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1、Diagnostic and Therapeutic Potential of Small Diagnostic and Therapeutic Potential of Small Nucleic Acid MoleculesNucleic Acid Molecules Mark E.Sobel,M.D.,Ph.D.Mark E.Sobel,M.D.,Ph.D.Executive OfficerExecutive Officer American Society for Investigative PathologyAmerican Society for Investigative Pat
2、hology Penn State Milton S.Hershey Medical CenterPenn State Milton S.Hershey Medical Center August 11,2009August 11,2009 This presentation will be available at This presentation will be available at Part I:The New GeneticsPart I:The New Genetics Part II:Small RNAsPart II:Small RNAsPart III:miRNA and
3、 Hodgkin LymphomaPart IV:Small Molecule Gene-Silencing StrategiesDark Matter is 96%of the mass of the universeThe New Genetics:The Human GenomeThe New Genetics:The Human Genome 3.1 x 103.1 x 109 9 bpbp 500,000 stretches of DNA that are conserved 500,000 stretches of DNA that are conserved through ev
4、olutionthrough evolution 22,000 genes based on current algorithms=22,000 genes based on current algorithms=5%of genome5%of genome 30%have instructions to make proteins30%have instructions to make proteins 70%have instructions to regulate the 70%have instructions to regulate the protein-coding genesp
5、rotein-coding genes The American Society for Investigative PathologyComplexity of the Human GenomeComplexity of the Human Genome The other 70%(regulating)The other 70%(regulating)Ribosomal(Ribosomal(rRNArRNA)Transfer RNA(Transfer RNA(tRNAtRNA)Small Small nucleolarnucleolar RNA(RNA(snoRNAsnoRNA)Micro
6、 RNA(Micro RNA(miRNAmiRNA)95%Junk DNA:95%Junk DNA:PseudogenesPseudogenes Other functions?Other functions?American Society for Investigative PathologyThe Human Genome is NOT“The Human Genome is NOT“GreenGreen”Inefficiencies can be an advantageInefficiencies can be an advantage Adapt quickly to rare,l
7、ife-threatening Adapt quickly to rare,life-threatening circumstancescircumstances Fill up your gas tank and have it ready Fill up your gas tank and have it ready at all times for a long tripat all times for a long trip BleedingBleeding StarvationStarvation Extreme heat or coldExtreme heat or cold Am
8、erican Society for Investigative PathologySmall RNAsSmall RNAs20-30 ntAssociated with Argonaute(Ago)Family ProteinsThree classes of small RNAs miRNA:microRNAs control mRNA stability and translationsiRNA:small interfering RNAs suppress repetitive genes by cleaving their transcriptspiRNA:Piwi-interact
9、ing RNAs mediate RNA cleavage or heterochromatin formation of transposons American Society for Investigative PathologysiRNARISCsiRNA is bound by RISC and unwound by RNA helicaseRISCSense RNA strand degraded American Society for Investigative PathologyRISCAAAAAA.ATarget mRNAmRNA cleavage and degradat
10、ionRISC-bound antisense strand directed to target mRNAAAAAAA.ATarget mRNARISC American Society for Investigative PathologyMicroRNA(miRNA,miR)MicroRNA(miRNA,miR)Pleiotropic:each miR can regulate more than one target mRNA“Silent”mutations in mRNAs such as conservative nucleotide changes may have effec
11、tsTwo main mechanisms of action:Degradation of mRNAEfficiency of mRNA translation American Society for Investigative PathologyMicroRNA(miRNA,miR)MicroRNA(miRNA,miR)Conserved through evolutionWhy did it take so long to discover?Too small to isolate with usual techniquesLethal mutations for developmen
12、tCoded throughout the genome,often at chromosomal breakpoints American Society for Investigative PathologyABCDELJFGKICDEBAJIHFKGBADCADBECGCBDFAEBACDGFEHIGFEJDACBBEDCFAACFDBECGDABFEFBGAHEDCIBCADFEDCBEAABEDCABCDBCAABABBBAmiRs located at Fragile sitesmiRs located in other genomic regionsAdapted from Ca
13、rlo Croce,Ohio State UniversitymiRNA clusters in Chr.8q24.1-24.3Courtesy of Vladimir Valera-Romero and Maria MerinoMicroRNA as an Oncogene MicroRNA as an Oncogene Higher expression of the miR will decrease the stability and translatability of the target tumor suppressor mRNALeads to decreased expres
14、sion of the tumor suppressorIncreased tumor formationLower expression of the miR will increase the stability and translatability of the target oncogene mRNALeads to increased expression of the oncogeneIncreased tumor formation American Society for Investigative PathologyAn Approach to Identifying No
