RNAi and Databases

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Introduction to RNA interference
&
and computer tools used to design siRNAs with optimal potency
and specificity

by: Toumazos Theodorou
RNAi – History and Evolution

•Early 1990s unexpected results in
experiments with genetically modified
plants [1]

• Scientists recognized that certain genes
were down-regulated after “viral
infection” introducing short non-coding
RNA sequences in plants (Virus Induced
Gene Silencing) [2]

• In 1998 while working on C. elegance
Craig C. Mello and Andrew Fire uncovered
that dsRNA silenced target genes, by
interfering with mRNA translation , and
thus dubbed the process RNA interference
[3]

• They later went on to win the Nobel prize
in Physiology or Medicine in 2006
Introducing RNAi [4]

Click here to view movie
•RNA interference (RNAi) is a mechanism that inhibits gene expression at the stage of translation or by hindering the transcription of
specific genes.
• RNAi targets include RNA from viruses and transposons, and also plays a role in regulating development and genome maintenance.
• Small interfering RNA strands (siRNA) are key to the RNAi process, and have complementary nucleotide sequences to the targeted RNA
strand.
•Specific RNAi pathway proteins are guided by the siRNA to the targeted messenger RNA (mRNA), where they "cleave" the target,
breaking it down into smaller portions that can no longer be translated into protein.
•A type of RNA transcribed from the genome itself, microRNA (miRNA), works in the same way.
Proof that RNAi works in animal models
Since its discovery, numerous studies have been published demonstrating efficacious silencing of disease genes by local
or Systemic administration of siRNAs or shRNAs in animal models of human disease.[5] D. Bumcrot et. al.

•Lung •Spinocerebral ataxia •Digestive system
•RSV •ALS •Irritable bowel disease
•Flu •Neuropathic pain •HSV
•SARS •Encephalitis, West Nile virus •Influenza
•Eye •Tumor •Tumor
•AMD (wet) •Glioblastoma •Liver
•Nervous system •Human papillomavirus •HBV
•Depression •Prostate •Hypercholesterolemia
•Alzheimer disease •Adenocarcinoma •Joint
•Huntington disease •Rheumatoid arthritis

AMD Huntington’s Cancers

Hepatitis C RSV HIV
Human Clinical Trials for treatment of respiratory syncytial virus RSV

In June 2007, Alnylam Pharmaceuticals, Inc. initiated Part 2 of the human
experimental infection model (EIM) study, called "GEMINI.", on February 29th 2008
it announced that it had achieved human proof of concept with an RNAi
therapeutic.

Results from the company’s trial with ALN-RSV01, an RNAi therapeutic for the
treatment of respiratory syncytial virus that:

Subjects receiving ALN-RSV01 experienced a 38.1% reduction in infection rate as
measured by plaque assay (P<0.01); similar reductions in infection rate were
obtained with other measurements of viral infection, including RT-qPCR, spin-
enhanced culture, and RSV antigen assay; the overall infection rate by any of these
measures showed a statistically significant anti-viral effect for ALN-RSV01
(P<0.025); treatment with ALN-RSV01 also resulted in nearly a doubling -- a 95%
increase -- in the number of subjects who remained free of infection (P<0.01); 12 of
42 subjects receiving placebo were uninfected as compared with 24 of 43 subjects
treated with ALN-RSV01 [6]
Human Clinical Trials for AMD [7]

One of the first RNAi therapys to reach patients in clinical trials would aim at a debilitating eye
disease called acute macular degeneration.

Biotech firms such as Sirna Therapeutics, Inc. targeted VEGF that promotes blood vessel growth. In
patients with macular degeneration, too much of this protein leads to the sprouting of excess blood
vessels behind the retina.

The blood vessels leak, clouding and often entirely destroying vision. The new RNAi drugs shut down
genes that produce VEGF and allow it to make the leaky vessels.

The first clinical trial, involving about two dozen patients, was launched in the fall of 2004 and while
its primary intentions were to assess safety issues, it has shown and continues to show promising
results.

Two months after being injected with the drug, a quarter of the patients had significantly clearer
vision, and the other patients' vision had at least stabilized. If subsequent trials prove they are
effective, RNAi drugs for this condition could hit the market by 2009.
RNAi Therapeutics; 2 Major Technical Obstacles & Potential solutions [5]

Effective delivery methods of siRNA to affected Potency & Specificity
areas. Off-target silencing
Difficult and vary according to area . siRNA used not specific enough down-regulates
mRNAs with similar sequence

Local Delivery Specificity
•direct delivery of “naked” siRNA in saline solutions or Sequence homology to a specific gene/protein
conjugated to small molecules that allow them to enter “easily” achievable since decoding of human genome.
the target cell membranes Computational tools in combination with databases of
Pros- known protein sequences allow for quick
Controlled delivery & drug concentration, smaller drug doses identification of target areas.
achieve efficacy. i.e. BLAST
Cons.-
Toxicity issues when using siRNA associated with lipids or Potency[8]
polymers. Raynolds et al. recognised that only a limited fraction of
Effect wares of over time. siRNAs appear capable of producing highly effective RNAi.
They also managed to create 8 empirical rules that these
effective siRNA had in common and these serve as a basis
Systemic Delivery for screening siRNAs for potency, once homology is
Use of nonviral (lipids or conjugated polymers) & established.
lentiviral delivery. Through the blood stream.

