JP5468978B2 - Modified polynucleotides for use in RNA interference - Google Patents

Description

(Field of Invention)
The present invention relates to modified polynucleotides.

(background)
RNA-induced gene silencing in mammalian cells is currently thought to involve at least three different levels of regulation. (I) transcription inactivation (siRNA-induced DNA and histone methylation), (ii) degradation of mRNA induced by small interfering RNA (siRNA), and (iii) mRNA-induced transcription decay. RNA interference (RNAi) generated by siRNA is effective for a long time, and can be effective even if cell division is repeated many times. Thus, the ability to access gene function through siRNA-mediated methods, as well as developing therapeutics for overexpressed genes, stimulates gene function analysis, drug target validation, and extensive genome research Means a useful and useful tool. Furthermore, RNAi has a wide range of potential as a therapeutic tool.

  Relatively recent discoveries in the field of RNA metabolism have revealed that uptake of double stranded RNA (dsRNA) can induce RNAi.

  Under these circumstances, the type III RNase, called Dicer, processes long dsRNAs into siRNAs, which then cooperate with RNA-induced silencing complexes (RISCs) for sequence specific Results in degradation of the target transcript. This phenomenon was seen in a wide variety of organisms. Unfortunately, in mammalian cells, induction of RNAi with long dsRNAs has only achieved some success. Primarily, such ineffectiveness is due to the induction of an interferon response, which results in a general inhibition of protein synthesis as opposed to that targeted.

  Recently, when a synthetic siRNA is introduced into a mammalian cell in a culture solution, sequence-specific inhibition of the target mRNA can be achieved without inducing an interferon reaction. These short duplexes act catalytically at sub-molar concentrations and cleave more than 95% of the intracellular target mRNA. For a mechanism of siRNA activity, as well as some of its uses, see Provost et al. , Ribonuclease Activity and RNA Binding of Recombinant Human Dicer, E .; M.M. B. O. J. et al. , 2002 Nov. , 1, 21 (21): 5864-5874; Tabara et al. , The dsRNA Binding Protein RDE-4 Interacts with RDE-1, DCR-1 and a DexH-box Helicase to Direct RNAi in C. elegans, Cell. 2002, June 28, 109 (7): 861-71; Ketting et al. , Dicer Functions in RNA Interference and in Synthesis of Small RNA Involved in Development Timing in C. elegans; and Martinez et al. , Single-Stranded Antisense siRNAs Guide Target RNA Cleavage in RNAi, Cell 2002, Sept. 6, 110 (5): 563.

  When working with siRNA, there are four main problems that must be solved. (I) functionality, (ii) specificity, (iii) delivery method, and (iv) stability. Functionality refers to the ability of a particular siRNA to silence a desired target. Methods for improving functionality are, for example, the subject of US patent application Ser. No. 10 / 714,333. Specificity refers to the ability of a particular siRNA to silence a desired target and only that target. Thus, specificity means minimizing off-target effects. The delivery method is a means by which a user introduces a particular siRNA into a cell, and may include, for example, using a modification of the vector or siRNA itself. Stability refers to the ability of siRNA to suppress degradation by enzymes or other harmful substances present in the intracellular or extracellular environment. For example, when naked siRNA molecules are introduced into blood, serum, or serum-containing media, the naked siRNA molecules are not stable and most are rapidly degraded to reduce or eliminate the potency of the naked siRNA molecule.

  The present invention solves the second and fourth problems (ie, specificity and stability) by making modifications to siRNA that can increase or decrease specificity and / or increase stability. To do.

(Summary of the Invention)
The present invention relates to compositions and methods for performing RNA interference. In general, the siRNA chemical modifications described herein affect two critical properties of the molecule to which it is attached: stability and specificity. Such modifications that affect stability are used in applications that require exposure to blood, serum, serum-containing media, and other biological materials that contain nucleases or other factors that tend to degrade nucleic acids. Is particularly advantageous. Modifications that reduce the degree of off-target effects induced by siRNA directed to a particular target are particularly beneficial for research and therapeutic settings. A number of different combinations of modifications that substantially improve RNAi application have been disclosed, and these combinations are applicable to the design of optimal silencing reagents, transfection controls, and exaequo reagents.

  According to a first embodiment, the present invention provides a sense strand comprising a polynucleotide composed of at least one orthoester modified nucleotide and an antisense comprising a polynucleotide composed of at least one 2 ′ modified nucleotide unit. An siRNA having a strand is provided.

  According to a second embodiment, the present invention provides a sense strand comprising a polynucleotide composed of at least one orthoester modified nucleotide and an antisense comprising a polynucleotide composed of at least one 2 ′ modified nucleotide unit. An siRNA having a strand and a conjugate is provided.

  According to a third embodiment, the present invention provides an siRNA having a sense strand comprising at least one orthoester modified nucleotide, an antisense strand, and a conjugate.

  According to a fourth embodiment, the present invention provides an siRNA having a sense strand, an antisense strand, and a conjugate, wherein the sense strand and / or the antisense strand has at least one 2 ′ modified nucleotide. .

  According to a fifth embodiment, the present invention provides a sense strand comprising at least one orthoester modified nucleotide, a 2 ′ halogen modified nucleotide, a 2 ′ amine modified nucleotide, a 2′-O-alkyl modified nucleotide, and a 2 ′. An antisense strand comprising at least one 2′-modified nucleotide selected from the group consisting of alkyl-modified nucleotides, and amino acids, peptides, polypeptides, proteins, sugars, carbohydrates, lipids, polymers, nucleotides, polynucleotides, and combinations thereof And a conjugate selected from the group consisting of: siRNA comprising between 18 and 30 nucleotide base pairs of polyribonucleotides.

  According to a sixth embodiment, the present invention includes one of the following structures.

Wherein each of B ₁ and B ₂ is a nitrogen base, heterocycle, or carbocyclic compound, X is selected from the group consisting of O, S, C, and N, and W is OH, phosphorus Selected from the group consisting of acid salts, phosphate esters, phosphate diesters, phosphate triesters, modified internucleotide linkages, conjugates, nucleotides, and polynucleotides, R1 is an orthoester and R2 is 2'- Selected from the group consisting of O-alkyl groups, alkyl groups, amines, halogens, and Y is a nucleotide or polynucleotide. The dashed line between B ₁ and B ₂ indicates the interaction by hydrogen bonding between the nitrogen-containing bases.

  According to a seventh embodiment, the present invention provides a method for performing RNA interference. The method includes exposing the siRNA to a target nucleic acid, the siRNA being composed of a sense strand and an antisense strand, wherein at least one of the sense strand and the antisense strand comprises at least one orthoester modified nucleotide.

  According to an eighth embodiment, the present invention provides another method of performing RNA interference. The method includes exposing the siRNA to a target nucleic acid, the siRNA being composed of a sense strand, an antisense strand, and a conjugate. According to this embodiment, either the sense strand or the antisense strand is composed of 2 'modified nucleotides.

  The compositions of the first to eighth embodiments of the present invention can provide siRNAs that are resistant to nuclease degradation while retaining biological functionality. For example, using siRNA with at least one orthoester modified nucleotide (eg, on the sense strand) and at least one other modification (eg, at the appropriate position on the antisense strand) Stability can be increased while maintaining functionality in interference applications. Furthermore, in RNA interference applications, functionality can be achieved by using siRNA with one or more 2 ′ modifications and / or modified internucleotide linkages in conjunction with the conjugate, even in the absence of orthoester modifications on each strand. Stability can be increased while maintaining

  According to a ninth embodiment, the present invention provides a method for performing RNA interference, the method comprising exposing an siRNA to a target nucleic acid, the siRNA comprising a sense strand and an antisense strand. And the sense strand is substantially non-functional.

  According to a tenth embodiment, the present invention provides a method of performing RNA interference, the method comprising exposing the siRNA to a target nucleic acid, wherein the siRNA comprises (a) a conjugate, ( b) a sense strand comprising at least one 2′-O-alkyl modification and being substantially non-functional; and (c) an antisense strand comprising at least one 2′-fluorine modification, The antisense strand forms a 18-30 base pair duplex.

According to the eleventh embodiment, the present invention provides:
(A) (i) a first 5 'terminal sense nucleotide and a second 5' terminal sense nucleotide comprising a first 2'-O-alkyl sense modification A first 5 'terminal sense nucleotide and a second 5' terminal sense nucleotide, wherein the second 5 'terminal sense nucleotide comprises a nucleotide and a second 2'-O-alkyl sense modification;
(Ii) at least one 2′-O-alkyl pyrimidine modified sense nucleotide that is different from the first 5 ′ terminal sense nucleotide or the second 5 ′ terminal sense nucleotide; A sense strand composed of at least one 2′-O-alkyl pyrimidine modified sense nucleotide;
(B) (i) at least one 2 ′ halogen modified pyrimidine nucleotide, and (ii) a first 5 ′ terminal antisense nucleotide, the first 5 ′ terminal phosphorylated at the 5 ′ carbon position. An antisense strand composed of antisense nucleotides;
Consisting of
Provided is an siRNA in which the sense strand and the antisense strand can form a duplex of 18 to 30 base pairs.

  The molecules of the eleventh embodiment can also be used for target silencing and / or as a control. These molecules may further comprise a label, such as a fluorescent label, and / or a third 5 'terminal sense nucleotide comprising a third 2'-O-alkyl / sense modification.

According to a twelfth embodiment, the present invention
(A) (i) a first 5 'terminal sense nucleotide and a second 5' terminal sense nucleotide comprising a first 2'-O-alkyl sense modification A first 5 'terminal sense nucleotide and a second 5' terminal sense nucleotide, wherein the second 5 'terminal sense nucleotide comprises a nucleotide and a second 2'-O-alkyl sense modification;
(Ii) at least one 2′-O-alkyl pyrimidine modified sense nucleotide that is different from the first 5 ′ terminal sense nucleotide or the second 5 ′ terminal sense nucleotide; At least one 2′-O-alkyl pyrimidine modified sense nucleotide;
A sense strand composed of
(B) (i) a first 5 ′ terminal antisense nucleotide and a second 5 ′ terminal antisense nucleotide comprising the first 2′-O-alkyl antisense modification A first 5 ′ terminal antisense nucleotide and a second 5 which are a second 5 ′ terminal antisense nucleotide comprising a “terminal antisense nucleotide and a second 2′-O-alkyl antisense modification; 'Terminal antisense nucleotides;
(Ii) at least one 2′-O-alkyl pyrimidine modified antisense nucleotide that is different from the first 5 ′ terminal antisense nucleotide and the second 5 ′ terminal antisense nucleotide; At least one 2′-O-alkyl pyrimidine modified antisense nucleotide,
An antisense strand composed of
Consisting of
Provided is an siRNA in which the sense strand and the antisense strand can form a duplex of 16 to 28 base pairs.

  Preferably, the molecule of the twelfth embodiment also includes a label, which may be, for example, a fluorescent dye, and / or a third 5 'terminal sense-sense comprising a third 2'-O-alkyl sense modification. A third 5 ′ terminal antisense nucleotide comprising a nucleotide and / or a third 2′-O-alkyl antisense modification is included. The molecule of the twelfth embodiment can form a duplex of, for example, 19-25 base pairs, or of 19-28 base pairs, for example.

According to a thirteenth embodiment, the present invention provides:
(A) an antisense strand composed of a first 5 'terminal antisense nucleotide, wherein the first 5' terminal antisense nucleotide is 5 of the first 5 'terminal antisense nucleotide; 'Antisense strand phosphorylated at the carbon position,
(B) a sense strand composed of a first 5 ′ terminal sense nucleotide and a second 5 ′ terminal sense nucleotide, wherein the first 5 ′ terminal sense nucleotide is a first 2 ′ carbon sense; A sense strand having a modification and wherein the second 5 ′ terminal sense nucleotide has a second 2 ′ carbon sense modification;
An siRNA is provided comprising:

  Preferably, the 2'-modification is a '2'-O-alkyl modification. According to this embodiment, the sense strand and the antisense strand preferably comprise 18-30 base pairs that are at least substantially complementary.

