Posts Tagged ‘Small interfering RNA’

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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DNA Nanotechnology

Author: Tilda Barliya PhD

The field of DNA and RNA nanotechnologies  are considered one of the most dynamic research areas in the field of drug delivery in molecular medicine. Both DNA and RNA have a wide aspect of medical application including: drug deliveries, for genetic immunization, for metabolite and nucleic acid detection, gene regulation, siRNA delivery for cancer treatment (I), and even analytical and therapeutic applications.

Seeman (6,7) pioneered the concept 30 years ago of using DNA as a material for creating nanostructures; this has led to an explosion of knowledge in the now well-established field of DNA nanotechnology. The unique properties in terms of free energy, folding, noncanonical base-pairing, base-stacking, in vivo transcription and processing that distinguish RNA from DNA provides sufficient rationale to regard RNA nanotechnology as its own technological discipline. Herein, we will discuss the advantages of DNA nanotechnology and it’s use in medicine.

So What is the rational of using DNA nanotechnology(3)?

  • Genetic studies – its application in various biological fields like biomedicine, cancer research, medical devices  and genetic engineering.
  • Its unique properties of structural stability, programmability of sequences, and predictable self-assembly.
DNA origami

Structures made from DNA using the DNA-origami method (Rothemund, 2006)

Structural DNA nanotechnology rests on three pillars: [1] Hybridization; [2] Stably branched DNA; and [3] Convenient synthesis of designed sequences.


Hybridization. The self-association (self=assembly) of complementary nucleic acid molecules or parts of molecules, is implicit in all aspects of structural DNA nanotechnology. Individual motifs are formed by the hybridization of strands designed to produce particular topological species. A key aspect of hybridization is the use of sticky ended cohesion to combine pieces of linear duplex DNA; this has been a fundamental component of genetic engineering for over 35 years (7). Not only is hybridization critical to the formation of structure, but it is deeply involved in almost all the sequence-dependent nanomechanical devices that have been constructed, and it is central to many attempts to build structural motifs in a sequential fashion (7,8 ).

Stably Branched DNA

branched DNA molecules are central to DNA nanotechnology. It is the combination of in vitro hybridization and synthetic branched DNA that leads to the ability to use DNA as a construction material. Such branched DNA is thought to be intermediates in genetic recombination (such as Holliday junctions).

Convenient Synthesis of Designed Sequences

Biologically derived branched DNA molecules, such as Holliday junctions, are inherently unstable, because they exhibit sequence symmetry; i.e., the four strands actually consist of two pairs of strands with the same sequence. This symmetry enables an isomerization known as branch migration that allows the branch point to relocate.  DNA nanotechnology entailed sequence design that attempted to minimize sequence symmetry in every way possible.

One of the most remarkable innovations in structural DNA-nanotechnology in recent years is DNA origami, which was invented in 2006 by Paul Rothemund (1) (see Fig above). DNA origami utilizes the genome from a virus together with a large number of shorter DNA strands to enable the creation of numerous DNA-based structures (Figure 1). The shorter DNA strands forces the long viral DNA to fold into a pattern that is defined by the interaction between the long and the short DNA strands (1,2).

Rothemund believes that an  application of patterned DNA origami would be the creation of a ‘nanobreadboard’, to which diverse components could be added. The attachment of proteins23, for example, might allow novel biological experiments aimed at modelling complex protein assemblies and examining the effects of spatial organization, whereas molecular electronic or plasmonic circuits might be created by attaching nanowires, carbon nanotubes or gold nanoparticles (1).

DNA nanotechnology and Biological Application

The physical and chemical properties of nanomaterials such as polymers, semiconductors, and metals present diverse advantages for various in vivo applications (3,9 ). For example:

  • Therapeutics – In cancer for example, nanosystems that are designed from biological materials such as DNA and RNA are ‘programmed’ to be able to evade most, if not all, drug-resistance mechanisms. Based on these properties, most nanosystems are able to deliver high concentrations of drugs to cancer cells while curtailing damage to surrounding healthy cells (2b, 3, 9, 11, 15).
  • Biosensors – capable of picking up very specific biological signals and converting them into electrical outputs that can be analyzed for identification. Biosensors are efficient as they have a high ratio of surface area to volume as well as adjustable electronic, magnetic, optical, and biological properties (3, 12, 13, 14).
  • **Amin and colleagues have developed a biotinylated DNA thin film-coated fiber optic reflectance biosensor for the detection of streptavidin aerosols. DNA thin films were prepared by dropping DNA samples into a polymer optical fiber which responded quickly to the specific biomolecules in the atmosphere. This approach of coating optical fibers with DNA nanostructures could be very useful in the future for detecting atmospheric bio-aerosols with high sensitivity and specificity (3, 14)
  • Computing – Another aspect uses the programmability of DNA to create devices that are capable of computing. Here, the structure of the assembled DNA is not of primary interest. Instead, control of the DNA sequence is used in the creation of computational algorithms, like e.g. artificial neural networks. Qian et al for example, built on the richness of DNA computing and strand displacement circuitry, they showed how molecular systems can exhibit autonomous brain-like behaviours. Using a simple DNA gate architecture that allows experimental scale-up of multilayer digital circuits, they systematically transform arbitrary linear threshold circuits (an artificial neural network model) into DNA strand displacement cascades that function as small neural networks (3, 10).
  • Additional features: 3rd generation DNA sequencers (II), Biomimetic systems, Energy transfer and photonics etc


DNA nanotechnology is an evolving field that affects medicine, computation, material sciences, and physics. DNA nanostructures offer unprecedented control over shape, size, mechanical flexibility and anisotropic surface  modification. Clearly, proper control over these aspects can increase  circulation times by orders of magnitude, as can be seen for longcirculating particles such as erythrocytes and various pathogenic particles evolved to overcome this issue.  The use of DNA in DNA/protein-based matrices makes these structures inherently amenable to structural tunability. More research in this direction  will certainly be developed, making DNA a promising biomaterial  in tissue engineering. future development of novel ways in which DNA would be utilized to have a much more comprehensive role in biological computation and data storage is envisaged.


1. Paul W. K. Rothemund. Folding DNA to create nanoscale shapes and patterns. NATURE 2006 (March 16)|Vol 440: 297-302. http://www.nature.com/nature/journal/v440/n7082/full/nature04586.html


2. Andre V. Pinheiro, Dongran Han, William M. Shih and Hao Yan. Challenges and opportunities for structural DNA nanotechnology. Nature Nanotechnology 2011 Dec | VOL 6: 763-772.  http://www.nature.com/nnano/journal/v6/n12/pdf/nnano.2011.187.pdf

2b. Thi Huyen La, Thi Thu Thuy Nguyen, Van Phuc Pham, Thi Minh Huyen Nguyen and Quang Huan Le.  Using DNA nanotechnology to produce a drug delivery system. Adv. Nat. Sci.: Nanosci. Nanotechnol. 4 (2013) 015002 (7pp). http://iopscience.iop.org/2043-6262/4/1/015002http://iopscience.iop.org/2043-6262/4/1/015002/pdf/2043-6262_4_1_015002.pdf

3. Muniza Zahid, Byeonghoon Kim, Rafaqat Hussain, Rashid Amin and Sung H Park. DNA nanotechnology: a future perspective. Nanoscale Research Letters 2013, 8:119. http://www.nanoscalereslett.com/content/8/1/119

4.By: Cientifica Ltd 2007. The Nanotech Revolution in Drug Delivery.  http://www.cientifica.com/WhitePapers/054_Drug%20Delivery%20White%20Paper.pdf

5. Gemma Campbell. Nanotechnology and its implications for the health of the E.U citizen: Diagnostics, drug discovery and drug delivery. Institute of Nanotechnology and Nanoforum. http://www.nano.org.uk/nanomednet/images/stories/Reports/diagnostics,%20drug%20discovery%20and%20drug%20delivery.pdf

6.Peixuan Guo., Haque F., Brent Hallahan, Randall Reif and Hui Li. Uniqueness, Advantages, Challenges, Solutions, and Perspectives in Therapeutics Applying RNA Nanotechnology. Nucleic Acid Ther. 2012 August; 22(4): 226–245. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3426230/

7. SEEMAN N.C. Nanomaterials based on DNA. Annu. Rev. Biochem. 2010;79:65–87. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3454582/

8. Yin P, Choi HMT, Calvert CR, Pierce NA. Programming biomolecular self-assembly pathways. Nature.2008;451:318–323.  http://www.ncbi.nlm.nih.gov/pubmed/18202654

