Jon Karpilow

3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets

Off-target gene silencing can present a notable challenge in the interpretation of data from large-scale RNA interference (RNAi) screens. We performed a detailed analysis of off-targeted genes identified by expression profiling of human cells transfected with small interfering RNA (siRNA). Contrary to common assumption, analysis of the subsequent off-target gene database showed that overall identity makes little or no contribution to determining whether the expression of a particular gene will be affected by a given siRNA, except for near-perfect matches. Instead, off-targeting is associated with the presence of one or more perfect 3′ untranslated region (UTR) matches with the hexamer or heptamer seed region (positions 2–7 or 2–8) of the antisense strand of the siRNA. These findings have strong implications for future siRNA design and the application of RNAi in high-throughput screening and therapeutic development.

A protocol for designing siRNAs with high functionality and specificity

Effective gene silencing by the RNA interference (RNAi) pathway requires a comprehensive understanding of the elements that influence small interfering RNA (siRNA) functionality and specificity. These include (i) sequence space restrictions that define the boundaries of siRNA targeting, (ii) structural and sequence features required for efficient siRNA performance, (iii) mechanisms that underlie nonspecific gene modulation and (iv) additional features specific to the intended use (i.e., inclusion of native sugar or base chemical modifications for increased stability or specificity, vector design, etc.). Attention to each of these factors enhances siRNA performance and heightens overall confidence in the output of RNAi-mediated functional genomic studies. Here, we provide a detailed protocol explaining the methodologies used for manual and web-based design of siRNAs.

Different delivery methods-different expression profiles.

Recent publications by several groups of researchers have suggested that small interfering RNAs (siRNA) delivered by lipid-mediated transfection induce both sequence-specific effects1 and broad, class-specific changes in gene expression1, 2, 3, 4. These findings challenge convictions previously held in the RNA interference (RNAi) community that assert virtual sequence specificity of siRNA knockdown, and they bring into question the value of this methodology as a research and therapeutic tool.

Host gene targets for novel influenza therapies elucidated by high-throughput RNA interference screens

Influenza virus encodes only 11 viral proteins but replicates in a broad range of avian and mammalian species by exploiting host cell functions. Genome‐wide RNA interference (RNAi) has proven to be a powerful tool for identifying the host molecules that participate in each step of virus replication. Meta‐analysis of findings from genome‐wide RNAi screens has shown influenza virus to be dependent on functional nodes in host cell pathways, requiring a wide variety of molecules and cellular proteins for replication. Because rapid evolution of the influenza A viruses persistently complicates the effectiveness of vaccines and therapeutics, a further understanding of the complex host cell pathways coopted by influenza virus for replication may provide new targets and strategies for antiviral therapy. RNAi genome screening technologies together with bioinformatics can provide the ability to rapidly identify specific host factors involved in resistance and susceptibility to influenza virus, allowing for novel disease intervention strategies.—Meliopoulos, V. A., Andersen, L. E., Birrer, K. F., Simpson, K. J., Lowenthal, J. W., Bean, A. G. D., Stambas, J., Stewart, C. R., Tompkins, S. M., van Beusechem, V. W., Fraser, I., Mhlanga, M., Barichievy, S., Smith, Q., Leake, D., Karpilow, J., Buck, A., Jona, G., Tripp, R. A. Host gene targets for novel influenza therapies elucidated by high‐throughput RNA interference screens. FASEB J. 26, 1372‐1386 (2012). www.fasebj.org

Identifying siRNA-Induced Off-Targets by Microarray Analysis

RNA interference (RNAi) is an endogenous gene regulatory pathway that the research community has adopted to facilitate the creation of a functional map of the human genome. To achieve this, small interfering RNAs (siRNAs), short synthetic duplexes having complete homology to the intended target, are introduced into cells to silence gene expression via a posttranscriptional cleavage mechanism. While siRNAs can be designed to effectively knock down any target gene, recent studies have shown that these small molecules frequently trigger off-target effects. These unintended events can have a significant impact on experimental outcomes and subsequent data interpretation. As RNAi is envisioned to play a central role in developing a functional map of the human genome, the development of reliable protocols for identifying off-targeted genes is essential. This chapter focuses on the underlying features of siRNA-mediated off-targeting and the state-of-the-art methodology used to identify off-targeted genes via microarray-based gene expression analysis. Future adoption of standards in this field will allow a clean distinction between sequence-specific off-target gene regulation and other forms of gene modulation resulting from delivery effects and other events unrelated to the RNAi pathway.