15、vel Molecular Lesions and Potential Therapeutic TargetsMicroRNA-Mediated Down-Regulaton of PRDM1/Blimp-1 in Hodgkin/Reed-Sternberg Cells:A Potential Pathogenetic Lesion in Hodgkin LymphomasKui Nie,Mario Gomez,Pablo Pandgraf,Jose-Francisco Garcia,Yifang Liu,Leonard H.D.Tan,Amy Chadburn,Thomas Tuschl,
16、Daniel Knowles,and Wayne Tam Am J Pathol 2008 173:242-2252;DOI:10.2353/ajpath.2008.080009PRDM1/Blimp-1PRDM gene family of transcription repressorsContain Kruppel-type zinc fingers Expressed as two isoforms,and b,b,as a result of alternative promoter usageThe b b form is functionally impaired(lacks a
17、mino-terminal acidic domain and part of the PR domain)PRDM1 plays a critical role in:Terminal differentiation of lymphocytes and epidermal cellsFate specification of primordial germ cells(and other cell types)In B cells,key differentiation factor in post-germinal center(GC)cells-“master regulator”of
18、 plasma cell differentiationTumor suppressor gene in large B-cell lymphoma diffuse(DLBCL)inhibition of terminal B-cell differentiation may play a role in pathogenesisHRS cells and non-GCB DLBCL appear to be in similar differentiation stagesSequence analysis has not identified inactivating mutations
19、of PRDM1Decrease in accumulation of PRDM1 may occur through quantitative changes in synthesis or stabilityProfiles of miRNA Expression in Hodgkin Lymphoma Cell LinesNie,K.et al,Am J Pathol 2008,173:242-252Figure 1miR-9 and let-7aNie,K.et al,Am J Pathol 2008,173:242-252Figure 2BComplementarity Betwee
20、n miR-9 and Conserved Putative Binding Sites in the Target mRNA(PRDM1 3 UTR)Nie,K.et al,Am J Pathol 2008,173:242-252Figure 2CComplementarity Between let-7a and Conserved Putative Binding Sites in the Target mRNA(PRDM1 3 UTR)Real-time PCR of PRDM1 mRNANie,K.et al,Am J Pathol 2008,173:242-252Figure 2B
21、PEL-primary effusion lymphomaMM multiple myelomaHodgkin/Reed-Sternberg Cells Harbor Low Levels of PRDM1:Better Correlation with miR-9 and let-7a Expression than with PRDM1 mRNAPositive controls:PEL and MMNie,K.et al,Am J Pathol 2008,173:242-252Figure 5CReactive GC in ahyperplastic tonsilPRDM1-negati
22、ve HRS cellsWeakly positive HRS cellsIRF4/MUM1 positivePRDM1 Immunohistochemistry(HL Cases)Nie,K.et al,Am J Pathol 2008,173:242-252,Figure 5DNie,K.et al,Am J Pathol 2008,173:242-252Figure 6BTransfection with anti-micro RNA Increases PRDM1 Protein LevelsHypothetical Model of microRNA-mediated PRDM1 I
23、nactivation inPathogenesis of Hodgkin/Reed-Sternberg Cells:PRDM1 protein levels do not accumulate despite induction of PRDM1mRNA transcripts because of translation repression by miRNAsNie,K.et al,Am J Pathol 2008,173:242-252Figure 7Oncogene miR Expression:Neoplastic Progressiontranscriptiontarget mR
24、NAPhenotypemutationsamplificationtranslocation to strong promotertranslocation to weak promoterhomozygous deletionpromoter hypermethylationdeletion+mutationmiR genedown regulation of tumor suppressorover expression of oncogeneProliferationInvasionAngiogenesis American Society for Investigative Patho
25、logyMicroRNA as a Tumor SuppressorMicroRNA as a Tumor SuppressorLower expression of the miR will increase the stability and translatability of the target tumor suppressor mRNALeads to increased expression of the tumor suppressorSuppression of tumor formationHigher expression of the miR will decrease
26、 the stability and translatability of the target oncogene mRNALeads to decreased expression of the oncogeneSuppression of tumor formation American Society for Investigative PathologyGene-Silencing TherapeuticsGene-Silencing TherapeuticssiRNADNA enzymes(DNAzymes)Antisense oligonucleotidesDecoysRibozy
27、mesAptamers American Society for Investigative PathologysiRNA for Gene-Silencing Therapy siRNA for Gene-Silencing Therapy Major concerns:Tissue specificityAbility to withstand degradationClinical Trials:Prevent ocular neovascularizationAge-related macular degenerationCand5(VEGF siRNA)intravitreal in
28、jection(Phase II)SiRNA-027(Phase I)Diabetic retinopathy American Society for Investigative PathologyDNAzymesDNAzymesGene-specific molecular scissorsNot observed in natureAdvantages for therapeutics:Lower production costsSerum stability“10-23”subtype is best characterizedCation-dependent catalytic co
