Posts Tagged ‘RNA interference’


Nanoparticles can turn off genes in bone marrow cells

Reporter : Irina Robu, PhD

MIT engineers developed an alternative to turn off specific genes which play a vital role in producing blood cells of the bone marrow using specialized nanoparticles. These nanoparticles can be made-to-order to treat heart disease or increase the yield of stem cells in patience who need stem cell transplants. The particles are coated with lipids that help stabilize them, and they can target organs such as the lungs, heart, and spleen, depending on the particles’ composition and molecular weight. This genetic therapy, also known as RNA interference is difficult to target organs other than the liver, where most of the nanoparticles tend to collect.

RNA interference is an approach that could theoretically be used to treat a variety of diseases by delivering short strands of RNA that block specific genes from being turned on in a cell. Yet, the main obstacle to this kind of therapy has been  delivering it to the right part of the body. When injected into the bloodstream, nanoparticles carrying RNA tend to accrue in the liver, which various biotech companies have taken advantage of to develop new experimental treatments for liver disease.

In their recent study, scientists set out to adapt the nanoparticles so that they could reach the bone marrow which contains stem cells that produce different types of blood cells. Stimulating the process , they could enhance the yield of hematopoietic stem cells for stem cell transplantation and they created variants that have different arrangements of surface coating, polyethylene glycol. They were able to test 15 particles and determined one that was able to avoid being caught in the liver or the lungs, and that could effectively accumulate in endothelial cells of the bone marrow. They also showed that RNA carried by this particle could reduce the expression of a target gene by up to 80 percent.

The scientists then tested this approach with two genes. The first gene, SDF1 is a molecule that normally prevents hematopoietic stem cells from leaving the bone marrow. They realized that turning off the SDF1 gene could have the same effect as the drugs that are being used to induce hematopoietic stem cell release in patients who need undergo radiation treatments for blood cancers. These stem cells are later transplanted to repopulate the patient’s blood cells. By knocking down SDF1, they could boost the release of hematopoietic cells fivefold which is comparable to the levels achieved by the drugs that are now used to enhance stem cell release.

The second gene researchers use is MCP1, a molecule that plays a key role in heart disease.  They realized that when MCP1 is released by bone marrow cells after a heart attack, it stimulates a flood of immune cells to leave the bone marrow and travel to the heart. Researchers realize that by delivering RNA that targets MCP1 reduced the number of immune cells that went to the heart after a heart attack.

Using these new particles, researchers hypothesized that they could further develop treatments for heart disease and other conditions.



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long noncoding RNAs

Larry H. Bernstein, MD, FCAP, Curator


UPDATED 3/17/2020

What are lncRNAs?

Advances in RNA sequencing technologies have revealed the complexity of our genome. Non-coding RNAs make up the majority (98%) of the transcriptome, and several different classes of regulatory RNA with important functions are being discovered. Understanding the significance of this RNA world is one of the most important challenges facing biology today, and the non-coding RNAs within it represent a gold mine of potential new biomarkers and drug targets.
lncRNA sequences

Long non-coding RNAs (lncRNAs) are a large and diverse class of transcribed RNA molecules with a length of more than 200 nucleotides that do not encode proteins (or lack > 100 amino acid open reading frame). lncRNAs are thought to encompass nearly 30,000 different transcripts in humans, hence lncRNA transcripts account for the major part of the non-coding transcriptome. lncRNA discovery is still at a preliminary stage. There are many specialized lncRNA databases, which are organized and centralized throughRNAcentral.

lncRNAs can be transcribed as whole or partial natural antisense transcripts (NAT) to coding genes, or located between genes or within introns. Some lncRNAs originate from pseudogenes (Milligan & Lipovich, 2015). lncRNAs may be classified into different subtypes (Antisense, Intergenic, Overlapping, Intronic, Bidirectional, and Processed) according to the position and direction of transcription in relation to other genes (Peschansky & Wahlestedt, 2014, Mattick & Rinn, 2015).
lncRNA expression

Gene expression profiling and in situ hybridization studies have revealed that lncRNA expression is developmentally regulated, can be tissue- and cell-type specific, and can vary spatially, temporally, or in response to stimuli. Many lncRNAs are expressed in a more tissue-specific fashion and with greater variation between tissues compared to protein-coding genes (Derrien et al., 2012).

