RNA interference in specific gene silencing ('knockdown')

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RNA interference in specific gene silencing
('knockdown')

• Christopher V. Jones
• Jason Carter

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RNA Interference

• mRNA transcribed from DNA encodes for a protein
  expressed by a certain gene
• The presence of certain double-stranded RNA
  (dsRNA) interferes with expression of a gene by
  interfering w/ the translation of its mRNA
• dsRNAs direct the creation of small interfering
  RNAs (siRNAs) which target RNA-degrading enzymes
  (RNAses) to destroy mRNA transcripts
  complementary to the siRNAs

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Small interfering RNA (siRNA)

• dsRNA (usually 21-nt) with 2-nt overhangs on
  either end, including a 5' phosphate group and a
  3' hydroxy (-OH) group
• dsRNA enters RNAi pathway via enzyme Dicer
  producing siRNA
• siRNA molecules associate with a group of
  proteins termed the RNA-induced silencing complex
  (RISC), and directs the RISC to the target mRNA

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(No Transcript)
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Applications

• Typically, a single mRNA translates about 5,000
  protein copies
• RNAi can be used experimentally to "knockout"
  genes in organisms to help determine gene
  function
• dsRNAs that trigger RNAi may be usable as drugs
  to treat genetic disorders or cancers
• dsRNA can repress essential genes in pathogens or
  viruses that are dissimilar from any host genes

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Advantages

• Broad Applicability Diseases for which abnormal
  gene function is a cause or a contributing factor
  are potentially treatable with RNA interference
• Therapeutic Precision Side effects associated
  with traditional drugs may be reduced or avoided
  by using RNAi-based drugs designed to inhibit
  expression of only a targeted gene and no others
• Target RNA Destruction Most drugs only
  temporarily prevent targeted protein function,
  RNAi-based drugs are designed to destroy the
  target RNA stopping undesirable protein
  production required for disease progression

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Treatable diseases Macular Degeneration

• Eye disease caused by the growth of excess blood
  vessels
• Caused by protein VEGF that promotes blood vessel
  growth
• Vessels leak, clouding vision
• dsRNAs can be delivered locally via injection
• clinical trial of two dozen patients in 2004
• In two months
• ¼ improved, ¾ stabilized

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Treatable diseases HIV

• In 2002, scientists at MIT accounted they could
  interrupt various steps in the HIV life cycle
  using RNAi in cell cultures
• Mutates and evolves resistance too rapidly for
  any single target mRNA
• Molecular biologists at Colorado State University
  have engineered RNAi therapy aiming at multiple
  HIV genes
• Clinical trials may start as early as 2006

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Treatable diseases Cancer

• involves mutant genes that promote uncontrolled
  cell growth
• researchers have silenced more than a dozen known
  cancer-causing genes with RNAi in cell cultures
• delivery poses the key challenge for RNAi
  therapies how to reach and penetrate tumors
• Could stop production of P-glycoprotein which
  purges existing chemotherapy drugs from tumors,
  thus enhancing existing treatments

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siRNA Prediction

• Given a target gene, how to design an siRNA to
  knock it down?
• Select a candidate subsequence from the target
  gene
• Not all subsequences are recognizable by Dicer
• Arbitrary subsequence may knockdown unrelated
  gene(s)
• Identify siRNA patterns that are effective
  through experimentation
• Search entire genome to eliminate subsequences
  with off-target specificity

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siRNA Prediction Method fromsiDirect highly
effective target-specific siRNA design software
for mammalian RNA interference, (Naito, Yamada,
Ui-Tei, Morishita, Saigo, 2004)

• Studies of several genes led to these heuristics
• A/U at the 5' end of the antisense strand
• G/C at the 5' end of the sense strand
• AU richness in the 5' terminal 1/3rd of the
  antisense strand
• the absence of any G/C stretch exceeding 9 bp in
  length

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siRNA Prediction Method fromRational siRNA
design for RNA interference (Reynolds, Leake,
Boese, Scaringe, Marshall, Khvorova, 2004)

• At least 7 points are required to be scored as
  effective siRNA
• 30-52 GC content Add 1 point
• Three or more A/Us at positions 15-19 (sense) -
  Add 1 point for each A/U for a total up to 5
  points. At least 3 points are required.
• A at position 19 (sense) - Add 1 point
• A at position 3 (sense) - Add 1 point
• U at position 10 (sense) - Add 1 point
• No G/C at position 19 (sense) - Subtract 1 point
  for not satisfying this criterion.
• No G at position 13 (sense) - Subtract 1 point
  for not satisfying this criterion.