29、re of 15 dNTPsBinds to target mRNACleaves target RNA between unpaired purine and paired pyrimidineFlanking complementary binding arms6 to 12 dNTPsTarget mRNA specificity American Society for Investigative PathologyDNAzyme binds target mRNAmRNA cleavage and degradationDNAzymeAAAAAA.ATarget mRNAprotei
30、nX American Society for Investigative PathologyDNAzymesDNAzymesEnhance stabilityPrevent exonuclease degradation:Incorporate 3-3 inverted nucleotide at 3endExtend half-life with locked nucleic acidsLNA bases have a 2-O 4-C methylene bridgeConstraint on the ribose ringIncreases affinity for complement
31、ary sequencesIncreased solubilityEgr-1:master regulator zinc finger transcription factor Egr-1(animal models)Restenosis via inhibition of smooth muscle cell hyperplasiaIntracoronary administration reduces neointima formation after stent implantationReduce size of myocardial infarctVEGF:Inhibit tumor
32、 growth via inhibition of angiogenesis American Society for Investigative PathologyAntisense OligonucleotidesAntisense OligonucleotidesOver 50 Clinical StudiesInfection:Fomivirsen(Vitravene):targets immediate-early RNA of human CMV DNA-approved by US FDA to treat CMV retinitisInflammation:Alicaforse
33、n:targets ICAM-1 to treat inflammatory bowel diseaseCancer:Aprinocarsen:targets protein kinase C-a a to treat non-small cell lung carcinomaGenasense(oblimersen):targets bcl-2 to treat CLL,AML,myeloma American Society for Investigative PathologyDecoysDecoysShort double-stranded oligonucleotidesBindin
34、g elements for protein targetsCompetitively inhibit promoter bindingInhibit gene expressionReduction of neointima formation and atherosclerosis in animal models:E2F decoyEdifoligide(PREVENT IV Trial)to prevent vein graft failure in coronary artery bypass grafting American Society for Investigative P
35、athologyRibozymesRibozymesCatalytically active RNA molecules that cleave target mRNA in a site-specific mannerCan occur naturallyHEPTAZYME:targets 5-untranslated region of hepatitis C virus toxicCancer therapy:ANGIOZYME:targets VEGF receptor VEGF R1(Flt-1)in solid tumorsHERzyme:targets human epiderm
36、al growth factor-2 in breast and ovarian cell carcinomas American Society for Investigative PathologyAptamers(Aptamers(aptusaptus=to fit)=to fit)Synthetic oligonucleotidesBind target protein with high affinity and specificityChemical modifications for stabilityPegaptanib:RNA aptamer targets VEGF165
37、to treat neovascular age-related macular degeneration(US FDA approved)Inhibit HIV-1 American Society for Investigative PathologyReferences References 1)1)CalinCalin,GA and Croce,CM,GA and Croce,CM,MicroRNAMicroRNA signatures in human cancers.signatures in human cancers.2006;2006;Nature Reviews:Cance
38、rNature Reviews:Cancer 6:857-866.6:857-866.2)2)Cummins,JM and Cummins,JM and VelculescuVelculescu,VE,VE,Implications of micro-RNA Implications of micro-RNA profiling for cancer diagnosis.profiling for cancer diagnosis.2006;2006;OncogeneOncogene 25:6220-6227.25:6220-6227.3)3)BhindiBhindi,R,R,FahmyFah
39、my,RG,et al.,RG,et al.,Brothers in Arms:DNA Enzymes,Brothers in Arms:DNA Enzymes,Short Interfering RNA,and the Emerging Wave of Small-Molecule Short Interfering RNA,and the Emerging Wave of Small-Molecule Nucleic Acid-Based Gene-Silencing Strategies.Nucleic Acid-Based Gene-Silencing Strategies.2007;
40、2007;Am J Am J PatholPathol 171:1079-1088.171:1079-1088.4)4)NieNie,K,Gomez,M,K,Gomez,M,LandgrafLandgraf,P,Garcia,J-F,Tan,L.C,P,Garcia,J-F,Tan,L.C,ChadburnChadburn,A,A,TuschiTuschi,T,Knowles,DM;,and Tam,W,T,Knowles,DM;,and Tam,W,MicroRNAMicroRNA-Mediated Down-Mediated Down-Regulation of Regulation of
41、 PRDM1/Blimp-1PRDM1/Blimp-1 in Hodgkin/Reed-Sternberg Cells:A in Hodgkin/Reed-Sternberg Cells:A Potential Potential PathogeneticPathogenetic Lesion in Hodgkin Lymphomas Lesion in Hodgkin Lymphomas.2008;.2008;Am J Am J PatholPathol 173:242-252.173:242-252.American Society for Investigative PathologyThe Future:MicroRNA(MiRNA,miR)The Future:MicroRNA(MiRNA,miR)Expression profiles and mutation analysis of miRs will be standard procedure for determining the“molecular signature”of a diseased tissue.American Society for Investigative PathologyThis presentation will be available at