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DK1309726T4 - Rna-sekvensspecifikke formidlere af rna-interferens - Google Patents

Rna-sekvensspecifikke formidlere af rna-interferens Download PDF

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DK1309726T4
DK1309726T4 DK01922870.9T DK01922870T DK1309726T4 DK 1309726 T4 DK1309726 T4 DK 1309726T4 DK 01922870 T DK01922870 T DK 01922870T DK 1309726 T4 DK1309726 T4 DK 1309726T4
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rna
mrna
dsrna
gene
cell
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DK1309726T3 (da
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Phillip D Zamore
Phillip A Sharp
David P Bartel
Thomas Tuschl
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Whitehead Inst Biomedical Res
Max Planck Gesellschaft
Massachusetts Inst Technology
Univ Massachusetts
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Claims (14)

  1. RNA-SEKVENSSPECIFIKKE FORMIDLERE AF RNA-INTERFERENS
    1. Fremgangsmåde til frembringelse af dobbeltstrenget RNA med fra 21 til 23 nukleotider i længden, der omfatter: (a) kombination af dobbeltstrenget RNA med et opløseligt ekstrakt, der formidler RNA-interferens og derved frembringer en kombination; (b) opretholdelse af kombinationen (a) under forhold, hvor det dobbeltstrengede RNA bearbejdes til dobbeltstrenget RNA med fra 21 til 23 nukleotider i længden; og. (c) endvidere omfattende isolation af det dobbeltstrengede RNA med fra 21 til 23 nukleotider i længden fra kombinationen.
  2. 2. Fremgangsmåde ifølge krav 1, hvori det dobbeltstrengede RNA svarer til en sekvens af et gen, der skal nedbrydes; og RNA med fra 21 til 23 nukleotider i længden formidler RNA-interferens af mRNA af genet, der skal nedbrydes, og derved frembringer dobbeltstrenget RNA med fra 21 til 23 nukleotider i længden, der formidler RNA-interferens af genets mRNA.
  3. 3. Fremgangsmåde in vitro til formidling af RNA-interferens af et gens mRNA i en celle, der omfatter: (a) introduktion af et dobbeltstrenget RNA med fra 21 til 23 nukleotider i længden, der svarer til en sekvens af genet, der targeterer mRNA af genet, der skal nedbrydes ind i cellen in vitro; (b) opretholdelse af cellen, der er dannet i (a) under forhold, hvor nedbrydning af mRNA forekommer, hvorved der formidles RNA-interferens af mRNA af genet i cellen.
  4. 4. Fremgangsmåde til undersøgelse af et gens funktion i en testcelle, der omfatter formidling af RNA-interferens af et gens mRNA i en celle ifølge krav 3, hvori cellen er en testcelle; og (c) observation af fænotypen af testcellen, der er dannet i (b), og sammenligning af fænotypen observeret til den af en passende kontrolcelle, hvorved der tilvejebringes informationer om genets funktion.
  5. 5. Fremgangsmåde ifølge krav 3, der endvidere omfatter: (a) kombination af dobbeltstrenget RNA, der svarer til en sekvens af genet med et opløseligt ekstrakt, der formidler RNA-interferens, hvorved der dannes en kombination; (b) opretholdelse af kombinationen, der er dannet i (a) under forhold, hvor det dobbeltstrengede RNA bearbejdes til RNA med fra 21 til 23 nukleotider i længden, hvorved der frembringes dobbeltstrenget RNA med fra 21 til 23 nukleotider i længden; (c) isolation af det dobbeltstrengede RNA med fra 21 til 23 nukleotider i længden, der er frembragt i (b) forud for trinnet med introduktion af RNA isoleret i (c) ind i cellen.
  6. 6. Fremgangsmåde til vurdering af, hvorvidt et genprodukt er et egnet target for udviklingen af nye lægemidler, der omfatter formidling af RNA-interferens af et gens mRNA i en celle ifølge krav 3, hvori nedbrydning af mRNA resulterer i en reduceret genekspression; og: (c) bestemmelse af indvirkningen af den reducerede genekspression på cellen, hvori, hvis den reducerede ekspression har en indvirkning, genproduktet er et target for udvikling af nye lægemidler.
  7. 7. Fremgangsmåde ifølge et hvilket som helst af kravene 3-6, hvori genet indkoder et cellulært mRNA eller et viralt mRNA.
  8. 8. Fremgangsmåde ifølge et hvilket som helst af kravene 1-7, hvori det dobbeltstrengede RNA opnås fra dobbeltstrenget RNA, der er blevet spaltet til fragmenter med fra 21 til 23 nukleotider i længden.
  9. 9. Fremgangsmåde ifølge et hvilket som helst af kravene 1-8, hvori det dobbeltstrengede RNA med fra 21 til 23 nukleotider i længden omfatter en terminal 3'-hydroxylgruppe.
  10. 10. Fremgangsmåde ifølge et hvilket som helst af kravene 3-9, hvori det dobbeltstrengede RNA med fra 21 til 23 nukleotider i længden er kemisk syntetiseret RNA.
  11. 11. Fremgangsmåde ifølge et hvilket som helst af kravene 1-10, hvori RNA omfatter ét eller flere ikke-standard nukleotider, herunder ikke-naturligt forekommende nukleotider.
  12. 12. Fremgangsmåde ifølge et hvilket som helst af kravene 1-11, hvori det dobbeltstrengede RNA med fra 21 til 23 nukleotider i længden er isoleret ved anvendelse af gelelektroforese.
  13. 13. Fremgangsmåde ifølge et hvilket som helst af kravene 1-12, hvori det dobbeltstrengede RNA med fra 21 til 23 nukleotider i længden er komplementært til ét fra cellulært mRNA fra pattedyr eller ét viralt mRNA.
  14. 14. Fremgangsmåde ifølge et hvilket som helst af kravene 1, 2 eller 5, hvori det opløselige ekstrakt er afledt af syncytiale, blastoderme Drosophila-embryoer.
DK01922870.9T 2000-03-30 2001-03-30 Rna-sekvensspecifikke formidlere af rna-interferens DK1309726T4 (da)

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US26523201P 2001-01-31 2001-01-31
PCT/US2001/010188 WO2001075164A2 (en) 2000-03-30 2001-03-30 Rna sequence-specific mediators of rna interference

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