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Filtering out off-target hits

• Once we have predicted potentially effective
  candidate siRNAs, we must search the entire
  genome for off-target matches
• Exhaustive search is expensive, but accurate
• Smith-Waterman algorithm
• Approximate search BLAST algorithm
• Genes have introns that are spliced out of the
  mRNA
• Alternative-splicing means exons are spliced
  several ways we must search these areas also

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Exhaustive vs. Approximate search

• The human genome contains 3B nt
• Only 1.5 encodes proteins as genes
• Must search 45M nt, exon overlap sites, and
  alternative exon overlaps
• Must repeat search for each candidate siRNA
• Exhaustive search is O(nm) time and space
  complexity
• Smith-Waterman is a dynamic algorithm that finds
  optimal local alignment using a scoring system, a
  substitution matrix, and gap-scoring
• Approximate search BLAST can run 50 times faster
  using heuristic approach

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Approximate Search -Basic Local Alignment Search
Tool

• BLAST breaks a search into stages
• Searches for short matches of fixed length W
  between query and database
• If there is a matching word W, performs an
  ungapped alignment between the query and database
  sequence, extending the match in each direction
• High-scoring matches then subjected to a gapped
  alignment between the query sequence and the
  database sequence using a variation of the
  Smith-Waterman algorithm
• Statistically significant matches are returned
• Potential matches may get discarded due to
  heuristics

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siRNA specificity

• siRNA matches to any other gene of as few as 11
  residues can lead to off-target silencing
• High specificity has been observed with siRNAs
  that have at least 3 mismatches to all other
  genes
• Would be considered to have a mismatch tolerance
  of 3
• Higher mismatch tolerance indicates higher
  specificity
• Provides means to rank resulting siRNA candidates
  for study

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Conclusions

• Hundreds of successful experiments in cell
  cultures, and dozens in lab animals
• siRNA delivery methods major hurdle
• siRNA design will mature through competing
  prediction heuristics and better characterization
  of the RNAi machinery
• As RNAi databases mature, novel biocomputing
  approaches are likely
• Optimistic many RNAi therapies will enter
  clinical trials in next five years
• Possible FDA approvals within the next decade

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WebTools

• siDirect
• http//design.rnai.jp/
• Whitehead Institute siRNA
• http//jura.wi.mit.edu/bioc/siRNAext/
• Wistar Bioinformatics Gene-specific siRNA
  selector
• http//bioinfo.wistar.upenn.edu/siRNA/siRNA.htm
• Ambion siRNA design and databases
• http//www.ambion.com/techlib/misc/siRNA_tools.htm
  l
• Web RNAi databases
• http//www.rnainterference.org/
• http//nematoda.bio.nyu.edu/cgi-bin/rnai/index.cgi

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Bibliography

• Review Gene Silencing in mammals by small
  interfering RNAs, (McManus, Sharp) Genetics Vol.
  3 Oct. 2002, 737-747
• Rational siRNA design for RNA interference
  (Reynolds, Leake, Boese, Scaringe, Marshall,
  Khvorova) Nature Biotechnology Vol. 223 Mar.
  2004, 326-330.
• siDirect highly effective target-specific siRNA
  design software for mammalian RNA interference,
  (Naito, Yamada, Ui-Tei, Morishita, Saigo) Nucleic
  Acids Research Vol. 32 2004, 124-129.
• Guidelines for the selection of highly effective
  siRNA sequences for mammalian and chick RNA
  interference, (Ui-Tei, Naito, Takahashi,
  Haraguchi, Okhi-Hamazaki, Juni, Ueda, Saigo,
  2004) Nucleic Acids Research Vol. 323 2004
• Potent and Persistent in-vivo anti-HBV activity
  of chemically modified siRNAs, (Morrisey,
  Lockridge, et. al.) Nature Biotechnology July 2004