Pros-
In theory single administration could provide durable don-
regulation of target.

Cons.-
Toxicity issues due to lack of control of dosage.
Plethora of web based tools (software) for designing siRNAs

As new evidence appears giving us better understanding of
how RNAi works and how we can better target the
proteins/genes of interest engineers and computer
siVirus: web based antiviral siRNA
design software for highly
scientists develop new algorithms that interpret the existing
divergent viral sequences [10]
siRNA databases in the attempt to find the most
specific/potent siRNA for a given target . Explained in detail in further slides
Examples of such software are listed below:

DEQOR : web based siRNA design tool scanning dsCheck : Off-target search software for
through numerous genome databases [9] double stranded RNA-mediated RNAi [11]
siVirus webtool

siVirus searches for functional, off-target minimized siRNAs targeting
highly conserved regions of divergent viral sequences. These siRNAs are
expected to resist viral mutational escape, since their highly conserved
targets likely contain structurally/functionally constrained elements.
siVirus will be a useful tool for designing optimal siRNAs targeting highly
divergent pathogens,including human immunodeficiency virus (HIV),
hepatitis C virus (HCV), influenza virus and SARS coronavirus, all of which
pose enormous threats to global human health.
(A) The degree of conservation is calculated
for all possible siRNA candidates (total 4 417
157) targeting every other position of 495 HIV-
1 sequences.

(B) The efficacy predictions of these 4 417 157
siRNA candidates based on three different
guidelines: Ui-Tei et al. , Reynolds et al. and
Amarzguioui et al.
(C) Typical output of siVirus for designing anti-
HIV siRNAs. Sequence information, efficacy
predictions, off-target search results and the
degrees of conservation are shown.
Conclusion

In a very short time <10 years since the first time RNAi was identified, we see that it
has grown into a field of its own. It is undoubtedly a very strong tool with seemingly
unlimited potential in disease treatment once we get a better understanding of how
to harness its power while also limiting potential side effects.

I do believe though that the reason that such great progress has been achieved in
this short period of time is in effect the result of faster computing power and the
complete decoding of numerous genomes that allow for the creation of huge
datasets.

Datasets that can be then selectively queried by algorithms designed to find “a
needle in the hay stack”

Seeing the potential of RNAi and the multi-million dollar market that has been
created around it, I believe that it is only a matter of time until we are able to find
answers to questions (or cures to diseases) that are seemingly unanswerable at this
point.
Resources

[1] Napoli C, Lemieux C, Jorgensen R (1990). "Introduction of a [8] Reynolds A, Leake D, Boese Q, Scaring S, Marshall W, Khvorova
Chimeric Chalcone Synthase Gene into Petunia Results in A. Rational siRNA design for RNA interference. Nat
Reversible Co-Suppression of Homologous Genes in trans". Plant Biotechnol 2004;22(3):326—30.
Cell 2 (4): 279–289

[2] Van Blokland R, Van der Geest N, Mol JNM, Kooter JM (1994). [9] Henschel A, Buchholz F, Habermann B. Free in PMC DEQOR: a
"Transgene-mediated suppression of chalcone synthase expression web-based tool for the design and quality control of siRNAs.
in Petunia hybrida results from an increase in RNA turnover". Plant J Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W113-20.
6: 861–77
[10] Naito Y, Yamada T, Matsumiya T, Ui-Tei K, Saigo K, Morishita S.
[3] Fire A, Xu S, Montgomery M, Kostas S, Driver S, Mello C (1998). Free in PMC dsCheck: highly sensitive off-target search software
"Potent and specific genetic interference by double-stranded RNA for double-stranded RNA-mediated RNA interference.
in Caenorhabditis elegans". Nature 391 (6669): 806–11. Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W589-91

[4] PBS-NOVA presentation - [11] Naito Y, Ui-Tei K, Nishikawa T, Takebe Y, Saigo K.Free in PMC
http://www.pbs.org/wgbh/nova/sciencenow/3210/02.html siVirus: web-based antiviral siRNA design software for highly
divergent viral sequences. Nucleic Acids Res. 2006 Jul 1;34(Web
[5] Bumcrot D, Manoharan M, Koteliansky V, Sah DW. Abstract Server issue):W448-50
RNAi therapeutics: a potential new class of pharmaceutical drugs.
Nat Chem Biol. 2006 Dec;2(12):711-9. Review.

[6]
http://www.biospace.com/news_story.aspx?NewsEntityId=87731
http://www.alnylam.com/therapeutic-programs/programs.asp

[7]
http://www.biospace.com/news_story.aspx?NewsEntityId=2148742
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