In general, the expression level of lncRNA is at least one order of magnitude below that of mRNA. Many lncRNAs are located exclusively in the nucleus, but some are cytoplasmic or are located in both nucleus and cytoplasm.
lncRNA functions

To date, very few lncRNAs have been characterized in detail. However, it is clear that lncRNAs are important regulators of gene expression, and lncRNAs are thought to have a wide range of functions in cellular and developmental processes. lncRNAs may carry out both gene inhibition and gene activation through a range of diverse mechanisms, adding yet another layer of complexity to our understanding of genomic regulation. It is estimated that 25 – 40% of coding genes have overlapping antisense transcription, so the impact of lncRNAs on gene regulation is not to be underestimated.

Figure 1


Overview of some of the functions of long non-coding RNA. (Click for a larger image) lncRNAs are involved in gene regulation through a variety of mechanisms. The process of transcription of the lncRNA itself can be a marker of transcription and the resulting lncRNA can function in transcriptional regulation or in chromatin modification (usually via DNA and protein interactions) both in cis and in trans. lncRNAs can bind to complementary RNA and affect RNA processing, turnover or localization. The interaction of lncRNA with proteins can affect protein function and localization as well as facilitate formation of riboprotein complexes. Some lncRNAs are actually precursors for smaller regulatory RNAs such as microRNAs or piwi RNAs. Figure modified from Wilusz et al. Genes Dev. 2009. 23: 1494-1504. PMID: 19571179.


lncRNA mechanisms of gene regulation

lncRNAs are not defined by a common mode of action, and can regulate gene expression and protein synthesis in a number of different ways (Figure 1). Some lncRNAs are relatively highly expressed, and appear to function as scaffolds for specialized subnuclear domains. lncRNA possess secondary structures which facilitate their interactions with DNA, RNA and proteins. lncRNA may also bind to DNA or RNA in a sequence-specific manner. Gene regulation may occur in cis (e.g. in close proximity to the transcribed lncRNA) or in trans (at a distance from the transcription site). In the case of chromatin modulation, the effect of lncRNA is typically gene-specific, exerted at a local level (in cis) however regulation of chromatin can also occur in trans.

A few lncRNAs have had their functions experimentally defined and have been shown to be involved in fundamental processes of gene regulation including:

  • Chromatin modification and structure
  • Direct transcriptional regulation
  • Regulation of RNA processing events such as splicing, editing, localization, translation and turnover/degradation
  • Post-translational regulation of protein activity and localization
  • Facilitation of ribonucleoprotein (RNP) complex formation
  • Modulation of microRNA regulation
  • Gene silencing through production of endogenous siRNA (endo-siRNA)
  • Regulation of genomic imprinting

It has recently been attempted to categorize the various types of molecular mechanisms that may be involved in lncRNA function. lncRNAs may be defined as one or more of the following five archetypes:

  • The Signal archetype: functions as a molecular signal or indicator of transcriptional activity.
  • The Decoy archetype: binds to and titrates away other regulatory RNAs (e.g. microRNAs) or proteins (e.g. transcription factors).
  • The Guide archetype: directs the localization of ribonucleoprotein complexes to specific targets (e.g. chromatin modification enzymes are recruited to DNA).
  • The Scaffold archetype: has a structural role as platform upon which relevant molecular components (proteins and or RNA) can be assembled into a complex or spatial proximity.
  • The Enhancer archetype: controls higher order chromosomal looping in an enhancer-like model.

lncRNA and disease

With such a wide range of functions, it is not surprising that lncRNA play a role in the development and pathophysiology of disease. Interestingly, genome wide association studies have demonstrated that most disease variants are located outside of protein-coding genes.

lncRNAs have been found to be differentially expressed in various types of cancer including leukemia, breast cancer, hepatocellular carcinoma, colon cancer, and prostate cancer. Key oncogenes and tumor suppressors including PTEN and KRAS are now known to be regulated by corresponding lncRNA pseudogenes which also act as competing endogenous RNAs (ceRNAs) or microRNA sponges (Poliseno et al., 2010, Johnsson et al., 2013). This highlights the important role that lncRNAs play in oncogenesis.

Other diseases where lncRNAs are dysregulated include cardiovascular diseases, neurological disorders and immune-mediated diseases and genetic disorders. One of the first lncRNA to be discovered was the Xist lncRNA which plays an important role in X chromosome inactivation (Penny et al., 1996), an extreme case of genomic imprinting. lncRNAs are present at almost all imprinted loci, arguing for an important role for lncRNAs in this form of epigenetic regulation.

lncRNAs represent a gold mine of potential new biomarkers and drug targets, as well as a step change in the way we understand mechanisms of disease.
The challenges of studying lncRNA

Only a relatively small proportion of lncRNAs have so far been investigated and although we can start to classify different types of lncRNA functions, we are still far from being able to predict the function of new lncRNAs. This is mainly due to the fact that unlike protein-coding genes whose sequence motifs are indicative of their function, lncRNA sequences are not usually conserved and they don’t tend to contain conserved motifs. Other differences between lncRNA and mRNA are summarized in Table 1.