According to a fourteenth embodiment, the present invention relates to a unimolecular siRNA capable of forming a hairpin siRNA. This single molecule siRNA
(A) an antisense region, the antisense region being composed of a first 5′-end antisense nucleotide, wherein the first 5′-end antisense nucleotide is the first 5′-end antisense nucleotide; An antisense region that is phosphorylated at the 5 ′ carbon position of the sense nucleotide;
(B) a sense region comprising a first 5 'terminal sense nucleotide and a second 5' terminal sense nucleotide, wherein the first 5 'terminal sense nucleotide is a first 2' carbon sense modification A sense region wherein the second 5 'terminal sense nucleotide has a second 2' carbon sense modification;
(C) a loop region located between the sense region and the antisense region;
including.

  According to the fourteenth embodiment, the sense region and the antisense region are at least substantially complementary, and a stable duplex can be formed. Similar to the thirteenth embodiment, according to the fourteenth embodiment, the sense region and the antisense region preferably form a duplex comprising 18-30 base pairs. Preferably, the loop region is downstream of the antisense region.

According to a fifteenth embodiment, the invention relates to a method for minimizing off-target effects, which method is expressing or capable of expressing a target nucleic acid. Including exposing the cell to siRNA, wherein the siRNA comprises an antisense strand and a sense strand;
(A) the sense strand is composed of a first 5 'terminal sense nucleotide and a second 5' terminal sense nucleotide, wherein the first 5 'terminal sense nucleotide undergoes a first 2' carbon sense modification; The second 5 ′ terminal sense nucleotide has a second 2 ′ carbon sense modification, and (b) the antisense strand is composed of the first 5 ′ terminal antisense nucleotide. The first 5 ′ terminal antisense nucleotide is phosphorylated at the 5 ′ carbon position of the first 5 ′ terminal antisense nucleotide.

According to a sixteenth embodiment, the present invention relates to a method for minimizing off-target effects, which method is directed against a target nucleic acid or against a cell expressing or capable of expressing the target nucleic acid. Exposing a modified unimolecular siRNA capable of forming a hairpin, the unimolecular siRNA comprising an antisense region and a sense region;
(A) the sense strand is composed of a first 5 'terminal sense nucleotide and a second 5' terminal sense nucleotide, wherein the first 5 'terminal sense nucleotide undergoes a first 2' carbon sense modification; The second 5 'terminal sense nucleotide has a second 2' carbon sense modification;
(B) the antisense strand is composed of a first 5 ′ terminal antisense nucleotide, and the first 5 ′ terminal antisense nucleotide is 5 ′ of the first 5 ′ terminal antisense nucleotide; It is phosphorylated at the carbon position, and
(C) including a loop region located between the sense region and the antisense region.

  Preferably, the loop region is located downstream of the antisense region.

According to a seventeenth embodiment, the invention relates to siRNA, wherein the siRNA is
(A) an antisense strand comprising a first 5 ′ terminal antisense nucleotide and a second 5 ′ terminal antisense nucleotide, wherein the first 5 ′ terminal antisense nucleotide is a first The second 5 ′ terminal antisense nucleotide has a second 2 ′ carbon antisense modification, and the first 5 ′ terminal antisense nucleotide has An antisense strand in which the first 5 ′ terminal antisense nucleotide is phosphorylated at the 5 ′ carbon position;
(B) a sense strand comprising a first 5 'terminal sense nucleotide and a second 5' terminal sense nucleotide, wherein the first 5 'terminal sense nucleotide is a first 2' carbon sense modification A sense strand, wherein the second 5 ′ terminal sense nucleotide has a second 2 ′ carbon sense modification;
including.

  Preferably, the 2 'carbon modification is a 2'-O-alkyl modification. According to this seventeenth embodiment, the sense strand and the antisense strand preferably comprise 18-30 base pairs that are at least substantially complementary.

According to an eighteenth embodiment, the present invention relates to a unimolecular siRNA capable of forming a hairpin siRNA, the unimolecular siRNA comprising:
(A) an antisense region comprising a first 5 'terminal antisense nucleotide and a second 5' terminal antisense nucleotide;
The first 5 'terminal antisense nucleotide comprises a first 2' carbon antisense modification, the second 5 'terminal antisense nucleotide comprises a second 2' carbon antisense modification, and the first An antisense region wherein said first 5 'terminal antisense nucleotide is phosphorylated at the 5' carbon position of said 5 'terminal antisense nucleotide;
(B) a sense region comprising a first 5 'terminal sense nucleotide and a second 5' terminal sense nucleotide, wherein the first 5 'terminal sense nucleotide is a first 2' carbon sense A sense region comprising a modification, wherein the second 5 ′ terminal sense nucleotide comprises a second 2 ′ carbon sense modification;
(C) a loop region located between the sense region and the antisense region;
including.

  According to the eighteenth embodiment, the sense region and the antisense region are substantially complementary and can form a stable double strand. Similar to the fourteenth embodiment, according to this eighteenth embodiment, the sense region and the antisense region are preferably two comprising 18-30 base pairs that are at least substantially complementary. Form a chain. Preferably, the loop region is located downstream of the antisense region.

According to a nineteenth embodiment, the present invention relates to a method for minimizing off-target effects, which method is expressing or expressing the target nucleic acid. Exposing the siRNA to a cell capable of: wherein the siRNA comprises an antisense strand and a sense strand;
(A) the sense strand is composed of a first 5 'terminal sense nucleotide and a second 5' terminal sense nucleotide, wherein the first 5 'terminal sense nucleotide undergoes a first 2' carbon sense modification; The second 5 'terminal sense nucleotide has a second 2' carbon sense modification;
(B) a first 5 ′ terminal antisense nucleotide having a first 2 ′ carbon antisense modification and a second 5 ′ terminal antisense having a second 2 ′ carbon antisense modification; A nucleotide, and the first 5 ′ terminal antisense nucleotide is phosphorylated at the 5 ′ carbon position of the first 5 ′ terminal antisense nucleotide.

According to a twentieth embodiment, the present invention relates to a method for minimizing off-target effects, which method is expressing or capable of expressing the target nucleic acid. Exposing the cell to a unimolecular siRNA capable of forming a hairpin siRNA, wherein the unimolecular siRNA comprises an antisense region and a sense region;
(A) the sense region is composed of a first 5 ′ terminal sense nucleotide and a second 5 ′ terminal sense nucleotide, wherein the first 5 ′ terminal sense nucleotide has a first 2 ′ carbon sense modification; And the second 5 ′ terminal sense nucleotide has a second 2 ′ carbon sense modification,
(B) the antisense region is composed of a first 5 ′ terminal antisense nucleotide and a second 5 ′ terminal antisense nucleotide, and the first 5 ′ terminal antisense nucleotide is a first 2 ′ Having a carbon antisense modification, and wherein the second 5 ′ terminal antisense nucleotide has a second 2 ′ carbon antisense modification, and further comprises a 5 ′ carbon position of the first 5 ′ antisense nucleotide. And the first 5 ′ antisense nucleotide is phosphorylated,
(C) A loop region is located between the sense region and the antisense region.

  Preferably, the loop region is downstream of the antisense region.

  The present invention also provides control, tracking agents, and exaequo agents for siRNA applications. These agents comprise the molecules of the seventeenth and eighteenth embodiments, except that they do not contain phosphorylated 5 'terminal antisense nucleotides. Accordingly, these agents comprise (i) first and second (or first) each comprising a 2 ′ carbon antisense modification, preferably a 2′-O-alkyl modification, more preferably a 2′-O-methyl modification. , 2nd and 3rd) 5 'terminal antisense nucleotides, and (ii) each with a 2' carbon sense modification, preferably a 2'-O-alkyl modification, more preferably a 2'-O-methyl modification Including first and second (or first, second, and third) 5 ′ terminal sense nucleotides. In some embodiments, none of the other nucleotides are modified, for example a fluorescent label and other than the label located on the first 5 'terminal sense nucleotide. Also, modification of one or more sense pyrimidines with 2'-O-alkyl groups and / or modification of one or more sense pyrimidines with 2'-halogen groups, and / or sense and / or anti There may be a blocking group on the 5 ′ carbon of the 5 ′ terminal nucleotide of the sense strand.