Engineering Enhanced Vaccine Cell Lines To Eradicate Vaccine-Preventable Diseases: the Polio End Game

ABSTRACT Vaccine manufacturing costs prevent a significant portion of the worlds population from accessing protection from vaccine-preventable diseases. To enhance vaccine production at reduced costs, a genome-wide RNA interference (RNAi) screen was performed to identify gene knockdown events that enhanced poliovirus replication. Primary screen hits were validated in a Vero vaccine manufacturing cell line using attenuated and wild-type poliovirus strains. Multiple single and dual gene silencing events increased poliovirus titers >20-fold and >50-fold, respectively. Host gene knockdown events did not affect virus antigenicity, and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-mediated knockout of the top candidates dramatically improved viral vaccine strain production. Interestingly, silencing of several genes that enhanced poliovirus replication also enhanced replication of enterovirus 71, a clinically relevant virus to which vaccines are being targeted. The discovery that host gene modulation can markedly increase virus vaccine production dramatically alters mammalian cell-based vaccine manufacturing possibilities and should facilitate polio eradication using the inactivated poliovirus vaccine. IMPORTANCE Using a genome-wide RNAi screen, a collection of host virus resistance genes was identified that, upon silencing, increased poliovirus and enterovirus 71 production by from 10-fold to >50-fold in a Vero vaccine manufacturing cell line. This report provides novel insights into enterovirus-host interactions and describes an approach to developing the next generation of vaccine manufacturing through engineered vaccine cell lines. The results show that specific gene silencing and knockout events can enhance viral titers of both attenuated (Sabin strain) and wild-type polioviruses, a finding that should greatly facilitate global implementation of inactivated polio vaccine as well as further reduce costs for live-attenuated oral polio vaccines. This work describes a platform-enabling technology applicable to most vaccine-preventable diseases.

Development of improved vaccine cell lines against rotavirus

Rotavirus is a major cause of severe gastroenteritis among very young children. In developing countries, rotavirus is the major cause of mortality in children under five years old, causing up to 20% of all childhood deaths in countries with high diarrheal disease burden, with more than 90% of these deaths occurring in Africa and Asia. Rotavirus vaccination mimics the first infection without causing illness, thus inducing strong and broad heterotypic immunity against prospective rotavirus infections. Two live vaccines are available, Rotarix and RotaTeq, but vaccination efforts are hampered by high production costs. Here, we present a dataset containing a genome-wide RNA interference (RNAi) screen that identified silencing events that enhanced rotavirus replication. Evaluated against several rotavirus vaccine strains, hits were validated in a Vero vaccine cell line as well as CRISPR/Cas9 generated cells permanently and stably lacking the genes that affect RV replication. Knockout cells were dramatically more permissive to RV replication and permitted an increase in rotavirus replication. These data show a means to improve manufacturing of rotavirus vaccine.

MicroRNA screening identifies miR-134 as a regulator of poliovirus and enterovirus 71 infection

MicroRNAs (miRNAs) regulate virus replication through multiple mechanisms. Poliovirus causes a highly debilitating disease and though global efforts to eradicate polio have sharply decreased polio incidence, unfortunately three countries (Afghanistan, Nigeria and Pakistan) remain polio-endemic. We hypothesize that understanding the host factors involved in polio replication will identify novel prophylactic and therapeutic targets against polio and related viruses. In this data set, employing genome wide screens of miRNA mimics and inhibitors, we identified miRNAs which significantly suppressed polio replication. Specifically, miR-134 regulates poliovirus replication via modulation of ras-related nuclear protein (RAN), an important component of the nuclear transport system. MiR-134 also inhibited other Picornaviridae viruses including EV71, a growing concern and a high priority for vaccination in Asian countries like China. These findings demonstrate a novel mechanism for miRNA regulation of poliovirus and other Picornaviridae viruses in host cells, and thereby may provide a novel approach in combating infection and a potential approach for the development of anti-Picornaviridae strategies.