The main challenges of working with lncRNA are the fact that they can be present in very low amounts (typically an order of magnitude lower than mRNA expression levels), can overlap with coding transcripts on both strands and are often restricted to the nucleus.

Table 1

Tissue-specific expression Tissue-specific expression
Form secondary structure Form secondary structure
Undergo post-transcriptional processing, i.e. 5’cap, polyadenylation, splicing Undergo post-transcriptional processing, i.e. 5’cap, polyadenylation, splicing
Important roles in diseases and development Important roles in diseases and development
Protein coding transcript Non-protein coding, regulatory functions
Well conserved between species Poorly conserved between species
Present in both nucleus and cytoplasm Many predominantly nuclear, others nuclear and/or cytoplasmic
Total 20-24,000 mRNAs Currently ~30,000 lncRNA transcripts, predicted 3-100 fold of mRNA in number
Expression level: low to high Expression level: very low to moderate

Similarities and differences (dark) between mRNA and lncRNA


ncRNA discovery and profiling using Next Generation Sequencing

Expression profiling is one way to start to uncover the function of lncRNA. Identifying lncRNAs that are differentially expressed during development or in particular situations can shed light on their potential functions. Alternatively, looking for lncRNAs and protein-coding genes whose expression is correlated, can perhaps indicate co-regulation or related functions.

Whole transcriptome RNA sequencing is the method of choice for comprehensive lncRNA expression profiling, including the discovery of novel lncRNAs. Whole transcriptome sequencing enables the characterization of all RNA transcripts, including both the coding mRNA and non-coding RNA larger than 170 nucleotides in length, regardless of whether they are polyadenylated or not.

Exiqon offers a comprehensive whole transcriptome NGS Service including everything from RNA isolation to the final report including advanced data analysis and interpretation.
Advantages of LNA™-enhanced research tools for lncRNA

Exiqon offer a broad range of sensitive and specific tools specifically designed to address the challenges faced when investigating lncRNA expression and function. Exiqon’s tools are based on the Locked nucleic acid (LNA™) technology. LNA™ is a class of high-affinity RNA analogs that exhibit unprecedented thermal stability when hybridized to a complementary DNA or RNA strand. Hence, LNA™ enables superior sensitivity and specificity in any hybridization-based approach. We continue to use the LNA™ technology to develop new and innovative ways to improve our understanding of lncRNAs in this rapidly developing field.

Functional analysis of lncRNAs has been revolutionized by the development of Antisense LNA™ GapmeRs which enable efficient silencing of lncRNA both in vitro and in vivo. Exiqon also offer tools to investigate lncRNA function in other ways, for example using LNA™ oligos to block interactions between lncRNA and DNA, RNA or proteins. ExiLERATE LNA™ qPCR assays have been developed to enable robust detection of even low abundance and challenging lncRNAs by qPCR. Precise subcellular localization of lncRNAs can be studied using LNA™ probes for in situ hybridization.
Silencing lncRNA to disrupt their function

One strategy to study the function of lncRNA is to silence them using specific and potent antisense oligonucleotides (Antisense LNA™ GapmeRs).

The nuclear localization of many lncRNAs has meant that siRNA approaches to knockdown lncRNA, have met with limited success. The double-stranded siRNA duplex has difficulty crossing the nuclear membrane and the passenger strand (non-targeting sequence) of the duplex can often elicit its own effect, confounding interpretation of results.

Antisense LNA™ GapmeRs overcome this challenge by enabling highly efficient RNase H mediated silencing of all lncRNA. RNase H is present both in the cytoplasm and in the nucleus and it has been shown that LNA™ Gapmers offer significantly better knockdown of nuclear targets than siRNA mediated silencing. In addition, the single stranded LNA™ GapmeRs are an advantage for lncRNAs that are transcribed as antisense transcripts to coding genes because there is no second strand that could compromise specificity.