The present invention also provides the following items.
(Item 1)
A method of performing RNA interference comprising exposing a siRNA to a target nucleic acid, wherein the siRNA is comprised of a sense strand and an antisense strand, and wherein the sense strand is substantially non-functional, RNA interference Implementation method.
(Item 2)
The method of item 1, wherein the sense strand comprises at least one 2'-O-alkyl modification.
(Item 3)
3. The method of item 2, wherein the sense strand comprises at least one cytosine-containing nucleotide base or uracil-containing nucleotide base, and wherein the at least one cytosine-containing nucleotide base or uracil-containing nucleotide base has a 2′-O-methyl modification.
(Item 4)
Item 3. The method according to Item 2, wherein the 2'-O-alkyl modification is 2'-O-methyl modification.
(Item 5)
5. The method of item 4, wherein the at least one 2′-O-methyl modification is on the first, second, eighteenth and / or nineteenth nucleotide base.
(Item 6)
The method of item 1, wherein the sense strand further comprises a 5 'conjugate.
(Item 7)
Item 7. The method according to Item 6, wherein the conjugate is cholesterol.
(Item 8)
Item 2. The method according to Item 1, wherein the sense strand has a cap at the 3 'end.
(Item 9)
9. The method of item 8, wherein the cap is an inverted deoxythymidine or two consecutive 2'-O-methyl modified nucleotides.
(Item 10)
The method of item 1, wherein the antisense strand comprises at least one modified nucleotide.
(Item 11)
11. The method of item 10, wherein the at least one modified nucleotide is a 2′-halogen modified nucleotide.
(Item 12)
Item 12. The method according to Item 11, wherein the 2'-halogen modified nucleotide is a 2'-fluorine modified nucleotide.
(Item 13)
The sense strand includes one or more cytosine-containing nucleotide bases and / or uracil-containing nucleotide bases, and each of the one or more cytosine-containing nucleotide bases and / or uracil-containing nucleotide bases is 2′-fluorine modified. The method according to Item 1.
(Item 14)
A method of performing RNA interference comprising exposing an siRNA to a target nucleic acid, wherein the siRNA comprises:
(A) a conjugate;
(B) a sense strand comprising at least one 2′-O-alkyl modification and being substantially non-functional;
(C) an antisense strand comprising at least one 2′-fluorine modification;
A method of performing RNA interference, wherein the sense strand and the antisense strand form a 18-30 base pair duplex.
(Item 15)
15. The method of item 14, wherein the at least one 2'-O-alkyl modification is on the first, second, eighteenth, and / or nineteenth nucleotide base.
(Item 16)
15. A method according to item 14, wherein the conjugate is a 5 'conjugate.
(Item 17)
Item 15. The method according to Item 14, wherein the conjugate is cholesterol.
(Item 18)
Item 15. The method according to Item 14, wherein the sense strand further has a cap at the 3 'end.
(Item 19)
19. The method of item 18, wherein the cap is an inverted deoxythymidine or two consecutive 2'-O-methyl modified nucleotides.
(Item 20)
A method of performing RNA interference comprising exposing an siRNA to a target nucleic acid, wherein the siRNA is composed of a sense strand and an antisense strand, wherein at least one of the sense strand and the antisense strand is at least one A method of performing RNA interference comprising orthoester-modified nucleotides.
(Item 21)
21. The method of item 20, wherein the at least one orthoester modified nucleotide is located on the sense strand.
(Item 22)
Item 21. The antisense strand comprises at least one nucleotide selected from the group consisting of 2 ′ halogen modified nucleotides, 2 ′ amine modified nucleotides, 2′-O-alkyl modified nucleotides, and 2 ′ alkyl modified nucleotides. The method described.
(Item 23)
24. The method of item 22, wherein the antisense strand comprises at least one 2 ′ halogen modified nucleotide and the halogen is fluorine.
(Item 24)
Item 22. The method according to Item 21, wherein the siRNA further comprises a conjugate.
(Item 25)
25. The method of item 24, wherein the conjugate is selected from the group consisting of amino acids, peptides, polypeptides, proteins, sugars, hydrocarbons, lipids, polymers, nucleotides, polynucleotides, and combinations thereof.
(Item 26)
25. A method according to item 24, wherein the conjugate is cholesterol.
(Item 27)
25. A method according to item 24, wherein the conjugate is polyethylene glycol.
(Item 28)
21. The method of item 20, wherein the siRNA comprises 18-30 nucleotide base pairs.
(Item 29)
29. The method of claim steel 28, wherein the siRNA comprises 19 nucleotide base pairs.
(Item 30)
21. The method of item 20, wherein the siRNA has an overhang of at least one nucleotide unit on at least one of the sense strand and the antisense strand.
(Item 31)
21. The method of item 20, wherein at least one strand of the siRNA comprises at least one modified internucleotide linkage.
(Item 32)
32. The method of item 31, wherein the modified internucleotide linkage is selected from the group consisting of a phosphorothioate bond and a phosphorodithioate bond.
(Item 33)
Item 21. The method according to Item 20, wherein at least one strand of the siRNA is a polyribonucleotide.
(Item 34)
A method of performing RNA interference comprising exposing an siRNA to a target nucleic acid, wherein the siRNA comprises:
(A) a sense strand;
(B) an antisense strand;
(C) a conjugate;
Wherein at least one of the sense strand and the antisense strand is
A method of performing RNA interference, comprising 2 'modified nucleotides.
(Item 35)
siRNA,
(A) a sense strand comprising a polynucleotide composed of at least one orthoester modified nucleotide;
(B) an antisense strand comprising a polynucleotide composed of at least one 2 ′ modified nucleotide;
SiRNA containing.
(Item 36)
Item 35. The antisense strand comprises at least one nucleotide selected from the group consisting of 2 ′ halogen modified nucleotides, 2 ′ amine modified nucleotides, 2′-O-alkyl modified nucleotides, and 2 ′ alkyl modified nucleotides. The siRNA as described.
(Item 37)
Item 37. The siRNA according to Item 36, wherein the 2′-modified nucleotide is a 2 ′ halogen-modified nucleotide, and the halogen is fluorine.
(Item 38)
36. The siRNA of item 35, further comprising a conjugate.
(Item 39)
40. The siRNA of item 38, wherein the conjugate is selected from the group consisting of amino acids, peptides, polypeptides, proteins, sugars, carbohydrates, lipids, polymers, nucleotides, polynucleotides, and combinations thereof.
(Item 40)
40. The siRNA of item 38, wherein the conjugate is cholesterol.
(Item 41)
40. The siRNA according to item 38, wherein the conjugate is polyethylene glycol.
(Item 42)
36. The siRNA according to item 35, wherein the siRNA is composed of 18 to 30 nucleotide base pairs.
(Item 43)
43. The siRNA according to item 42, wherein the siRNA is composed of 19 nucleotide base pairs.
(Item 44)
36. The siRNA of item 35, further comprising an overhang of at least one nucleotide unit on at least one of the sense strand and the antisense strand.
(Item 45)
36. The siRNA of item 35, wherein at least one of the sense strand and the antisense strand comprises at least one modified internucleotide linkage.
(Item 46)
46. The siRNA according to item 45, wherein the modified internucleotide linkage is selected from the group consisting of a phosphorothioate bond and a phosphorodithioate bond.
(Item 47)
36. The siRNA according to item 35, wherein at least one of the sense strand and the antisense strand is a polyribonucleotide.
(Item 48)
siRNA,
(A) a sense strand comprising a polynucleotide composed of at least one orthoester modified nucleotide;
(B) an antisense strand comprising a polynucleotide composed of at least one 2 ′ modified nucleotide;
(C) a conjugate;
SiRNA containing.
(Item 49)
49. The siRNA of item 48, wherein the conjugate is located on the sense strand.
(Item 50)
49. The siRNA of item 48, wherein the conjugate is located on the antisense strand.
(Item 51)
Item 48. The antisense strand comprises at least one nucleotide selected from the group consisting of 2 ′ halogen modified nucleotides, 2 ′ amine modified nucleotides, 2′-O-alkyl modified nucleotides, and 2 ′ alkyl modified nucleotides. The siRNA as described.
(Item 52)
52. The siRNA according to Item 51, wherein the sense strand is composed of 2 ′ halogen-modified nucleotides, and the halogen is fluorine.
(Item 53)
49. The siRNA of item 48, wherein the conjugate is selected from the group consisting of amino acids, peptides, polypeptides, proteins, sugars, carbohydrates, lipids, polymers, nucleotides, polynucleotides, and combinations thereof.
(Item 54)
49. The siRNA of item 48, wherein the conjugate is cholesterol.
(Item 55)
49. The siRNA according to item 48, wherein the conjugate is polyethylene glycol.
(Item 56)
49. The siRNA of item 48, wherein the siRNA is composed of 18 to 30 nucleotide base pairs.
(Item 57)
57. The siRNA of item 56, wherein the siRNA is composed of 19 nucleotide base pairs.
(Item 58)
49. The siRNA of item 48, further comprising an overhang of at least one nucleotide unit on at least one of the sense strand and the antisense strand.
(Item 59)
49. The siRNA of item 48, wherein at least one of the sense strand and the antisense strand comprises at least one modified internucleotide linkage.
(Item 60)
60. The siRNA according to item 59, wherein the modified internucleotide linkage is selected from the group consisting of a phosphorothioate bond and a phosphorodithioate bond.
(Item 61)
49. The siRNA according to item 48, wherein at least one of the sense strand and the antisense strand is a polyribonucleotide.
(Item 62)
siRNA,
(A) a sense strand composed of at least one orthoester modified nucleotide;
(B) an antisense strand;
(C) a conjugate;
SiRNA containing.
(Item 63)
63. siRNA according to item 62, wherein the conjugate is located on the sense strand.
(Item 64)
63. The siRNA of item 62, wherein the is located on the antisense strand.
(Item 65)
Item 62. The antisense strand comprises at least one nucleotide selected from the group consisting of 2 ′ halogen modified nucleotides, 2 ′ amine modified nucleotides, 2′-O-alkyl modified nucleotides, and 2 ′ alkyl modified nucleotides. The siRNA as described.
(Item 66)
68. The siRNA according to item 65, wherein the antisense strand is composed of 2 ′ halogen-modified nucleotides, and the halogen is fluorine.
(Item 67)
63. siRNA according to item 62, wherein the conjugate is selected from the group consisting of amino acids, peptides, polypeptides, proteins, sugars, carbohydrates, lipids, polymers, nucleotides, polynucleotides, and combinations thereof.
(Item 68)
63. siRNA according to item 62, wherein the conjugate is cholesterol.
(Item 69)
63. The siRNA according to item 62, wherein the conjugate is polyethylene glycol.
(Item 70)
63. siRNA according to item 62, wherein the siRNA is composed of 18 to 30 nucleotide base pairs.
(Item 71)
The siRNA according to item 70, wherein the siRNA is composed of 19 nucleotide base pairs.
(Item 72)
63. The siRNA of item 62, further comprising an overhang of at least one nucleotide unit on at least one of the sense strand and the antisense strand.
(Item 73)
63. siRNA according to item 62, wherein at least one of the sense strand and the antisense strand comprises at least one modified internucleotide linkage.
(Item 74)
63. The siRNA according to Item 62, wherein at least one of the sense strand and the antisense strand is a polyribonucleotide.
(Item 75)
siRNA,
(A) a sense strand;
(B) an antisense strand;
(C) a conjugate;
SiRNA, wherein the sense strand and / or the antisense strand comprises at least one 2 'modified nucleotide.
(Item 76)
76. The siRNA of item 75, wherein the 2 'modified nucleotide is selected from the group consisting of 2' halogen modified nucleotides, 2 'amine modified nucleotides, 2'-O-alkyl modified nucleotides, and 2' alkyl modified nucleotides.
(Item 77)
77. The siRNA according to Item 76, wherein the 2′-modified nucleotide is a 2 ′ halogen-modified nucleotide and the halogen is fluorine.
(Item 78)
76. The siRNA of item 75, wherein the conjugate is selected from the group consisting of amino acids, peptides, polypeptides, proteins, sugars, carbohydrates, lipids, polymers, nucleotides, polynucleotides, and combinations thereof.
(Item 79)
76. siRNA according to item 75, wherein the conjugate is cholesterol.
(Item 80)
76. siRNA according to item 75, wherein the conjugate is polyethylene glycol.
(Item 81)
The siRNA according to Item 75, wherein the siRNA is composed of 18 to 30 nucleotide base pairs.
(Item 82)
76. The siRNA of item 75, wherein the siRNA is composed of 19 nucleotide base pairs.
(Item 83)
76. The siRNA of item 75, further comprising an overhang of at least one nucleotide unit on at least one of the sense strand and the antisense strand.
(Item 84)
76. The siRNA of item 75, wherein at least one of the sense strand and the antisense strand comprises at least one modified internucleotide linkage.
(Item 85)
85. siRNA according to item 84, wherein the modified internucleotide linkage is selected from the group consisting of a phosphorothioate bond and a phosphorodithioate bond.
(Item 86)
The siRNA according to Item 75, wherein at least one of the sense strand and the antisense strand is a polyribonucleotide.
(Item 87)
siRNA,
(A) a sense strand composed of at least one 2 ′ orthoester modified nucleotide;
(B) an antisense composed of at least one 2 ′ modified nucleotide selected from the group consisting of 2 ′ halogen modified nucleotides, 2 ′ amine modified nucleotides, 2′-O-alkyl modified nucleotides, and 2 ′ alkyl modified nucleotides chain and,
(C) a conjugate selected from the group consisting of amino acids, peptides, polypeptides, proteins, sugars, carbohydrates, lipids, polymers, nucleotides, polynucleotides, and combinations thereof;
And the polynucleotide comprises between 18 and 30 nucleotide base pairs.
(Item 88)
A composition comprising:

Including
B ₁ And B ₂ Each is a nitrogen-containing base, heterocyclic, or carbocyclic compound;
X is selected from the group consisting of O, S, C, and N;
W is selected from the group consisting of OH, phosphate, phosphate ester, phosphate diester, phosphate triester, modified internucleotide linkage, conjugate, nucleotide, and polynucleotide;
R1 is an orthoester;
R2 is selected from the group consisting of a 2′-O-alkyl group, an alkyl group, and an amine, and further a halogen, and
A composition wherein Y is a nucleotide or a polynucleotide.
(Item 89)
siRNA,
(A) a sense strand,
(I) a first 5 'terminal sense nucleotide comprising a first 2'-O-alkyl sense modification and a second 5' terminal sense nucleotide comprising a second 2'-O-alkyl sense modification And
(Ii) at least one 2′-O-alkyl pyrimidine modified sense nucleotide that is a nucleotide that is different from both the first 5 ′ terminal sense nucleotide and the second 5 ′ terminal sense nucleotide;
A sense strand composed of
(B) an antisense strand,
(I) at least one 2 ′ halogen-modified pyrimidine nucleotide, and
(Ii) a first 5 'terminal antisense nucleotide phosphorylated at the 5' carbon position;
An antisense strand composed of
Consisting of
An siRNA in which the sense strand and the antisense strand can form a duplex between 18 and 30 base pairs.
(Item 90)
90. siRNA according to item 89, wherein the sense strand and the antisense strand can form a duplex between 19 base pairs and 25 base pairs.
(Item 91)
90. siRNA according to item 89, further comprising a label.
(Item 92)
92. The siRNA of item 91, wherein the label is attached to the first 5 ′ terminal sense nucleotide.
(Item 93)
93. The siRNA according to item 92, wherein the label is a fluorescent dye.
(Item 94)
90. siRNA according to item 89, wherein all pyrimidines on the sense strand contain a 2'-O-alkyl modification.
(Item 95)
90. siRNA according to item 89, wherein all pyrimidines on the antisense strand contain a 2'-fluorine modification.
(Item 96)
Each of the first 2'-O-alkyl sense modification, the second 2'-O-alkyl sense modification, and the at least one 2'-O-alkyl pyrimidine modified sense nucleotide is 2 ' 90. siRNA according to item 89, comprising -O-methyl.
(Item 97)
90. siRNA according to item 89, wherein the halogen is fluorine.
(Item 98)
The first 2'-O-alkyl sense modification is 2'-O-methyl, the second 2'-O-alkyl sense modification is 2'-O-methyl, and the at least one Item 89. The 2′-O-alkyl pyrimidine modified sense nucleotide comprises 2′-O-methyl, the halogen is fluorine, and the first 5 ′ terminal sense nucleotide further comprises a fluorescent dye. The siRNA as described.
(Item 99)
90. siRNA according to item 89, further comprising at least one phosphorothioate.
(Item 100)
90. siRNA according to item 89, further comprising at least one methylphosphonate.
(Item 101)
siRNA,
(A) a sense strand,
(I) a first 5 'terminal sense nucleotide comprising a first 2'-O-alkyl sense modification and a second 5' terminal sense nucleotide comprising a second 2'-O-alkyl sense modification And
(Ii) at least one 2′-O-alkyl pyrimidine modified sense nucleotide that is a nucleotide that is different from both the first 5 ′ terminal sense nucleotide and the second 5 ′ terminal sense nucleotide;
A sense strand composed of
(B) an antisense strand,
(I) a first 5'-end antisense nucleotide comprising a first 2'-O-alkyl antisense modification and a second 5'-end comprising a second 2'-O-alkyl antisense modification Antisense nucleotides, and
(Ii) at least one 2′-O-alkyl pyrimidine modified antisense nucleotide that is a different nucleotide from either the first 5 ′ terminal antisense nucleotide or the second 5 ′ terminal antisense nucleotide;
An antisense strand composed of
SiRNA, wherein the sense strand and the antisense strand are capable of forming a duplex between 16 and 28 base pairs.
(Item 102)
102. The siRNA of item 101, wherein the sense strand and the antisense strand can form a 19-28 base pair duplex.
(Item 103)
102. The siRNA of item 101, wherein the sense strand and the antisense strand can form a 19 to 25 base pair duplex.
(Item 104)
102. The siRNA of item 101, wherein all pyrimidines on the sense strand contain a 2′-O-alkyl modification.
(Item 105)
102. The siRNA of item 101, wherein all pyrimidines on the antisense strand contain a 2′-O-alkyl modification.
(Item 106)
The first 2'-O-alkyl sense modification, the second 2'-O-alkyl sense modification, the first 2'-O-alkyl antisense modification, the second 2'- Each of the O-alkyl antisense modification, the at least one 2'-O-alkyl pyrimidine modified sense nucleotide, and the at least one 2'-O-alkyl pyrimidine modified antisense nucleotide is 2'- 102. The siRNA of item 101, comprising O-methyl.
(Item 107)
102. The siRNA of item 101, further comprising a label.
(Item 108)
108. The siRNA of item 107, wherein the label is attached to the first 5 ′ terminal sense nucleotide.
(Item 109)
108. siRNA according to item 107, wherein the label is a fluorescent dye.
(Item 110)
102. The siRNA of item 101, further comprising at least one phosphorothioate.
(Item 111)
102. The siRNA of item 101, further comprising at least one methylphosphonate.
(Item 112)
90. A gene silencing method comprising introducing the siRNA of item 89 into a cell expressing or capable of expressing the target gene.
(Item 113)
A method of performing gene silencing comprising using a control reagent comprising:
Using a control reagent which is the siRNA of item 101;
Introducing the control reagent into a cell expressing a target gene or capable of expressing the target gene;
A method for performing gene silencing, comprising:
(Item 114)
siRNA,
(A) an antisense strand composed of a first 5 ′ terminal antisense nucleotide, wherein the 5 ′ carbon position of the first 5 ′ terminal antisense nucleotide is phosphorylated;
(B) a sense strand comprised of a first 5 'terminal sense nucleotide having a first 2' carbon sense modification and a second 5 'terminal sense nucleotide having a second 2' carbon sense modification;
SiRNA containing.
(Item 115)
115. The siRNA of item 114, wherein each of the first 2 ′ carbon sense modification and the second 2 ′ carbon sense modification is a 2′-O-alkyl modification.
(Item 116)
116. The siRNA of item 115, further comprising a third 5 'terminal sense nucleotide, wherein the third 5' terminal sense nucleotide comprises a 2'-O-alkyl modification.
(Item 117)
116. The siRNA of item 115, wherein at least one of the 2′-O-alkyl modifications is a 2′-O-methyl modification.
(Item 118)
116. The siRNA of item 115, wherein each of the 2′-O-alkyl modifications is a 2′-O-methyl modification.
(Item 119)
116. The siRNA of item 115, wherein the siRNA is composed of between 18 and 30 base pairs.
(Item 120)
120. The siRNA of item 119, wherein the siRNA is composed of base pairs between 19 and 25.
(Item 121)
120. The siRNA of item 119, wherein the antisense strand and the sense strand are substantially complementary and form a duplex that does not contain an overhang.
(Item 122)
A method for reducing off-target effects in RNAi, comprising exposing siRNA to a target nucleic acid, wherein the siRNA comprises an antisense strand and a sense strand;
(A) the sense strand is comprised of a first 5 'terminal sense nucleotide having a first 2' carbon modification and a second 5 'terminal sense nucleotide having a second 2' carbon modification; And
(B) Off-target effect, wherein the antisense strand is composed of a first 5 ′ terminal antisense nucleotide, and the 5 ′ carbon position of the first 5 ′ terminal antisense nucleotide is phosphorylated Decrease method.
(Item 123)
123. The method of item 122, wherein each of the first 2 'carbon sense modification and the second 2' carbon sense modification is a 2'-O-alkyl modification.
(Item 124)
124. The method of item 123, further comprising a third 5 'terminal sense nucleotide having a 2'-O-alkyl modification.
(Item 125)
124. The method of item 123, wherein at least one of the 2′-O-alkyl modifications is a 2′-O-methyl modification.
(Item 126)
124. The method of item 123, wherein each of the 2′-O-alkyl modifications is a 2′-O-methyl modification.
(Item 127)
124. The method of item 122, wherein the siRNA consists of between 18 and 30 base pairs.
(Item 128)
128. siRNA according to item 127, wherein the siRNA is composed of base pairs between 19 and 25.
(Item 129)
128. The method of item 127, wherein the antisense strand and the sense strand form a duplex that is substantially complementary and free of overhangs.
(Item 130)
A unimolecular siRNA capable of forming a hairpin, comprising:
(A) an antisense region composed of a first 5 'terminal antisense nucleotide phosphorylated at the 5' carbon position;
(B) a sense region comprised of a first 5 'terminal sense nucleotide having a first 2' carbon sense modification and a second 5 'terminal sense nucleotide having a second 2' carbon sense modification;
(C) a loop region located between the sense region and the antisense region;
SiRNA containing.
(Item 131)
130. The siRNA of item 130, wherein each of the first 2 ′ carbon sense modification and the second 2 ′ carbon sense modification is a 2′-O-alkyl modification.
(Item 132)
130. The siRNA of item 130, further comprising a third 5 'terminal sense nucleotide comprising a 2'-O-alkyl modification.
(Item 133)
134. The siRNA of item 131, wherein at least one of the 2′-O-alkyl modifications is a 2′-O-methyl modification.
(Item 134)
134. The siRNA of item 131, wherein each of the 2′-O-alkyl modifications is a 2′-O-methyl modification.
(Item 135)
130. The siRNA of item 130, wherein the siRNA is composed of between 18 and 30 base pairs.
(Item 136)
140. The siRNA of item 135, wherein the siRNA is composed of base pairs between 19 and 25.
(Item 137)
A method for reducing off-target effects in RNAi comprising:
Exposing the target nucleic acid to a single molecule siRNA capable of forming a hairpin, the single molecule siRNA comprising an antisense region and a sense region;
(A) The sense region comprises a first 5 'terminal sense nucleotide having a first 2' carbon sense modification and a second 5 'terminal sense nucleotide having a second 2' carbon sense modification And
(B) the antisense region is composed of a first 5 ′ terminal antisense nucleotide phosphorylated at the 5 ′ carbon position;
(C) A method for reducing the off-target effect, including a loop region located between the sense region and the antisense region.
(Item 138)
140. The method of item 137, wherein each of the first 2 ′ carbon sense modification and the second 2 ′ carbon sense modification is a 2′-O-alkyl modification.
(Item 139)
139. The method of item 138, further comprising a third 5 'terminal sense nucleotide having a 2'-O-alkyl modification.
(Item 140)
140. The method of item 138, wherein at least one of the 2'-O-alkyl modifications is a 2'-O-methyl modification.
(Item 141)
139. The method of item 138, wherein each of the 2'-O-alkyl modifications is a 2'-O-methyl modification.
(Item 142)
140. The method of item 137, wherein the siRNA is composed of between 18 and 30 base pairs.
(Item 143)
And further comprising a second 5 ′ terminal antisense nucleotide, wherein the first 5 ′ terminal antisense nucleotide further comprises a first 2 ′ carbon terminal antisense modification, and wherein the second 5 ′ terminal antisense nucleotide comprises 115. siRNA according to item 114, wherein the nucleotide comprises a second 2 ′ carbon-terminal antisense modification.
(Item 144)
