RNA interference in RNA interference in specific gene specific gene silencing silencing ('knockdown')('knockdown')

Christopher V. JonesChristopher V. Jones

Jason CarterJason Carter

RNA InterferenceRNA 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

Small interfering RNA (siRNA)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

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

Applications

AdvantagesAdvantages

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

Treatable diseases Treatable diseases ——Macular DegenerationMacular Degeneration

Eye disease caused by the Eye disease caused by the growth of excess blood vesselsgrowth 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

Treatable diseases Treatable diseases ——HIVHIV

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

Treatable diseases Treatable diseases ——CancerCancer

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

siRNA PredictionsiRNA 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

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

Studies of several genes led to these heuristics: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

siRNA Prediction Method from:siRNA Prediction Method from:Rational siRNA design for RNA Rational siRNA design for RNA interference (Reynolds, Leake, Boese, interference (Reynolds, Leake, Boese, Scaringe, Marshall, Khvorova, 2004)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.

Filtering out off-target hits

Once we have predicted potentially effective Once we have predicted potentially effective candidate siRNAs, we must search the entire candidate siRNAs, we must search the entire genome for off-target matchesgenome for off-target matches

Exhaustive search is expensive, but accurate:Exhaustive search is expensive, but accurate:

Smith-Waterman algorithmSmith-Waterman algorithm Approximate search: BLAST algorithmApproximate search: BLAST algorithm Genes have introns that are spliced out of the Genes have introns that are spliced out of the

mRNAmRNA Alternative-splicing means exons are spliced Alternative-splicing means exons are spliced

several ways – we must search these areas alsoseveral ways – we must search these areas also

Exhaustive vs. Approximate Exhaustive vs. Approximate searchsearch

The human genome contains ~3B ntThe human genome contains ~3B nt Only 1.5% encodes proteins as genesOnly 1.5% encodes proteins as genes Must search ~45M nt, exon overlap sites, and Must search ~45M nt, exon overlap sites, and

alternative exon overlapsalternative exon overlaps Must repeat search for each candidate siRNAMust repeat search for each candidate siRNA Exhaustive search is O(nm) time and space Exhaustive search is O(nm) time and space

complexitycomplexitySmith-Waterman is a dynamic algorithm that finds Smith-Waterman is a dynamic algorithm that finds optimal local alignment using a scoring system, a optimal local alignment using a scoring system, a substitution matrix, and gap-scoringsubstitution matrix, and gap-scoring

Approximate search BLAST can run ~50 times faster Approximate search BLAST can run ~50 times faster using heuristic approachusing heuristic approach

Approximate Search -Approximate Search -Basic Local Alignment Search Basic Local Alignment Search Tool Tool BLAST breaks a search into stagesBLAST breaks a search into stages Searches for short matches of fixed length W Searches for short matches of fixed length W

between query and databasebetween query and database If there is a matching word W, performs an ungapped If there is a matching word W, performs an ungapped

alignment between the query and database alignment between the query and database sequence, extending the match in each directionsequence, extending the match in each direction

High-scoring matches then subjected to a gapped High-scoring matches then subjected to a gapped alignment between the query sequence and the alignment between the query sequence and the database sequence using a variation of the Smith-database sequence using a variation of the Smith-Waterman algorithmWaterman algorithm

Statistically significant matches are returnedStatistically significant matches are returned Potential matches may get discarded due to Potential matches may get discarded due to

heuristicsheuristics

siRNA specificitysiRNA specificity

siRNA matches to any other gene of as few siRNA matches to any other gene of as few as 11 residues can lead to off-target silencingas 11 residues can lead to off-target silencing

High specificity has been observed with High specificity has been observed with siRNAs that have at least 3 mismatches to all siRNAs that have at least 3 mismatches to all other genesother genes

Would be considered to have a mismatch Would be considered to have a mismatch tolerance of 3tolerance of 3

Higher mismatch tolerance indicates higher Higher mismatch tolerance indicates higher specificityspecificity

Provides means to rank resulting siRNA Provides means to rank resulting siRNA candidates for studycandidates for study

ConclusionsConclusions

Hundreds of successful experiments in cell Hundreds of successful experiments in cell cultures, and dozens in lab animalscultures, and dozens in lab animals

siRNA delivery methods major hurdlesiRNA delivery methods major hurdle siRNA design will mature through competing siRNA design will mature through competing

prediction heuristics and better prediction heuristics and better characterization of the RNAi machinerycharacterization of the RNAi machinery

As RNAi databases mature, novel As RNAi databases mature, novel biocomputing approaches are likelybiocomputing approaches are likely

Optimistic many RNAi therapies will enter Optimistic many RNAi therapies will enter clinical trials in next five yearsclinical trials in next five years

Possible FDA approvals within the next decadePossible FDA approvals within the next decade

WebToolsWebTools

siDirect:siDirect:http://design.rnai.jp/http://design.rnai.jp/

Whitehead Institute siRNA:Whitehead Institute siRNA:http://jura.wi.mit.edu/bioc/siRNAext/http://jura.wi.mit.edu/bioc/siRNAext/

Wistar Bioinformatics Wistar Bioinformatics Gene-specific siRNA selector:Gene-specific siRNA selector:http://bioinfo.wistar.upenn.edu/siRNA/siRNA.htmhttp://bioinfo.wistar.upenn.edu/siRNA/siRNA.htm

Ambion Ambion siRNA design and databases:siRNA design and databases:http://www.ambion.com/techlib/misc/siRNA_tools.htmlhttp://www.ambion.com/techlib/misc/siRNA_tools.html

Web RNAi databasesWeb RNAi databases

http://www.rnainterference.org/http://www.rnainterference.org/http://nematoda.bio.nyu.edu/cgi-bin/rnai/index.cgihttp://nematoda.bio.nyu.edu/cgi-bin/rnai/index.cgi

BibliographyBibliography

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Guidelines for the selection of highly effective siRNA sequences for Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference, (Ui-Tei, Naito, Takahashi, mammalian and chick RNA interference, (Ui-Tei, Naito, Takahashi, Haraguchi, Okhi-Hamazaki, Juni, Ueda, Saigo, 2004) Haraguchi, Okhi-Hamazaki, Juni, Ueda, Saigo, 2004) Nucleic Acids Nucleic Acids Research Research Vol. 32:3 2004Vol. 32:3 2004

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