Antisense LNA™ GapmeRs show high potency in a broad range of tissues in vivo when administered systemically without formulation in animal models. This makes LNA™ GapmeRs very promising antisense drugs for lncRNA targets in the future.
Studying lncRNA interactions with DNA, RNA and proteins

The fact that the molecular mechanism of lncRNAs often relies on sequence specific interaction with DNA, RNA or proteins means that it is possible to design highly specific LNA™-oligonucleotides that can be used to inhibit these interactions and thereby reveal the details of how lncRNAs function. Please contact us and our experts can help you with the design of custom LNA™ oligonucleotides for studying lncRNA interactions.
lncRNA analysis by qPCR

Short, high affinity, LNA™-enhanced qPCR primers offer an advantage for the detection of low abundance targets. In addition, the use of LNA™ to adjust primer melting temperature provides greater flexibility in primer design which is important for qPCR analysis of overlapping transcripts. The ExiLERATE LNA™ qPCR System offers a sophisticated primer design tool combined with highly sensitive and specific qPCR assays for any RNA target.

ExiLERATE LNA™ qPCR assays are ideal to monitor the efficiency of LNA™ GapmeR-mediated RNA knockdown. Validated LNA™ qPCR primer sets are available to detect the lncRNA targeted by Antisense LNA™ GapmeR positive controls.

ExiLERATE LNA™ qPCR primer sets also provide a convenient way to validate RNA sequencing data. Our advanced online design algorithm can design LNA™ qPCR assays for novel lncRNA transcripts, isoforms or splice variants. LNA™qPCR assays for multiple lncRNAs can easily be designed using the batch mode function in our online design algorithm.
Subcellular localization of lncRNA expression by in situ hybridization

Understanding the subcellular localization of a lncRNA is important information when starting to hypothesize the potential functions that the lncRNA may be performing. LNA™-enhanced probes for in situ hybridization have increased affinity for their target sequence and offer increased sensitivity and increased signal to noise ratio, which is important for detection of rare targets such as lncRNA.


UPDATED 3/17/2020

From the journal Science :

Coding function of “noncoding” RNAs

Lian-Huan WeiJunjie U. Guo

Science  06 Mar 2020:
Vol. 367, Issue 6482, pp. 1074-1075
DOI: 10.1126/science.aba6117



High-throughput RNA sequencing studies have revealed pervasive transcription of the human genome, which generates a variety of long noncoding RNAs (lncRNAs) that have no apparent protein-coding functions (1). Subsequent studies that globally monitor translation have similarly identified numerous translation events outside of canonical protein-coding sequences (24), suggesting pervasive translation of the transcriptome. However, only a few examples of functional peptides encoded by RNA regions previously thought to be noncoding have been reported to regulate distinct biological processes (59). On page 1140 of this issue, Chen et al. (10) provide evidence for an expanded repertoire of functional peptides encoded by lncRNAs and other “untranslated” RNA regions.

The researchers sequenced ribosome-protected messenger RNA fragments (RFPs) to identify global translated open reading frames (ORFs).  RFPs are considered noncanonical ORFs and found in many lncRNAs and untranslated regions of RNA.  Therefore Chen et al. used genome-wide loss of function screens to assess noncanonical ORFs affect on cell growth.  They filtered RFPs obtained from several human cell types and identified over 5000 previously unannotated ORFs which also include many variants if canonical ORFs, upstream ORFs in 5′ UTRs, and ORFs within transcripts that were annotated as lncRNAs.

Using CRISPR-Cas9, Chen et al. disrupted 2353 unannotated ORFs and identified over 400 RFPs that promoted cell growth in human  leukemic cells and stem cells.  However there were only a few lncRNAs, when disrupted, showed consistent effects on growth suggesting noncoding functions of the remaining lncRNA loci.  Many of the lncRNA ORFs encoded for small peptides.  These microproteins present a challenge to identify such proteins that don’t have much evolutionary conservation.

This noncanonical translation has been linked to many neurological diseases such as short tandem repeats diseases such as fragile X sydrome and other polyglutamate diseases such as Huntington’s Chorea.

References cited within this paper include

1. I. Ulitsky, D. P. Bartel, Cell154, 26 (2013).
2. A. A. Bazzini et al., EMBO J. 33, 981 (2014).
3. N.T. Ingolia et al., Cell Rep. 8, 1365 (2014).
4. Z. Ji, R. Song, A. Regev, K. Struhl, eLife 4, e08890 (2015).
5. D. M. Anderson et al., Cell160, 595 (2015).
6. T. Kondo et al., Nat. Cell Biol. 9, 660 (2007).
7. A. Pauli et al., Science 343, 1248636 (2014).
8. E. G. Magny et al., Science 341, 1116 (2013).
9. S. R. Starck et al., Science 351, aad3867 (2016).
10. J. Chen et al., Science 367, 1140 (2020).
11. M. Guttman, P. Russell, N. T. Ingolia, J. S. Weissman, E. S. Lander, Cell154, 240 (2013).
12. T.G. Johnstone, A. A. Bazzini, A. J. Giraldez, EMBO J. 35, 706 (2016).
13. W. F. Doolittle, T. D. Brunet, S. Linquist, T. R. Gregory,
Genome Biol.Evol. 6, 1234 (2014).
14. F. B. Gao, J. D. Richter, D. W. Cleveland, Cell171, 994 (2017).
15. M. G. Kearse et al., Mol. Cell 62, 314 (2016).

