to further RIT's goal of combining traditional laboratory work and classroom instruction with experiential learning. The symposium now hosts over 40 presentations a year and includes representatives from industry leaders including Eastman Kodak and Xerox

The RNA interference (RNAi) is a biological process by which a double stranded RNA (dsRNA also called small interfering RNA-siRNA) triggers the sequence-dependent degradation of a target RNA within the cellular environment. Thus siRNAs can be used to combat the expression of deleterious gene(s) causing disease or to destroy invading pathogen RNAs. Despite their enormous therapeutic potential, the use of siRNA as drugs presents two major problems: the difficulties to identify optimal delivery systems and the possible induction of different unwanted side effects. In this review, after presenting an overview about the mechanisms ruling the process of RNAi, we focus the attention on the description of the strategies developed to optimise systemic siRNA delivery; in this sense, considerations about the attempts to improve siRNA stability in the biological environment, the development of synthetic vectors for siRNA delivery, the siRNA bio-distribution and pharmacokinetics together with the selection of siRNA targeted delivery systems, are discussed. Since in the optimisation of the siRNA delivery systems the minimization of siRNA side effects should not be neglected, in the last part of the review we consider the problems related to the possible induction of siRNA mediated side effects focusing on the so called mi-croRNA like off-targeting.

Small interfering RNAs (siRNAs) conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand are being evaluated in investigational clinical studies for a variety of indications. The typical development candidate selection process includes evaluation of the most active compounds for toxicity in rats at pharmacologically exaggerated doses. The subset of GalNAc-siRNAs that show rat hepatotoxicity is not advanced to clinical development. Potential mechanisms of hepatotoxicity can be associated with the intracellular accumulation of oligonucleotides and their metabolites, RNA interference (RNAi)-mediated hybridization-based off-target effects, and/or perturbation of endogenous RNAi pathways. Here we show that rodent hepatotoxicity observed at supratherapeutic exposures can be largely attributed to RNAi-mediated off-target effects, but not chemical modifications or the perturbation of RNAi pathways. Furthermore, these off-target effects can be mitigated by modulating seed-pairing usin...

The Morbillivirus genus includes measles virus (MV), peste des petits ruminants virus (PPRV) and rinderpest virus (RPV). Although preventive vaccines are available against these three viruses, efficient therapeutics for virus control under emergency situations are desirable. Inhibition of morbillivirus replication can be achieved by post-transcriptional silencing of the nucleoprotein (N) gene by RNA interference (RNAi). The viral N protein, a well conserved protein among the genus, plays a central role in the replication of the virus. Using a comprehensive siRNA-based screening of the conserved sequences of the N gene, we have identified, three common positions on the N gene, for the design of siRNA evenly effectives for PPRV, RPV and MV. siRNA silencing resulted in more than 80% decrease of the viral replication in infected Vero cell, as shown by real-time quantitative PCR, flow cytometry and virus titration. In a second step, a recombinant replication-defective human type 5 adenov...

Background: The hepatitis C virus (HCV) is an attractive target for RNA interference (RNAi) based therapeutics because its genome consists of a single, positive stranded RNA and its replication occurs strictly within the cytoplasm. TT-034 is a DNA-directed RNAi (ddRNAi) based gene therapy product for the treatment of chronic HCV infection and is currently in a phase I/IIa clinical trial. TT-034 is comprised of a self-complementary recombinant adeno-associated virus (AAV) serotype 8 vector for transduction of hepatic tissues and is systemically administered as a single intravenous dose. Inside the hepatocyte, TT-034 uses the cell's own transcriptional machinery to continuously express three independent short hairpin RNAs (shRNAs) that simultaneously target the 5' UTR and NS5B regions of the HCV genome. Results: Following a single administration of up to 3.75E12 vg/ kg of TT-034, biodistribution analyses demonstrated that greater than 90% of the quantified vector is found in the Cynomolgus monkey liver tissues. In situ hybridization analyses demonstrated almost 100% transduction of the hepatocytes when dosed at 1.25E12 vg/kg. Furthermore, qPCR data revealed that shRNA levels persisted for the duration of the 180-day experiment, demonstrating the durability of expression following a single injection. We examined the impact of continuous TT-034 expression on endogenous miRNAs in liver and cardiac tissues because previous reports have suggested that high expression of shRNA may cause global dysregulation of endogenous microRNA (miRNA) levels within cells. Non-human primates were dosed with saline control or either 1.00E11, 1.00E12, 1.00E13 vg/kg of TT-034. Expression profiles of mature miRNAs were generated from liver biopsies taken 15 days after TT-034 administration. Additionally, samples from liver and heart tissues were collected 60 and 180 days after administration. Approximately 260 different miRNAs were reliably detected in each sample type and used for the analyses. The day 15 liver samples showed significant differences in the expression levels of 27 miRNAs. When analyzing all 260 miRNAs, the day 60 and day 180 liver samples showed no significant statistical differences from the control group at any dose. In the heart tissues, no significant statistical differences between the TT-034 treated group and the control group were noted at any timepoint. Conclusions: These data suggest that a single, intravenous infusion of TT-034 results in almost complete transduction of Cynomolgus monkey hepatocytes. Furthermore, TT-034 results in durable expression of three anti-HCV shRNAs. These doses of TT-034 do not cause long-term perturbation of endogenous miRNA levels.