9. Yan Lee P, Wong KY: Nanomedicine: a new frontier in cancer therapeutics. Curr Drug Deliv 2011, 8(3):245-253. OpenURLhttp://www.eurekaselect.com/73728/article

10. Qian, L.L., Winfree, E., and Bruck, J. Neural Network Computation with DNA Strand Displacement Cascades. Nature 2011 475, 368-372.  http://www.nature.com/nature/journal/v475/n7356/full/nature10262.html

11. Acharya S, Dilnawaz F, Sahoo SK: Targeted epidermal growth factor receptor nanoparticle bioconjugates for breast cancer therapy. Biomaterials 2009, 30(29):5737-5750. http://www.sciencedirect.com/science/article/pii/S0142961209006929

12. Bohunicky B, Mousa SA: Biosensors: the new wave in cancer diagnosisNanotechnology, Science and Applications 2011, 4:1-10. http://www.dovepress.com/biosensors-the-new-wave-in-cancer-diagnosis-peer-reviewed-article-NSA-recommendation1

13. Sanvicens N, Mannelli I, Salvador J, Valera E, Marco M: Biosensors for pharmaceuticals based on novel technologyTrends Anal Chem 2011, 30:541-553. http://www.sciencedirect.com/science/article/pii/S016599361100015X

14. Amin R, Kulkarni A, Kim T, Park SH: DNA thin film coated optical fiber biosensorCurr Appl Phys 2011, 12(3):841-845. http://www.sciencedirect.com/science/article/pii/S1567173911005888

15. Choi, Y.; Baker, J. R. Targeting Cancer Cells with DNA Assembled Dendrimers: A Mix and Match Strategy for Cancer. Cell Cycle 2005, 4, 669–671. http://www.ncbi.nlm.nih.gov/pubmed/15846063  http://www.landesbioscience.com/journals/cc/article/1684/

Other related articles on this Open Access Online Scientific Journal include the following

I. By: Ziv Raviv PhD. The Development of siRNA-Based Therapies for Cancer. http://pharmaceuticalintelligence.com/2013/05/09/the-development-of-sirna-based-therapies-for-cancer/

II. By: Tilda Barliya PhD. Nanotechnology, personalized medicine and DNA sequencing. http://pharmaceuticalintelligence.com/2013/01/09/nanotechnology-personalized-medicine-and-dna-sequencing/

III. By: Larry Bernstein MD FACP. DNA Sequencing Technology. http://pharmaceuticalintelligence.com/2013/03/03/dna-sequencing-technology/

IV. By: Venkat S Karra PhD. Measuring glucose without needle pricks: nano-sized biosensors made the test easy. http://pharmaceuticalintelligence.com/2012/09/04/measuring-glucose-without-needle-pricks-nano-sized-biosensors-made-the-test-easy/

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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





Related Videos:

RNA interference mechanism of action

RNA interference (RNAi): by Nature video

RNAi Therapeutics and Cancer Treatment

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Reporter: Aviva Lev-Ari, RN

With New Microfludic Technique, MIT Team Aims to ‘Squeeze’ siRNAs into Cells

January 31, 2013

Researchers from the Massachusetts Institute of Technology last week reported on the development of a new microfluidic-based approach to delivering macromolecules, including functional siRNAs, into cells without the need for a vector.

According to the investigators, who published their findings in the Proceedings of the National Academy of Sciences, the technique involves compressing cells by passing them through a constriction, which opens up temporary holes in their membranes that permit the diffusion of materials in surrounding buffer to enter the cytosol.

“By providing flexibility in application and obviating the need for exogenous materials or electrical fields, this method could potentially enable new avenues of disease research and treatment,” they wrote.

Although intracellular delivery of macromolecules is a key step in therapeutic and research applications, the cellular membrane is largely impermeable to such compounds, according to the PNAS paper. Existing methods to overcome this hurdle, which has proven to be a major stumbling block for RNAi drugs, typically involve the use of polymeric nanoparticles, liposomes, or chemical modifications of the target molecules to facilitate membrane poration or endocytotic delivery.

When it comes to nucleic acids, which are relatively structurally uniform, these approaches can be efficient. Still, the “endosome escape mechanism that most of these methods rely on is often inefficient; hence, much material remains trapped in endosomal and lysosomal vesicles,” the MIT team pointed out. “More effective gene delivery methods, such as viral vectors, however, often risk chromosomal integration.