Other articles of note on lncRNAs on this Online Open Access Journal Include

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Reporter: Aviva Lev-Ari, PhD,RN


Functional Genomics Screening Strategies: Part One

Utilizing RNA Interference (RNAi) Screens

to Explore Drug Targets and Cellular Pathways

Boston, MA | September 24-25, 2013

Dr. Scott Martin, Team Leader for RNAi Screening at NIH’s Chemical Genomics Center, to Present “Swimming in the Deep End – Sources Leading to a False Sense of Security in RNAi Screen Data” at Functional Genomics Screening Strategies Conference

There has been a growing skepticism surrounding RNAi data and the validity of hits arising from largescale RNAi screens. Much of this comes from a lack of agreement between screens conducted in similar biological systems and difficulty in validating published screen hits. In light of these realities, we must rethink some widely held beliefs about screening and validation strategies. These issues and relevant data will be discussed.


Functional Genomics Screening Strategies: Part Two

Exploring Novel Screening Platforms and Cellular

Models for Next-Generation Screens

Boston, MA | September 25-26, 2013

The second half of Functional Genomics Screening Strategies will explore the use of chemical genomics screens, microRNA (miRNA) and long non-coding RNA (lncRNA) screens and the transition into advanced cellular models such as, 3D cell cultures, co-cultures and stem cells that will launch the next generation of functional screens. Screening experts from pharma/biotech as well as from academic and government labs will share their experiences leveraging the utility of such diverse screening platforms and models for a wide range of applications.



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Reporter: Aviva Lev-Ari, PhD, RN

September 24 – 26, 2013

Westin Boston Waterfront

Boston, MA

About the Functional Genomics Screening Event:

In the screening world there is definitely no one-size fits- all and no dearth of options to choose from in terms of assay platforms, protocols, cells or reagents. So how do you decide which screening strategy will work best for you? Can different screening techniques be utilized in tandem or be staggered to better validate results and overcome inherent shortcomings? Which type of screen will provide information that is most accurate and physiologically relevant to your biological query? Cambridge Healthtech Institute’s tenth annual conference on Functional Genomics Screening Strategies will cover the latest in the use of RNA interference (RNAi) screens, combination (RNAi and small molecule) screens, chemical genomics and phenotypic screens, for identifying and validating new drug targets and exploring unknown cellular pathways. The first half of the conference will focus on the design and use of RNAi screens, while the second half will explore the use of chemical genomics and long non-coding RNA (LncRNA) screens and the transition into advanced cellular models such as, 3D cell cultures and stem cells that will launch the next-generation of functional screens. Screening experts from pharma/biotech as well as from academic and government labs will share their experiences leveraging the utility of such diverse screening platforms and models for a wide range of applications


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Discovery on Target: Industry’s Preeminent Event on Novel Drug Targets 

Reporter: Aviva Lev-Ari, PhD, RN



Cambridge Healthtech Institute’s tenth annual conference on Functional Genomics Screening Strategies will cover the latest in the use of RNA interference (RNAi) screens, combination (RNAi and small molecule) screens, chemical genomics and phenotypic screens, for identifying and validating new drug targets and exploring unknown cellular pathways. The first half of the conference will focus on the design and use of RNAi screens, while the second half will explore the use of chemical genomics screens, microRNA (miRNA) and long non-coding RNA (LncRNA) screens and the transition into advanced cellular models such as, 3D cell cultures, co-cultures and stem cells that will launch the next-generation of functional screens. Screening experts from pharma/biotech as well as from academic and government labs will share their experiences leveraging the utility of such diverse screening platforms and models for a wide range of applications.