And further comprising a second 5 ′ terminal antisense nucleotide, wherein the first 5 ′ terminal antisense nucleotide further comprises a first 2 ′ carbon terminal antisense modification, and wherein the second 5 ′ terminal antisense nucleotide comprises 131. A unimolecular siRNA according to item 130, wherein the nucleotide comprises a second 2 ′ carbon-terminal antisense modification.
(Item 145)
144. A method for reducing off-target effects induced in RNAi, comprising introducing the siRNA of any one of items 114 or 143 into a cell.
(Item 146)
A method for reducing an off-target effect in RNAi, comprising introducing the siRNA of item 144 into a cell.
(Item 147)
siRNA,
(A) consisting of a first 5 'terminal antisense nucleotide having a first 2' carbon antisense modification and a second 5 'terminal antisense nucleotide having a second 2' carbon antisense modification An antisense strand,
(B) a sense strand comprised of a first 5 'terminal sense nucleotide having a first 2' carbon sense modification and a second 5 'terminal sense nucleotide having a second 2' carbon sense modification; ,
SiRNA containing.
(Item 148)
148. The siRNA of item 147, wherein the sense strand and the antisense strand form a duplex of 16 to 28 base pairs.
(Item 149)
149. siRNA according to item 148, wherein the sense strand and the antisense strand form a 18 to 25 base pair duplex.
(Item 150)
Each of the first 2 'carbon antisense modification, the second 2' carbon antisense modification, the first 2 'carbon sense modification, and the second 2' carbon sense modification is 2'-O 147. siRNA of item 148, comprising an alkyl modification.
(Item 151)
150. The siRNA of item 150, wherein the 2′-O-alkyl modification is 2′-O-methyl.
(Item 152)
The siRNA of item 150, further comprising a label.
(Item 153)
A unimolecular siRNA capable of forming a hairpin, comprising:
(A) consisting of a first 5 'terminal antisense nucleotide having a first 2' carbon antisense modification and a second 5 'terminal antisense nucleotide having a second 2' carbon antisense modification An antisense region,
(B) a sense region comprised of a first 5 'terminal sense nucleotide having a first 2' carbon sense modification and a second 5 'terminal sense nucleotide having a second 2' carbon sense modification; ,
(C) a loop region located between the sense region and the antisense region;
SiRNA containing.
(Item 154)
154. The siRNA of item 153, wherein the sense region and the antisense region form a duplex of 16 to 28 base pairs.
(Item 155)
155. siRNA of item 154, wherein the sense region and the antisense region form a 18-25 base pair duplex.
(Item 156)
Each of the first 2 'carbon antisense modification, the second 2' carbon antisense modification, the first 2 'carbon sense modification, and the second 2' carbon sense modification is 2'-O 155. siRNA of item 154, comprising an alkyl modification.
(Item 157)
156. siRNA according to item 156, wherein the 2′-O-alkyl modification is 2′-O-methyl.
(Item 158)
156. siRNA of item 156, further comprising a label.
(Item 159)
An siRNA comprising a sense strand and an antisense strand, wherein each of the sense strand and the antisense strand has at least one orthoester modification at the 2 ′ position.
(Item 160)
siRNA comprising a sense strand and an antisense strand, wherein the antisense strand is at least one orthoester modification and / or 2′-halogen modification, 2′-alkyl modification, 2′-O-alkyl modification, 2 An siRNA having at least one modification selected from the group consisting of a '-amine modification and a 2'-deoxy modification.
(Item 161)
siRNA comprising a sense strand and an antisense strand, wherein the sense strand and / or the antisense strand comprises at least one orthoester modification, and wherein the sense strand and / or the antisense strand is 2′- An siRNA comprising at least one 2 'modification selected from the group consisting of halogen modification, 2'-alkyl modification, 2'-O-alkyl modification, 2'-amine modification, and 2'-deoxy modification.
(Item 162)
siRNA,
(A) a sense strand comprised of a first 5 'terminal sense nucleotide having a first 2' carbon sense modification and a second 5 'terminal sense nucleotide having a second 2' carbon sense modification; ,
(B) consisting of a first 5 ′ terminal antisense nucleotide having a first 2 ′ carbon antisense modification and a second 5 ′ terminal antisense nucleotide having a second 2 ′ carbon antisense modification. An antisense strand,
SiRNA containing.
(Item 163)
siRNA comprising a sense strand and an antisense strand, wherein the antisense strand has at least one 2′-orthoester modification, and the antisense strand further comprises 2′-orthoester modification, 2 ′ -Modified by one or more modifications selected from the group consisting of alkyl modification, 2'-halogen modification, 2'-O-alkyl modification, 2'-amine modification, and 2'-deoxy modification, An siRNA wherein the alkyl modification, the 2′-O-methyl modification, and the 2′-halogen modification are in one or more pyrimidines of the antisense strand, and the siRNA further comprises a 3 ′ cap.
(Item 164)
164. The siRNA of item 162, wherein the 2 ′ carbon modification is a 2′-O-alkyl modification.
(Item 165)
165. siRNA according to item 164, wherein the 2′-O-alkyl modification is 2′-O-methyl modification.
(Item 166)
163. siRNA according to item 162, wherein the first 5 ′ terminal sense nucleotide and / or the first 5 ′ terminal antisense nucleotide is further modified by a 5 ′ blocking group.
(Item 167)
173. siRNA according to item 166, wherein the 5 ′ blocking group is selected from the group consisting of 5′-methyl modification, 5′-O-methyl modification, and 5′-azide modification.
(Item 168)
The first and second 2′-O-alkyl modifications of the 5 ′ terminal sense nucleotide and / or the 5 ′ terminal antisense nucleotide are 2′-O-methyl modifications, and the first 5 ′ terminal 111. The siRNA of any of paragraphs 101-111, wherein the 'terminal sense nucleotide and / or the first 5' terminal antisense nucleotide further comprises a 5 'blocking group.
(Item 169)
169. siRNA according to item 168, wherein the 5 ′ blocking group is selected from the group consisting of 5′-methyl modification, 5′-O-methyl modification, or 5 ′ azide modification.
(Item 170)
164. The siRNA of any one of items 35-111, 114-121, 130-136, 143-144, and 147-162, further comprising a 3 ′ cap.
(Item 171)
170. The siRNA of item 170, wherein the 3 ′ cap is an inverted deoxythymidine.
(Item 172)
Further, according to any one of items 35-111, 114-121, 130-136, 143-144, and 147-161, comprising at least one of a 2′-deoxy modification and / or a methylphosphonate internucleotide linkage The siRNA as described.
(Item 173)
Item 35-111, 114-121, 130, further comprising a modification selected from the group consisting of a 2′-alkyl modification, a 2′-O-alkyl modification, and a 2′-halogen modification in one or more pyrimidines. The siRNA according to any one of ˜136, 143 to 144, and 147 to 161.
(Item 174)
175. siRNA according to item 175, wherein the 2′-alkyl modification is a 2′-methyl modification.
(Item 175)
175. siRNA according to item 175, wherein the 2′-O-alkyl modification is 2′-O-methyl modification.
(Item 176)
175. siRNA according to item 175, wherein the 2′-halogen modification is 2′-fluorine modification.
(Item 177)
181. The siRNA of any one of items 87-111, 114-121, 130-136, 143-144, and 147-178, further comprising a conjugate.
(Item 178)
180. The siRNA of item 179, wherein the conjugate is selected from the group consisting of amino acids, peptides, polypeptides, proteins, sugars, carbohydrates, lipids, polymers, nucleotides, polynucleotides, and combinations thereof.
(Item 179)
179. siRNA according to item 179, wherein the conjugate comprises cholesterol or PEG.
(Item 180)
179. siRNA according to item 179, wherein the conjugate further comprises a label.
(Item 181)
184. siRNA according to item 182, wherein the label is a fluorescent label.
(Item 182)
184. siRNA according to item 183, wherein the fluorescent label is selected from the group consisting of TAMRA, BODIPY, Cy3, Cy5, fluorescein, and Dabsyl.
(Item 183)
87. siRNA according to any one of items 38-41 and 48-86, wherein the conjugate further comprises a label.
(Item 184)
185. siRNA according to item 185, wherein the label is a fluorescent label.
(Item 185)
187. siRNA according to item 186, wherein the fluorescent label is selected from the group consisting of TAMRA, BODIPY, Cy3, Cy5, fluorescein, and Dabsyl.
(Item 186)
90. siRNA according to any one of items 36, 51, 65, 76, 87, and 88, wherein the 2'-alkyl modified nucleotide is a 2'-methyl modified nucleotide.
(Item 187)
89. siRNA according to any one of items 36, 51, 65, 76, 87, and 88, wherein the 2'-O-alkyl modified nucleotide is a 2'-O-methyl modified nucleotide.
(Item 188)
The 2'-amine modified nucleotide is 2'-NH ₂ 90. siRNA according to any one of items 36, 51, 65, 76, 87, and 88, which is a modified nucleotide.
(Item 189)
89. siRNA according to any one of items 87 and 88, wherein the 2′-halogen modified nucleotide is a 2′-fluorine modified nucleotide.
(Item 190)
89. siRNA according to any one of items 45, 59, 73, 84, and 88, wherein the modified internucleotide linkage is methylphosphonate.
(Item 191)
90. siRNA according to item 89, further comprising at least one modified internucleotide linkage.
(Item 192)
195. siRNA according to item 193, wherein the at least one modified internucleotide linkage is selected from the group consisting of a phosphorothioate bond, a phosphorodithioate bond, a methylphosphonate bond, and combinations thereof.
(Item 193)
164. The siRNA of any one of 88-111, 114-121, 130-136, 147-158, and 162, further comprising at least one 2'-orthoester modification.
(Item 194)
195. siRNA according to item 195, wherein the 2′-orthoester modification is 2′-bis (hydroxyethyl) orthoester modification.
(Item 195)
164. siRNA according to any one of items 35-74, 87-88, 159-161, and 163, wherein the 2 'orthoester modification is a 2'-bis (hydroxyethyl) orthoester.
(Item 196)
190. A method for performing RNA interference, comprising administering to a cell the siRNA of any one of items 35-74, 87-88, 159-161, and 163-194.
(Item 197)
199. The method of item 198, wherein the orthoester modification is 2'-bis (hydroxyethyl) orthoester.
(Item 198)
A method for performing RNA interference, comprising administering to the cell the siRNA according to any one of items 195 to 199.
(Item 199)
siRNA,
(A) an antisense strand;
(B) a sense strand;
Including
The sense strand is comprised of a first 5 'terminal sense nucleotide and a second 5' terminal sense nucleotide, wherein the first 5 'terminal sense nucleotide has a first 2' carbon sense modification; And the second 5 ′ terminal sense nucleotide has a second 2 ′ carbon sense modification.
(Item 200)
siRNA,
(A) composed of a first 5 'terminal sense nucleotide and a second 5' terminal sense nucleotide, wherein the first 5 'terminal sense nucleotide has a first 2' carbon sense modification; and A sense strand wherein the second 5 ′ terminal sense nucleotide has a second 2 ′ carbon sense modification;
(B) composed of a first 5 ′ terminal antisense nucleotide and a second 5 ′ terminal antisense nucleotide, wherein the first 5 ′ terminal antisense nucleotide has a 5 ′ phosphorylation modification, and An antisense strand wherein the second 5 ′ terminal antisense nucleotide has a 2 ′ carbon antisense modification;
SiRNA containing.
  For a better understanding of other advantages and embodiments of the present invention and further advantages and embodiments, reference is made to the following description in conjunction with the examples, the scope of which is set forth in the appended claims. Yes.