Meantime, electroporation has proven effective, even in difficult to transfect primary cells, but has limited applicability and can cause cell death. Microinjection, too, has certain advantages in settings such as the creation of transgenic organisms, but its low throughput hamstrings many therapeutic and research applications, the researchers noted.

To overcome the limitations of existing delivery techniques, the MIT group had initially been attempting to “shoot” molecules of interest into cells, Armon Sharei, an MIT graduate student in chemical engineering and lead author of the PNAS paper, told Gene Silencing News.

“That system had its own challenges, and through the course of that work, we stumbled upon this effect where if you squeeze the cells rapidly enough, it will temporarily disrupt their membrane,” he said.

More specifically, the researchers found that the “rapid mechanical deformation of a cell, as it passes through a constriction with a minimum dimension smaller than the cell diameter, results in the formation of transient membrane disruptions or holes,” they wrote in PNAS. “The size and frequency of these holes would be a function of the shear and compressive forces experienced by the cell during its passage through the constriction. Material from the surrounding medium may then diffuse directly into the cell cytosol throughout the life span of these holes.”

To test this idea, the researchers constructed devices, each consisting of 45 identical, parallel microfluidic channels containing one or more constrictions, etched onto a silicon chip and sealed in glass. The width of each constriction ranged from 4 to 8 micrometers, and the lengths ranged from 10 to 40 micrometers.

“Before use, the device is first connected to a steel interface that connects the inlet and outlet reservoirs to the silicon device,” the researchers wrote. “A mixture of cells and the desired delivery material is then placed into the inlet reservoir and Teflon tubing is attached at the inlet. A pressure regulator is then used to adjust the pressure at the inlet reservoir and drive the cells through the device. Treated cells are collected from the outlet reservoir.”

The system was tested with a variety of molecules, including carbon nanotubes and proteins, as well as siRNAs targeting GFP. According to Sharei, when the siRNAs were delivered into GFP-expressing HeLa cells using the microfluidic platform, the investigators were able to achieve 80 to 90 percent target knockdown.

He noted that the knockdown effects weren’t as robust as with Lipofectamine 2000, but “we were still encouraged because something like Lipofectamine is known to be toxic and therefore inapplicable for humans.” Notably, the microfluidic device and operating parameters were not optimized for siRNAs, further limiting its ability to compete with the transfection reagent in these studies.

“The other good thing was that we seem to work just as well for primary cells, whereas existing methods like Lipofectamine don’t translate well once you start moving out of the standard cell models you have in the lab,” he added.

The MIT team also successfully delivered 3 kilodalton dextran molecules — which are approximately the same size as a standard siRNA molecule and a “pretty accurate” surrogate for the gene-silencing molecules — into newborn human foreskin fibroblasts, primary murine dendritic cells, and embryonic stem cells, suggesting that the method could be used with siRNAs into a variety of cell types, Sharei said.

Buoyed by the positive data, he and his colleagues are now further testing the platform with siRNAs against “easy readout genes” in primary cells including immune cells and stem cells, he said. “Once we establish that, we’d try to go for an application where there’s an siRNA that’s going to knock down something functional.

“I can’t say exactly what we’ve been up to because it’s not published, but it has been going pretty well,” he added.

Ultimately, the MIT group aims to develop the microfluidic platform not only for research applications, where it could be “incorporated into a larger integrated system consisting of multiple pre-treatment and post-treatment modules” that could take advantage of its average throughput rate of 20,000 cells a second, but also therapeutic ones, too.

A number of investigational stem cell-based therapies, for instance, involve the ex vivo manipulation of the cells, Sharei said. The delivery platform could theoretically be used to “enhance or facilitate that manipulation.”

“Such an approach would take advantage of the potentially increased delivery efficiency of therapeutic macro- molecules and could be safer than existing techniques because it would obviate the need for potentially toxic vector particles and would mitigate any potential side effects associated with reticuloendothelial clearance and off-target delivery,” the study authors wrote in PNAS.

Doug Macron is the editor of GenomeWeb’s Gene Silencing News. He covers research and therapeutic applications of RNAi, miRNA, and other gene-silencing technologies. E-mail Doug Macron or follow his GenomeWeb Twitter account at@Genesilencing.


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