September 23: Setting Up Effective RNAi Screens: Getting From Design to Data Short Course
September 24 – 25: Functional Genomic Screening Strategies Conference Part One
September 25: Setting Up Effective Functional Screens Using 3D Cell Cultures Dinner Short Course
September 25 – 26: Functional Genomic Screening Strategies Conference Part Two



Comparative Analysis of Arrayed RNAi Screening Performance of siRNA versus shRNA at Genome-Scale

Hakim Djaballah, Ph.D., Director, HTS Core Facility, Molecular Pharmacology and Chemistry Program, Memorial Sloan Kettering Cancer Center


RNAi Screening: Strategies, Examples and Outcomes

David Root, Ph.D., Director, RNAi Platform and Project Leader, The RNAi Consortium, The Broad Institute of MIT and Harvard


Swimming in the Deep End – Sources Leading to a False Sense of Security in RNAi Screen Data

Scott Martin, Ph.D., Team Leader, RNAi Screening, NIH Chemical Genomics Center, NIH Center for Translational Therapeutics, National Institutes for Health


Rebuilding the RNAi Screen

Eugen Buehler, Ph.D., Group Leader, Informatics, National Center for Advancing Translational Sciences, National Institutes of Health


Genetic Strategies for Investigating Host-Virus Interactions

Abraham Brass, M.D., Ph.D., Assistant Professor, Department of Microbiology and Physiology Systems, University of Massachusetts Medical School


PANEL DISCUSSION: Advanced RNAi Screening: Strengths, Caveats and Pitfalls at Reaching the 14-Year Milestone

Moderator: Christophe Echeverri, Ph.D., CEO & CSO, Cenix BioScience USA, Inc.


Caroline Shamu, Ph.D., Director, ICCB-Longwood Screening Facility, Harvard Medical School

David Root, Ph.D., Director, RNAi Platform and Project Leader, The Broad Institute

Hakim Djaballah, Ph.D., Director, HTS Core Facility, Memorial Sloan Kettering Cancer Center

Scott Martin, Ph.D., Team Leader, RNAi Screening, NIH Chemical Genomics Center




RNAi Screening to Enable Translational R&D For Oncology and Immuno-Oncology Target Discovery

Namjin Chung, Ph.D., Senior Research Investigator, Applied Genomics, Bristol-Myers Squibb Co.


Target Identification and Validation of Novel Ion Channels in Cancer

Alex Gaither, Ph.D., Research Investigator II, Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research


Cell-Based Functional Profiling of Lipid-Traits and Cardiovascular Disease

Heiko Runz, M.D., Group Leader, Molecular Metabolic Disease Unit, Institute of Human Genetics; Group Leader, University of Heidelberg


Use of Functional Genomics to Identify Patients at High Risk for Recurrence of Hepatitis C Following Liver Transplantation

Robert Carithers, M.D., Professor of Medicine, Director, Liver Care Line; Medical Director, Liver Transplant Program, University of Washington Medical Center

CellectaPooled RNAi Genetic Screening to Identify Functional Genes and Novel Drug Targets

Paul Diehl, Director, Business Development, Cellecta, Inc.


TECHNOLOGY PANEL: Tools for Next-Generation Functional Genomics Screens

Moderator: Christophe Echeverri, Ph.D., CEO & CSO, Cenix BioScience USA, Inc.

This panel will bring together 4-5 technical experts from leading technology and service companies to discuss screening trends and improvements in assay platforms and reagents that users can expect to see in the near future.

(Opportunities Available for Sponsoring Panelists)




siRNA Screening and RNA-seq for Identification of Targets for the Treatment of Alzheimer’s Disease

Paul Kassner, Ph.D., Director, Research, Amgen, Inc.


Fusing RNAi Screening and Gene Expression Analyses to Reveal Pathway Responses

Alexander Bishop, Ph.D., Assistant Professor, Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio




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The Development of siRNA-Based Therapies for Cancer

Author: Ziv Raviv, PhD


The use of gene regulation technology in research and medicine had evolved rapidly since the discovery of post transcriptional gene silencing using RNA interference (RNAi). RNAi was first described in C. elegance in the 90s of the previous century. RNAi post transcriptional gene regulation is carried out by small non-coding RNA double strand RNA (dsRNA) molecules such as microRNA (miRNA; miR) and small interference RNA (siRNA), and has an important role in defending cells against parasitic nucleotide sequences (e.g. viruses) as well as in gene expression regulation.