  The preferred embodiments of the present invention are not intended to limit the scope of the invention in any way, but are chosen for illustration and description purposes. The advantages of preferred embodiments of some aspects of the invention are illustrated in the accompanying drawings.

FIG. 1A illustrates the functionality of orthoester modification to the sense and / or antisense strand measured 24 hours after transfection. See “Preferred Embodiments” for a description of modified duplexes. The degree of SEAP silencing induced by each siRNA was determined by comparing the level of SEAP expression in cells co-transfected with siRNA + SEAP expression plasmids to cells transfected with SEAP expression plasmid alone. FIG. 1B shows the functionality of the orthoester modification (on the sense and / or antisense strand) measured 48 hours after transfection. FIG. 2A shows the functionality of the orthoester modification (on the sense and / or antisense strand) along with other modifications as measured 24 hours after transfection. FIG. 2B shows the functionality of the orthoester modification on the sense and / or antisense strand along with other modifications as measured 72 hours after transfection. FIG. 2C shows the functionality of the orthoester modification on the sense and / or antisense strand, along with other modifications, as measured 144 hours after transfection. FIG. 3 shows the effect of sense and antisense strand modifications on siRNA functionality. FIG. 4 illustrates the effect of sense and antisense strand modifications on siRNA functionality. FIG. 5 shows the effect of thio modification of the antisense strand in combination with various modifications on the sense strand. FIG. 6 shows the effect of phosphorothioate modification on both the sense and antisense strands. FIG. 7 shows the effect of 2'-O-methyl modifications (in both sense and antisense strands) in combination with various other modifications. FIG. 8 shows the effect of siRNA, a 2'-deoxy-RNA hybrid (either strand) in combination with other chemical modifications. FIG. 9 shows the functionality of the 5 'end of the cholesterol conjugate at the 5' end of the sense strand in combination with other chemical modifications. FIG. 10 shows the functionality of the PEG conjugate at the 5 ′ end of the sense strand in combination with other chemical modifications. FIG. 11 shows the increased potency of the modified siRNA with a cholesterol conjugate at the 5 ′ end of the sense strand. FIG. 12 shows a protected RNA nucleotide phosphoramidite that can be used in Dharmacon 2'-ACE RNA synthesis chemistry. FIG. 13 shows an overview of the Dharmacon RNA synthesis cycle. FIG. 14 shows the structure of a preferred 2'-ACE protected RNA. FIG. 15A shows the functional consequences of a single 2'-deoxy modification (sense or antisense strand) on other naked siRNAs. FIG. 15B shows the functional impact of two tandem 2'-deoxy modifications (sense or antisense strand) on other naked siRNAs. FIG. 15C shows the functional impact of three tandem 2'-deoxy modifications (sense or antisense strand) on other naked siRNAs. FIG. 16A shows the functional effects of single 2'-O-methyl modifications (sense or antisense strand) on other naked siRNAs. FIG. 16B shows the functional impact of two tandem 2'-O-methyl modifications (sense or antisense strand) on other naked siRNAs. FIG. 16C shows the functional impact of three tandem 2'-O-methyl modifications (sense or antisense strand) on other naked siRNAs. FIG. 17 shows the effect of modification with sense and antisense strands on duplex stability. FIG. 18 shows the effect of a conjugate comprising a 5 'cholesterol moiety on the passive uptake of naked and modified siRNA. FIG. 19 shows the functional impact of two tandem 2'-deoxy modifications at various positions within the sense strand. FIG. 20 shows the functional impact of three tandem 2'-deoxy modifications at various positions within the sense strand. FIG. 21 shows the functional impact of a single 2'-deoxy modification at various positions within the antisense strand. FIG. 22 shows the functional impact of two tandem 2'-deoxy modifications at various positions within the antisense strand. FIG. 23 shows the functional impact of three tandem 2'-deoxy modifications at various positions within the antisense strand. FIG. 24 shows the functional impact of two tandem 2'-O-methyl modifications at various positions within the sense strand. FIG. 25 shows the functional impact of three tandem 2'-O-methyl modifications at various positions within the sense strand. FIG. 26 shows the functional impact of a single 2'-O-methyl modification at various positions on the antisense strand. FIG. 27 shows the functional impact of two tandem 2'-O-methyl modifications at various positions in the antisense strand. FIG. 28 shows the functional impact of three tandem 2'-O-methyl modifications at various positions in the antisense strand. FIG. 29 shows two (1) 5 ′ sense, (2) 5 ′ antisense, or (3) 5 ′ sense and two on position 1 and 2 of the antisense strand using siRNA against the human cyclophilin gene. Shows the functional impact of '-O-methyl modification. FIG. 30 shows (1) 5 ′ sense, (2) 5 ′ antisense, or (3) 2 ′ on positions 1 and 2 of the 5 ′ sense and antisense strands using siRNA against the firefly luciferase gene. Shows the functional impact of '-O-methyl modification. Note: In this figure, phosphorylation of the 5 'end of the antisense strand is denoted by "P". FIG. 31 shows two (1) 5 ′ sense, (2) 5 ′ antisense, or (3) 5 ′ sense and two on position 1 and 2 of the antisense strand using siRNA against the firefly luciferase gene. Shows the functional impact of '-O-methyl modification. Note: In this figure, phosphorylation of the 5 'end of the antisense strand is denoted by "P". FIG. 32 shows the stability of the modified siRNA in human serum. Modification "a" = 2'-O-methyl modification of all Cs and Us in the sense strand (combined with 3'idT capping). Modification "b" = 2'F modification of all Cs and Us in the sense strand (combined with 3'idT capping). FIG. 33 shows the affinity of modified siRNA-cholesterol conjugates for albumin and other serum proteins. FIG. 34 shows the effect that small molecule conjugates have on siRNA potency. FIG. 35 shows the stability of siRNA conjugates in human serum. siRNA carries on the sense strand (1) a 2′-O-methyl group on all Cs and Us, and (2) a 3′idT cap, and siRNAa-conjugates on the sense strand (1 It carries a 2'-O-methyl group on all Cs and Us, (2) a 3'idT cap, and (3) a 5 'cholesterol conjugate. FIG. 36 shows the effect that cholesterol conjugates have on passive siRNA uptake. FIG. 37A represents the results obtained from a typical serum stability test showing an ethidium bromide stained gel containing siRNA exposed to serum (upper unmodified, lower modified with “molecule 1 modified”). . For the purposes of this figure, the term “molecule 1 modification” refers to 2′-O-methyl modifications on positions 1 and 2 of the sense strand, 2′-O on all Cs and Us of the sense strand. -Refers to a molecule comprising a methyl modification, a 2'-fluoro (Fl) modification on all Cs and Us of the antisense strand, and a phosphate modification on the 5 'end of the antisense strand. FIG. 37B is a set of line graphs plotting the relative stability of four human cyclophilin B siRNAs in modified and unmodified forms (U 1 = 5′GAA AGA GCA UCU ACG GUG A (sequence No. 315), U2 = 5′GAA AGG AUU UGG CUA CAA A (SEQ ID NO: 316), U3 = 5′ACA GCA AAU UCC AUC GUG U (SEQ ID NO: 317), and U4 = 5′GGA AAG AGU AGU AGU AU (SEQ ID NO: 318), sense strand). The X axis represents time (hr). The Y axis represents the ratio of double strands that keep the original state. Black squares indicate unmodified sequences. Open squares indicate the modified sequences. FIG. 38 compares the ability of siRNAs (U1 and U3) that are naked or modified (molecular 1 modification) to silence a given target at various concentrations. The Y axis represents the degree of expression compared to the control group (untransfected cells). The X-axis represents the concentration of siRNA during the transfection procedure. Black bars represent unmodified sequences. The white bar represents the modified sequence. For the purposes of this figure, the term “molecule 1 modification” refers to 2′-O-methyl modifications on positions 1 and 2 of the sense strand, 2′-O on all Cs and Us of the sense strand. -Refers to a molecule comprising a methyl modification, a 2'-fluoro (Fl) modification on all Cs and Us of the antisense strand, and a phosphate modification on the 5 'end of the antisense strand. FIG. 39 assesses the degree of silencing induced over 7 days by modified siRNA (with “molecule 1 modification”) and unmodified siRNA. The Y-axis represents the degree of gene expression when compared to the control group (untransfected cells). The X axis represents the number of days after transfection. Black bars represent unmodified sequences. White bars represent modified sequences. For the purposes of this figure, the term “molecule 1 modification” refers to 2′-O-methyl modifications on positions 1 and 2 of the sense strand, 2′-O on all Cs and Us of the sense strand. -Refers to a molecule comprising a methyl modification, a 2'-fluoro (Fl) modification on all Cs and Us of the antisense strand, and a phosphate modification on the 5 'end of the antisense strand. FIG. 40 compares the degree of cell death induced by four individual modified siRNAs (based on molecular 1 modification) and unmodified siRNA transfected into cells at various concentrations. The Y axis represents the relative viability of cells (based on untransfected cells). The X axis represents the concentration of siRNA when performing the transfection procedure. Cultures were tested 24-48 hours after transfection using the Alamar Blue assay. Black bars represent unmodified sequences. White bars represent modified sequences. For the purposes of this figure, the term “molecule 1 modification” refers to 2′-O-methyl modifications on positions 1 and 2 of the sense strand, 2′-O on all Cs and Us of the sense strand. -Refers to a molecule comprising a methyl modification, a 2'-fluoro (Fl) modification on all Cs and Us of the antisense strand, and a phosphate modification on the 5 'end of the antisense strand. FIG. 41 compares the degree of off-targeting by four individual human cyclophilin B siRNAs, which are a modified form (based on molecule 1 modification) and an unmodified form. The data was divided into 6 different groups. That is, the number of targets in which expression decrease is more than 4 times (4x), the number of targets in which expression reduction is 3-4 times (3-4x), 2.5-3 times (2.5-3x) The number of targets with reduced expression of 4x (> 4x), the number of targets with increased expression greater than 4x (> 4x), the number of targets with 3-4x (3-4x) increased expression, and an additional 2.5 It is the number of targets where an expression increase of ˜3 times (2.5-3 ×) is seen Black bars represent unmodified sequences. White bars represent modified sequences. For the purposes of this figure, the term “molecule 1 modification” refers to 2′-O-methyl modifications on positions 1 and 2 of the sense strand, 2′-O on all Cs and Us of the sense strand. -Refers to molecules containing methyl modifications, 2'-fluoro (Fl) modifications on all Cs and Us of the antisense strand, and phosphate modifications on the 5 'end of the antisense strand. FIG. 42 shows a histogram comparing the gene silencing ability of cycl14 siRNA comprising (1) molecular 2 modifications, (2) naked molecule modification with Cy3, and (3) naked NSC9 molecule modified with Cy3. Show. The Y axis shows the degree of human cyclophilin B expression compared to the control gene (GAPDH). “Lipid” refers to control cells treated with the transfection reagent Lipofectamine 2000. “Control” refers to untreated cells. NS9 is a non-specific sequence # 9. For the purposes of this figure, the term “molecule 2 modification” refers to 2′-O-methyl modifications on positions 1 and 2 of the sense strand, 2′-O on all Cs and Us of the sense strand. -Methyl modification, Cys3 label at the 5 'end of the sense region, 2'-fluoro (Fl) modification on all C and U of the antisense strand, and phosphate modification on the 5' end of the antisense strand. It refers to the molecule that contains it. Figures 43A and B show the relationship between function and modification for 2'-O-methylated SEAP-2217 siRNA. These figures show single strand 2′-O-methyl modifications (black bars) and paired 2′-O-methyl modifications (gray) of the sense strand (FIG. 43A) and antisense strand (FIG. 43B) of SEAP-2217. ) Shows the effect of gene silencing. The X axis represents the relative position of the modification along the siRNA (5 '→ 3'). The Y axis represents the expression ratio relative to the control group. 44A and 44B show the effect of addition of 2'-deoxy groups on SEAP2217 function. In particular, the 2'-deoxy group modifies three consecutive nucleotides (eg, positions 1, 2, and 3). An identification number is associated with each group (eg, S3D-16 is decoded as follows: “S” = sense strand modification, “3” = number of consecutively modified nucleotides, ““ D ”= 2 ′ deoxy modification,“ 16 ”= first position of modification). FIG. 44A shows sense strand modification. FIG. 44B shows antisense strand modification. Control groups include no siRNA and non-specific (ns) RNA. 45A-F shows 2′-O-methylation with or without 5 ′ phosphorylation on the antisense strand of six luciferase specific siRNAs (luc8, 18, 56, 58, 63, and 81). The effect of “S” = sense strand. “AS” = antisense strand. “*” Indicates 2′-O-methylation at positions 1 and 2 of the designated strand. “P” indicates 5 ′ phosphorylation of the designated strand. The Y axis shows the percent expression of control (non-transfected) cells. “Control” = mock-transfected cells. Figures 46A-E represent the dose dependence of five luciferase specific siRNAs (luc8, 56, 58, 63, 81) produced according to one embodiment of the present invention. “S” = sense strand. “AS” = antisense strand. “M” indicates 2′-O-methylation at positions 1 and 2 of the designated strand. “P” indicates 5 ′ phosphorylation of the designated strand. The Y-axis represents the expression rate of control (non-transfected) cells. The X-axis represents the concentration of siRNA during the transfection with 1 = 200 mM, 2 = 100 nM, 3 = 10 nM, 4 = 1 nM, 5 = 0.1 nM, and 6 = 0.01 nM. Figures 46A-E represent the dose dependence of five luciferase specific siRNAs (luc8, 56, 58, 63, 81) produced according to one embodiment of the present invention. “S” = sense strand. “AS” = antisense strand. “M” indicates 2′-O-methylation at positions 1 and 2 of the designated strand. “P” indicates 5 ′ phosphorylation of the designated strand. The Y-axis represents the expression rate of control (non-transfected) cells. The X-axis represents the concentration of siRNA during the transfection with 1 = 200 mM, 2 = 100 nM, 3 = 10 nM, 4 = 1 nM, 5 = 0.1 nM, and 6 = 0.01 nM. Figures 46A-E represent the dose dependence of five luciferase specific siRNAs (luc8, 56, 58, 63, 81) produced according to one embodiment of the present invention. “S” = sense strand. “AS” = antisense strand. “M” indicates 2′-O-methylation at positions 1 and 2 of the designated strand. “P” indicates 5 ′ phosphorylation of the designated strand. The Y-axis represents the expression rate of control (non-transfected) cells. The X-axis represents the concentration of siRNA during the transfection with 1 = 200 mM, 2 = 100 nM, 3 = 10 nM, 4 = 1 nM, 5 = 0.1 nM, and 6 = 0.01 nM. FIG. 47 shows the expected kinetics of the sense and antisense strand-RISC interaction and (1) 2′-O-methylation (*) and (2) the sensitivity of the interaction to the relative functionality of the molecule. Represents. For highly functional molecules (eg,> F90, functionality greater than 90%), RISC is highly selective for the antisense strand (K _overall = 100). Modification of the sense strand with 2'-O-methyl groups at nucleotides 1 and 2 further tends to AS strand selection, but the relative rate of increase and decrease is mild. Sense and antisense strands derived from poor or moderately functional duplexes show a better balanced equilibrium with RISC (K _{overall ˜1} ). In these cases, modification of S essentially removes the ability to bind to RISC and strongly tilts its selectivity for RISC-AS interaction. FIGS. 48A and 48B show a study of competition between cyclo4 siRNA and NS4 siRNA. A-I: Cells were transfected with various concentrations of cyclo4 siRNA. A-II: cyclo4 (at various concentrations), 2′-O-methyl group at positions 1 and 2 of the sense and antisense strands, 2′-O-methyl group at Cs and Us of the sense strand, anti Cy3 conjugated 19 bp NS4 duplex modified by a 2′F1 group on the Cs and Us of the sense strand and a Cy3 group on the 5 ′ end of the sense strand. A-III: cyclo4 (at various concentrations), a 2′-O-methyl group at positions 1 and 2 of the sense and antisense strands, a 2′-O-methyl group at Cs and Us of the sense strand, and Cys3 conjugated 17 bp NS4 duplex modified by 2′F1 group attached to Cs and Us of the antisense strand. BI: cyclo4 (at various concentrations) and 19 bp NS4 duplex. B-II: cyclo4 (at various concentrations), a 2′-O-methyl group at positions 1 and 2 of the sense and antisense strands, a 2′-O-methyl group at Cs and Us of the sense strand, and 19 bp NS4 duplex modified by 2′F1 groups on Cs and Us of the antisense strand. B-III: cyclo4 (at various concentrations), 2'-O-methyl groups at positions 1 and 2 of the sense and antisense strands, 2'-O-methyl groups at Cs and Us of the sense strand, and 17 bp NS4 duplex modified by 2'Fl groups on Cs and Us of the antisense strand. Note: All transfections were performed using Liphofectamine 2000 (manufacturer's instructions) and a nucleic acid concentration of 100 nM. Data were normalized to internal GAPDH levels. FIGS. 48A and 48B show a study of competition between cyclo4 siRNA and NS4 siRNA. A-I: Cells were transfected with various concentrations of cyclo4 siRNA. A-II: cyclo4 (at various concentrations), 2′-O-methyl group at positions 1 and 2 of the sense and antisense strands, 2′-O-methyl group at Cs and Us of the sense strand, anti Cy3 conjugated 19 bp NS4 duplex modified by a 2′F1 group on the Cs and Us of the sense strand and a Cy3 group on the 5 ′ end of the sense strand. A-III: cyclo4 (at various concentrations), a 2′-O-methyl group at positions 1 and 2 of the sense and antisense strands, a 2′-O-methyl group at Cs and Us of the sense strand, and Cys3 conjugated 17 bp NS4 duplex modified by 2′F1 group attached to Cs and Us of the antisense strand. BI: cyclo4 (at various concentrations) and 19 bp NS4 duplex. B-II: cyclo4 (at various concentrations), a 2′-O-methyl group at positions 1 and 2 of the sense and antisense strands, a 2′-O-methyl group at Cs and Us of the sense strand, and 19 bp NS4 duplex modified by 2′F1 groups on Cs and Us of the antisense strand. B-III: cyclo4 (at various concentrations), 2'-O-methyl groups at positions 1 and 2 of the sense and antisense strands, 2'-O-methyl groups at Cs and Us of the sense strand, and 17 bp NS4 duplex modified by 2'Fl groups on Cs and Us of the antisense strand. Note: All transfections were performed using Liphofectamine 2000 (manufacturer's instructions) and a nucleic acid concentration of 100 nM. Data were normalized to internal GAPDH levels. FIG. 49 shows a competition experiment between GAPDH and non-specific control # 2. (1) non-competing siRNA cycl14, (2) competing siRNA (non-specific sequence # 2), or (3) labeled with 2'-O-methyl groups at positions 1 and 2 of both sense and antisense strands GAPDH siRNA was introduced into HeLa cells at various concentrations (0.781 to 100 nM) along with the competing siRNA.