Background Small interfering (si)RNA mediated inhibition of oncogenes or viral genes may offer great opportunities for the treatment of several diseases such as hepatocellular carcinoma and viral hepatitis. However, the development of siRNAs as therapeutic agents strongly depends on the availability of safe and effective intracellular delivery systems. Poly(β-amino esters) (PbAEs) are, in contrast to many other cationic polymers evaluated in siRNA delivery, biodegradable into smaller, nontoxic molecules. We show for the first time that PbAE : siRNA complexes, containing 1,4-butanediol (PbAE1) or 1,6-hexanediol (PbAE2) diacrylate-based polymers, induced efficient gene silencing in both hepatoma cells and primary hepatocytes without causing significant cytotoxicity. Furthermore, carriers that slowly release the siRNA into the cytoplasm and hence induce a prolonged gene silencing are of major clinical interest, especially in fast dividing tumour cells. Therefore, we also studied the duration of gene silencing in the hepatoma cells and found that it was maintained for at least 5 days after siRNA delivery with PbAE2, the polymer with the slowest degradation kinetics. Conclusions From the time-dependent cellular distribution of these PbAE : siRNA complexes, we suggest that the slowly degrading PbAE2 causes a sustained endosomal release of siRNA during a much longer period than PbAE1. This may support the hypothesis that the endosomal release mechanism of PbAE : siRNA complexes is based on an increase of osmotic pressure in the endosomal vesicles after polymer hydrolysis. In conclusion, our results show that both PbAEs, and especially PbAE2, open up new perspectives for the development of efficient biodegradable siRNA carriers suitable for clinical applications.

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism preserved during evolution. This mechanism, recently described, is mediated by small double-stranded RNAs (dsRNAs) that can specifically recognize a target mRNA sequence and mediate its cleavage or translational repression. The use of RNAi as a tool for gene therapy has been extensively studied, especially in viral infections, cancer, inherited genetic disorders, cardiovascular and rheumatic diseases. Together with data from human genome, the knowledge of gene silencing mediated by RNAi could allow a functional determination of virtually any cell expressed gene and its involvement in cellular functioning and homeostasis. Several in vitro and in vivo therapeutic studies with autoimmune disease animal models have been carried out with promising results. The pathways of tolerance breakage and inflammation are potential targets for RNAi therapy in inflammatory autoimmune diseases. This review will present the basic principles of RNAi and discuss several aspects of RNAi-based therapeutic approaches, from in vitro tool design and target identification to in vivo pre-clinical drug delivery, and tests of autoimmune diseases in human cells and animal models. Finally, this review will present some recent clinical experience with RNAi-based therapy.

The Undergraduate Research Symposium was founded to honor student achievement in scientific research and to further RIT's goal of combining traditional laboratory work and classroom instruction with experiential learning. The symposium now hosts over 40 presentations a year and includes representatives from industry leaders including Eastman Kodak and Xerox

Immune stimulation is a significant hurdle in the development of effective and safe RNA interference therapeutics. Here, we address this problem in the context of a mimic of microRNA-122 by employing novel nucleobase and known 2 0 -ribose modifications. The nucleobase modifications are analogues of adenosine and guanosine that contain cyclopentyl and propyl minor-groove projections. Via a site-by-site chemical modification analysis, we identify several immunostimulatory 'hot spots' within the miRNA guide strand at which single base modifications significantly reduce immune stimulation. A duplex containing one base modification on each strand proved to be most effective in preventing immune stimulation.

Objectives: Interleukin-27 (IL-27) is known as an anti-HIV cytokine. We have recently demonstrated that IL-27-pretreatment promotes phytohemagglutinin-stimulated CD4(+) T cells into HIV-1-resistant cells by inhibiting an uncoating step. Purpose: To further characterize the function of the HIV resistant T cells, we investigated profiles of microRNA in the cells using microRNA sequencing (miRNA-seq) and assessed anti-HIV effect of the microRNAs. Methods: Phytohemagglutinin-stimulated CD4(+) T cells were treated with or without IL-27 for 3 days. MicroRNA profiles were analyzed using miRNA-seq. To assess anti-HIV effect, T cells or macrophages were transfected with synthesized microRNA mimics and then infected with HIVNL4.3 or HIVAD8. Anti-HIV effect was monitored by a p24 antigen enzyme-linked immunosorbent assay kit. interferon (IFN)-a, IFN-b, or IFN-l production was quantified using each subtype-specific enzyme-linked immunosorbent assay kit. Results: A comparative analysis of microRNA profiles indicated that expression of known miRNAs was not significantly changed in IL-27-treated cells compared with untreated T cells; however, a total of 15 novel microRNAs (miRTC1 ; miRTC15) were identified. Anti-HIV assay using overexpression of each novel microRNA revealed that 10 nM miRTC14 (GenBank accession number: MF281439) remarkably suppressed HIV infection by (99.3 6 0.27%, n = 9) in macrophages but not in T cells. The inhibition was associated through induction of .1000 pg/mL of IFN-as and IFN-l1. Conclusion: We discovered a total of 15 novel microRNAs in T cells and characterized that miRTC14, one of the novel micro-RNAs, was a potent IFN-inducing anti-HIV miRNA, implicating that regulation of the expression of miRTC14 may be a potent therapeutic tool for not only HIV but also other virus infection.