In RNAi-mediated gene regulation, short dsRNA molecules are being transcribed in the nucleus (in the case of miRs) or introduced exogenously into the cell (in the case of synthetic siRNA or viruses), and are processed in the cytoplasm by an enzyme called Dicer that cleaves long dsRNA and pre-microRNA to produce short double-stranded RNA fragments of 21 base pairs long. The 21 nucleotides long double strand RNA is then being incorporated into the RNA-induced silencing complex (RISC) where it is unwound into two single strands RNA (ssRNA). The “guide” strand is then paired with its complementary targeted messenger RNA (mRNA) that is subsequently cleaved by Argonaute RISC-associated endonuclease. Consequently, the targeted gene protein expression is blocked, leading to its substantial reduced levels in the cell. This so called gene silencing or gene knockdown, hitting the message not the gene itself, will last as long as RNAi molecules are present. The mechanism of action of RNAi is illustrated in the following Video.

RNAi technology was then massively adapted for research allowing the evaluation of functional involvement of genes in various cellular processes because introducing synthetic siRNA into cells can selectively suppress any specific gene of interest.  Not only that RNAi serves as a valuable research tool both in cell culture and in vivo, RNAi has an extremely high potential for specific gene-targeting therapy, as many diseases consist gene deregulation. Synthetic siRNAs are perfectly and completely base pairing to a target (in contrast to endogenous miRs), leading to mRNA-induced cleavage in a single-specific manner that allows treatment without non-specific off-target side effects.

RNAi as therapeutic tool for cancer

All malignant conditions consist of gene deregulations in the form of mutations causing protein misfunction that lead to loss of cell growth regulation and consequently to cancer. Therefore, the fact that siRNA can selectively and specifically target any gene of interest creates a powerful tool to downregulate cancer-associated genes, that eventually will lead to a decrease and even abolishment of the malignant condition.

The advantages of using siRNA for therapy thus are:

  • RNAi technology represents a 3rd revolutionary step for pharmaceutics after small molecules and monoclonal antibodies (mAb), and has a strong commercial potential similar to mAb and even beyond.
  • The ability to target any gene of interest, by blocking specifically the message from DNA to protein consequently the protein is not allowed to be expressed and thus is not functioning.
  • Specificity – siRNA have strong potential to bind specifically to target mRNA, thus lowering unwanted side effects.
  • siRNAs are double stranded oligonucleotides, which are resistant to nucleases.
  • Fast pre-clinical development

General considerations for developing anti-cancer RNAi-based treatment

Given the great potential of siRNA as a therapeutic tool for cancer, one should bring into consideration some general aspects for the development of a siRNA anti-cancer drug:

  • Choosing the gene of interest to be silenced – A wide spectrum of genes could be considered as targets based upon gene of interest role in the cancer cell, type of cancer, and condition of the disease: (i) Oncogenes or central signaling molecules that are crucial for cancer cell growth (ii) Anti-apoptotic deregulated genes (iii) Cancer metabolism associated genes (iv) Angiogenic related genes (v) Metastatic condition related genes.
  • Considering the option of hitting combined target genes consist of different functions (e.g. an oncogene and an anti-apoptotic gene).
  • Basic research evaluation – To examine the effect of silencing the gene of interest in cancer cell based assays and in animal models.
  • Chemical modifications of the siRNA molecule – Modifications such as 2′OMe to increase protection from nuclease, decrease the immunogenicity, lower the incidence of off-target effects, and improve pharmacodynamics of the siRNA.
  • Drug delivery formulation – For an efficient transport of the siRNA. Such delivery system could be formulated using liposome-based nanoparticles (NP) or other nanocarriers to facilitate the siRNA effective systemic distribution.
  • PEGylation – PEGylation of the NPs carriers to reduce non-specific tissue interactions, increase serum stability and half life, and reduce immunogenicity of the siRNA molecule.
  • Site specific targeting – Target tissue-specific distribution of the siRNA drug could be performed by attaching on the outer surface of the nanocarrier a ligand that will direct the siRNA drug to the tumor site.
  • Preclinical – Efficiency and validity, as well as toxicity and pharmacokinetic studies for the siRNA-transporter formulation should be evaluated in animal models.
  • Personalized treatment – In first stages clinical trials, biomarkers should be developed and detected to direct the selection criteria for further treatment of patients with the selected siRNA.
  • Combined therapy – Conduct clinical trials using a combination of the siRNA drug together with a chemotherapy drug that is in-clinical use. Such combined therapy can result in synergism actions of the two combined drugs, and could lower the dosage and thus the side effects of the drugs. In addition, the use of established contemporary agents has practical industrial-related advantages as it is much easier to introduce a new mode of treatment on the background of an existing one.