(Detailed explanation)
The invention will now be described in connection with a preferred embodiment. These embodiments are presented to aid the understanding of the present invention and are not intended and should not be construed as limiting the present invention in any way. All alternatives, modifications, and equivalents that can be readily understood by one of ordinary skill in the art upon reading this disclosure are encompassed within the spirit and scope of the present invention.

  This disclosure is not a primer on compositions and methods for performing RNA interference. The basic concepts known to those skilled in the art were not described in detail.

  The present invention relates to compositions and methods for performing RNA interference, including siRNA-induced gene silencing. By using the present invention, modified polynucleotides, and derivatives thereof, the efficiency of RNA interference applications can be improved.

  Unless otherwise stated, the following terms and expressions have the meanings defined below.

(Alkyl)
The term “alkyl” refers to a hydrocarbyl moiety that is saturated or unsaturated, and further substituted or unsubstituted. It may be linear, branched, cyclic, and / or heterocyclic and composed of a moiety containing a functional group such as ether, ketone, aldehyde, carboxylate.

  Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl , Nonadecyl, eicosyl, and higher alkyl groups, as well as 2-methylpropyl, 2-methyl-4-ethylbutyl, 2,4-diethylproyl, 3-propylbutyl, 2,8-dibutyldecyl, 6, Examples include 6-dimethyloctyl, 6-propyl-6-butyloctyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, and 2-ethylhexyl. The term alkyl also includes alkenyl groups such as vinyl, allyl, aralkyl, and alkynyl groups.

  Substitution within an alkyl group can include any atom or group. This is acceptable for alkyl moieties, including but not limited to halogens, sulfur, thiols, thioethers, thioesters, amines (primary, secondary, or tertiary), amides, ethers, esters, alcohols, and oxygen. Can be mentioned. As an example, an alkyl group includes an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro, nitroso, or a nitrile group, and a heterocycle such as an imidazole, hydrazino, or hydroxylamino group, an isocyanate or cyanate group, a sulfoxide, Sulfur-containing groups such as disulfides can also be included.

  In addition, alkyl groups may also include hetero substitutions, in which carbon atoms are substituted, for example, with nitrogen, oxygen, or sulfur. Heterocyclic substitution refers to an alkyl ring having one or more heteroatoms. Examples of heterocyclic moieties include, but are not limited to, morpholino, imidazole, and pyrrolidone.

(2′-O-alkyl modified nucleotide)
The phrase “2′-O-alkyl modified nucleotide” refers to a nucleotide unit, which is a sugar moiety, eg, a carbon located at the 2 ′ position of the sugar. It has a deoxyribosyl moiety that is modified at the 2 'position so that an oxygen atom is attached to both the atom and the alkyl group.

(Amine and 2 'amine modified nucleotides)
The term “amine” refers to being derived directly or indirectly from ammonia, for example, by replacing one, two, or three hydrogen atoms with another group (eg, an alkyl group). Primary amines have the general structure RNH ₂ and secondary amines have the general structure R ₂ NH. The phrase “2 ′ amine modified nucleotide” refers to a nucleotide unit having a sugar moiety that is modified by an amine or nitrogen containing group attached to the 2 ′ position of the sugar.

  The term amine includes, but is not limited to, methylamine, ethylamine, propylamine, isopropylamine, aniline, cyclohexylamine, benzylamine, polycyclic amine, heteroatom-substituted aryl and alkylamine, dimethylamine, diethylamine, Diisopropylamine, dibutylamine, methylpropylamine, methylhexylamine, methylcyclopropylamine, ethylcyclohexylamine, methylbenzylamine, methylcyclohexylmethylamine, butylcyclohexylamine, morpholine, thiomorpholine, pyrrolidine, piperidine, 2,6- Examples include dimethylpiperidine, piperazine, and heteroatom-substituted alkyl or aryl secondary amines.

(Antisense strand)
As used herein, the phrase “antisense strand” refers to a polynucleotide or a region of a polynucleotide that is substantially or 100% complementary to a target nucleic acid of interest. Say. The antisense strand can be composed of a polynucleotide region (ie, RNA, DNA, or chimeric RNA / DNA). For example, the antisense strand may be wholly or partially complementary to a single molecule of messenger RNA, RNA that is not mRNA (eg, tRNA, rRNA, and hnRNA), or a coding or non-coding DNA sequence. Also good. The phrase “antisense strand” includes the antisense region of both polynucleotides formed from two different strands and a unimolecular siRNA capable of forming a hairpin structure. The phrases “antisense strand” and “antisense region” are intended to be equivalent and are used interchangeably. The antisense strand can be modified by various groups of small molecules and / or conjugates.

(2 'carbon modification)
The phrase “2 ′ carbon modification” refers to a nucleotide unit having a sugar moiety (eg, a deoxyribosyl moiety modified at the 2 ′ position). At this position, the carbon atom located at the 2 'position of the sugar and 2'-O-methyl, 2'-O-ethyl, 2'-O-propyl, 2'-O-isopropyl, 2'-O-butyl Alkyl groups such as 2′-O-isobutyl, 2′-O-ethyl-O-methyl (—OCH ₂ CH ₂ OCH ₃ ), and 2′-O-ethyl-OH (—OCH ₂ CH ₂ OH); The “2′-O-alkyl modified nucleotide” is modified so that an oxygen atom is attached to both. “2 ′ carbon sense modification” refers to a modification at the 2 ′ carbon position of a nucleotide on the sense strand or within the sense region of a polynucleotide. A “2 ′ carbon antisense modification” refers to a modification at the 2 ′ carbon position of a nucleotide on the antisense strand or within the antisense region of a polynucleotide.