Development of transport methods for siRNA

As mentioned above, an important aspect in applying siRNA-based therapy is the development of a suitable delivery method that should carry the siRNA molecule systemically to the site of the tumor. In addition, the siRNA-transporter formulation should provide protection from serum nucleases to the siRNA and should decrease its immunogenicity by blocking response of the innate immune system. Examples of such NPs are illustrated in Figure 1. Indeed, several clinical trials were conducted to evaluate the efficacy, validity, and safety use of such transporters for clinical use (Table I).

Figure 1: Various types of nanoparticles for siRNA delivery

Taken from: Cho K et al. Clin Cancer Res 2008;14:1310-1316

Table IClinical trials examining siRNA delivery methods

T1Click on table to enlarge

Table resources: nmOK drug database and clinicaltrials.gov

Download table with active links: Development of siRNA-Based Therapies for Cancer_Table I

Current development status of RNAi-based cancer therapies  

The potential use of RNAi technology to treat cancer is versatile as for any gene of interest it is easy to synthesize a siRNA molecule and the pre-clinical development of siRNA agent is fast. Several companies specialized in siRNA technology have begun recently developing RNAi-based therapies to various cancer associated genes (as well as to other diseases) and to conduct clinical trials. Table II summaries the current clinical trials status of such siRNA-based anti-cancer agents.

Table II: Current clinical trials of siRNA therapies for cancer

T2Click on table to enlarge

Table resources: nmOK drug database, clinicaltrials.gov, and World Health Organization (WHO)

Download table with active links: Development of siRNA-Based Therapies for Cancer_Table II

Conclusion remarks

The power of siRNA-based therapeutics resides in the ability to target and silence any desired gene. Pharmaceutical and biotech companies have started to conduct clinical trials of siRNA therapies for cancer. Most of these clinical trials are in the early preclinical and phase I stages. The results expected from these experiments should further direct the development of siRNA-based anti-cancer therapies and phase II and III trials should consequently emerge. Other target genes should be evaluated as well for siRNA-anti cancer therapy in addition to those that are currently in evaluation, and accelerated efforts should be made in the direction of combining existing chemotherapy with the technology of siRNA. The next future to come will tell us if the potential of siRNA therapy for cancer had been fulfilled.

Related references:

  1. RNAi-Based Therapies for Cancer in Development. Anna Azvolinsky, PhD. Cancernetwork, March 3, 2011.
  2. siRNA-based approaches in cancer therapy. GR Devi. Cancer Gene Therapy (2006) 13, 819–829
  3. Therapeutic Effect of RNAi Gene Silencing Effective in Cancer Treatment, Study Suggests. Sciencedaily, Feb. 11, 2013.
  4. Kinesin Spindle Protein SiRNA Slows Tumor Progression. Marra E, Palombo F, Ciliberto G, Aurisicchio L. J Cell Physiol. 2013 Jan;228(1):58-64.
  5. First-in-Humans Trial of an RNA Interference Therapeutic Targeting VEGF and KSP in Cancer Patients with Liver Involvement. Josep Tabernero et al. Cancer Discov. 2013 Apr;3(4):406-417.

Chemical modification:

  1. Chemical Modification of siRNAs for In Vivo Use. Behlke MA. Oligonucleotides. 2008 Dec; 18(4):305-19.

Delivery Technology:

  1. Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle. Schiffelers RM et al. Nucleic Acids Res. 2004 Nov 1;32(19):e149.
  2. Therapeutic Nanoparticles for Drug Delivery in Cancer.  Kwangjae Cho, Xu Wang, Shuming Nie, et al. Clin Cancer Res 2008;14:1310-1316.
  3. Liposomes and nanoparticles: nanosized vehicles for drug delivery in cancer. Malam Y, Loizidou M, Seifalian AM. Trends Pharmacol Sci. 2009 Nov; 30(11):592-9.
  4. Silence-therapeutics delivery platform

Related articles on this Open Access Online Scientific Journal:

  1. MIT Team: Microfluidic-based approach – A Vectorless delivery of Functional siRNAs into Cells. Reporter: Aviva Lev-Ari, Ph.D., RN
  2. Targeted Tumor-Penetrating siRNA Nanocomplexes for Credentialing the Ovarian Cancer Oncogene ID4. Reporter and Curator: Sudipta Saha, Ph.D.
  3. Targeted delivery of therapeutics to bone and connective tissues: current status and challenges- Part II. Curator and Reporter: Aviral Vatsa Ph.D., MBBS
  4. Nanotechnology and HIV/AIDS treatment. Author: Tilda Barliya, PhD

To download tables of this post (with active links) :

  1. Development of siRNA-Based Therapies for Cancer_Table I
  2. Development of siRNA-Based Therapies for Cancer_Table II





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