(Complementarity)
The term “complementary” refers to the ability of polynucleotides to base pair with each other. Base pairs are generally formed by hydrogen bonds between nucleotide units within an antiparallel polynucleotide chain or region. Complementary polynucleotide strands or regions can base pair in a Watson-Crick fashion (eg, A and T, A and U, C and G), or a stable duplex Base pairs can be formed in any other way that allows them to be formed.

  Full complementarity or 100% complementarity refers to a situation where each nucleotide unit of one polynucleotide strand or region can form a hydrogen bond with each nucleotide of the other polynucleotide strand or region. Less than perfect complementarity means that some but not all of the nucleotide units of the two strands or two regions can hydrogen bond to each other. For example, for two 20-mers, if only two base pairs on each strand are capable of hydrogen bonding to each other, this indicates that their polynucleotide strands or regions are 10% complementary. In the same example, a polynucleotide strand exhibits 90% complementarity if 18 base pairs on each strand or region can hydrogen bond with each other. Substantial complementarity refers to a polynucleotide strand or region that exhibits 90% or greater complementarity.

(Conjugate and terminal conjugate)
The term “conjugate” refers to a molecule or moiety that alters the physical properties of the siRNA, such as increasing stability and / or promoting the uptake of the siRNA by itself. “Terminal conjugates can be attached to the 3 ′ and / or 5 ′ ends of the siRNA directly or via a linker. The internal conjugate can be directly or indirectly via a linker, It is possible to attach to the base, the 2 ′ position of ribose, or any other suitable position or positions, such as 5-aminoallyl uridine.

  For siRNAs with two different strands that are not attached to each other (ie, the siRNA is not a single molecule or a hairpin siRNA), one or both 5 ′ ends of the strand can have a conjugate, and / or One or both 3 ′ ends of the containing strand can have a conjugate.

  Conjugates are, for example, amino acids, peptides, polypeptides, proteins, antibodies, antigens, toxins, hormones, lipids, nucleotides, nucleosides, sugars, carbohydrates, polymers (eg, polyethylene glycol and polypropylene glycol), and also these substances Or any analog or derivative thereof. Additional examples of conjugates also include steroids (eg, cholesterol), phospholipids, di and triacylglycerols, fatty acids, carbohydrates with or without unsaturation or substitution, enzyme substrates, biotin, digoxigenin, and polysaccharides. . Still other examples include thioethers such as hexyl-S-tritylthiol, acyl chains such as thiocholesterol, dodecanediol or undecyl groups, dihexadecyl-rac-glycerol, triethylammonium, 1,2-di-O-hexadecyl-rac- Examples include glycero-3-H-phosphonate, polyamine, polyethylene glycol, adamantaneacetic acid, palmityl moiety, octadecylamine moiety, hexylaminocarbonyl-oxycholesterol, farnesyl, geranyl, and geranylgeranyl moieties.

  The conjugate may also be a detectable label. For example, the conjugate can be a fluorophore. Conjugates include fluorophores such as TAMRA, BODIPY, cyanine derivatives (eg, Cy3 or Cy5 dabsyl), fluorescein, or other known suitable fluorophores.

  Conjugates may be attached at any position on the terminal nucleotide that is convenient and does not substantially interfere with the desired activity of the siRNA carrying it (eg, at the 3 ′ or 5 ′ position of the ribosyl sugar). The gate is such that siRNA's ability to mediate RNA interference is reduced by more than 80% in an in vitro assay using cultured cells whose functionality is measured 24 hours after transfection. When adversely affecting functionality, it substantially interferes with the desired activity of the siRNA.

(Deoxynucleotide)
The term “deoxynucleotide” refers to a 2 ′ or 3 ′ sugar moiety having a suitable bond and / or a 2 ′, 3 ′ terminal dideoxy instead of having a hydrogen bonded to the 2 ′ and / or 3 ′ carbon. It refers to a nucleotide or polynucleotide lacking an OH group at the 'position.

(Deoxyribonucleotide)
The terms “deoxyribonucleotide” and “DNA” refer to a nucleotide or polynucleotide comprising at least one ribosyl moiety with H at the 2 ′ position of the ribosyl moiety.

(downstream)
One region of a single strand of nucleotides is the region where the 5 'most portion of the first region is closest to the 3' end of the second region. In some cases, it is considered downstream of the second region.

(Exaequio)
The phrase “exaequio agent” refers to a nucleic acid that is inactive or semi-inactive in terms of its potential to participate in the RNAi pathway or the ability to compete with other nucleic acids for its ability to participate in the RNAi pathway. Say. The molecule can be used as an exaequio agent, which is used to make the total amount of nucleic acids in solution uniform or equivalent.

(First 5 ′ terminal antisense nucleotide)
The phrase “first 5 ′ terminal antisense nucleotide” refers to the most position of the 5 ′ end of the strand with respect to the base of the antisense strand or region that has a corresponding complementary base on the sense strand or region. ) Nucleotides in the antisense strand or region located in Thus, in a siRNA composed of two different strands (ie, not a single molecule or hairpin siRNA), the 5 ′ end most different from the base that is part of any 5 ′ overhang on the antisense strand It is called a base. When the first 5 ′ terminal antisense nucleotide is part of a hairpin molecule, the term “terminal” refers to the relatively 5 ′ most terminal position within the antisense region; Therefore, it is the 5 ′ most terminal nucleotide of the antisense region.

(First 5 ′ terminal sense nucleotide)
The phrase “first 5 ′ terminal sense nucleotide” is defined in terms of an antisense nucleotide. For molecules composed of two different strands (ie, not a single molecule or hairpin siRNA), the 5 ′ extreme position of the strand with respect to the base of the sense strand with the corresponding complementary base on the antisense strand Refers to the nucleotide of the sense strand that is located. Thus, for siRNAs composed of two different strands (ie, not single molecules or hairpin siRNAs), the 5′-most base other than the base that is part of any 5 ′ overhang on the sense strand or region It is. When the first 5 ′ terminal sense nucleotide is part of a unimolecular siRNA molecule capable of forming a hairpin, the term “terminal” is complementary to the first 5 ′ terminal antisense nucleotide. The relative position within the sense strand or region measured by the distance from the base it has.

(Functionality)
siRNAs can be divided into five groups (non-functional, semi-functional, functional, highly functional, and superfunctional) based on the level or degree of silencing induced in cultured cell lines. is there. As used herein, these differences are based on a set of conditions that include that the siRNA is transfected into the cell line at a concentration of 100 nM, and that the level of silencing depends on the level of silencing. It is to be examined approximately 24 hours after transfer (but should not exceed 72 hours after transfection). Within this context, a “non-functional siRNA” is defined as an siRNA that induces less than 50% (<50%) of target silencing. A “semi-functional siRNA” induces 50-79% target silencing. A “functional siRNA” is a molecule that induces 80-95% gene silencing. “High-functional siRNA” is a molecule that induces gene silencing of more than 95%. “Hyperfunctional siRNA” is a special molecular species. For the purposes of this document, hyperfunctional siRNAs induce (1) silencing of specific targets at a rate of greater than 95% when transfected at sub-nanomolar concentrations (ie, less than 1 nanomolar); In addition and / or (2) is defined as a molecule that induces a functional (or good) level of silencing for over 96 hours. These relative functionalities (not intended to be absolute) are used to compare siRNAs with specific targets for applications such as functional genomic analysis, target identification, and drug therapy.

(Functional dose)
“Functional dose” refers to the dose of siRNA that has the effect of reducing the 100 nM concentration of mRNA by 95% or more at 24, 48, 72, and 96 hours after administration. On the other hand, the “slightly functional dose” of siRNA has the effect of reducing mRNA at 100 nM concentration by 50% or more 24 hours after administration, and further, the “non-functional dose” of RNA. "Indicates the effect of reducing the decrease in mRNA at 100 nM concentration to less than 50% 24 hours after administration.

(halogen)
The term “halogen” refers to any atom of fluorine, chlorine, bromine, iodine, or astatine. The phrase “2 ′ halogen modified nucleotide” refers to a nucleotide unit having a sugar moiety modified with a halogen at the 2 ′ position directly attached to the 2 ′ carbon.

(2 'halogen-modified pyrimidine)
The phrase “2 ′ halogen modified pyrimidine” refers to a pyrimidine (eg, cytosine or uracil) containing a halogen group attached to the 2 ′ carbon of the nucleotide sugar.

(Internucleotide linkage)
The phrase “internucleotide linkage” refers to the type of linkage or linkage that exists between two nucleotide units within a single siRNA, either modified or unmodified. There may be. The phrase “modified internucleotide linkage” encompasses all modified internucleotide linkages now known, now known, or inferred by one of ordinary skill in the art upon reading this disclosure. An internucleotide linkage may have an associated counterion, and the term is intended to include such a counterion and any coordination compound that can be formed by an internucleotide linkage. .

  Modifications of internucleotide linkages include, but are not limited to, phosphorothioates, phosphorodithioates, methyl phosphonates, 5′-alkylene phosphonates, 5′-methyl phosphonates, 3′-alkylene phosphonates, 3′-5 ′. Linked or 2′-5 ′ linked boron trifluoride, boranophosphate ester, and selenophosphate, phosphate triester, thionoalkylphosphotriester, hydrogen phosphonate linkage, alkyl phosphonate, alkylphosphonothioate, Arylphosphonthioate, phosphoroselenoate, phosphorodiselenoate, phosphinate, phosphoramidate, 3'-alkyl phosphoramidate, aminoalkyl phosphoramidate, thionophosphoramidate, phosphoropiperadida Phosphoroanilothioate, phosphoroanilide, ketone, sulfone, sulfonamide, carbonate, carbamate, methylene hydrazo, methylene dimethyl hydrazo, form acetal, thioform acetal, oxime, methylene imino, methylene methyl imino, thioamidate, Linkage with riboacetyl group, aminoethylglycine, silyl or siloxane linkage, alkyl containing or without heteroatoms consisting of 1 to 10 carbons containing heteroatoms and / or saturated or unsaturated and / or substituted Or a combination of a cycloalkyl linkage, a linkage with a morpholino structure, a polyamide to which a base can be attached directly or indirectly to the aza nitrogen of the main chain, and their modified internucleotide linkage within siRNA .

(Linker)
“A linker is a moiety that attaches to other moieties (eg, nucleotides and conjugates thereof). The conjugate enhances the stability and / or ability of the molecule to be taken up by the cell, while the linker is attached to the cell. It is possible to distinguish between a linker and a conjugate simply by attaching the conjugate to the molecule to be incorporated.

  By way of example, linkers may be modified or unmodified nucleotides, nucleotides, polymers, sugars and other carbohydrates, polyethers such as polyethylene glycol, polyalcohol, polypropylene, propylene glycol, mixtures of ethylene and propylene glycol, polyalkylamines, spermidine Such as polyamines, polyesters such as poly (ethyl acrylate), polyphosphodiesters, and alkylenes. An example of a conjugate and its linker is cholesterol-TEG-phosphoramidite, cholesterol is a conjugate, and tetraethylene glycol and phosphate are used as linkers.

(nucleotide)
The term “nucleotide” refers to ribonucleotides or deoxyribonucleotides, or modified forms thereof, as well as analogs thereof. Nucleotides include purines such as adanine, hypoxanthine, guanine, and derivatives and analogs thereof, and pyrimidines include, for example, cytosine, uracil, thymine, and derivatives and analogs thereof.

Nucleotide analogs include, but are not limited to, nucleotides with modifications in the chemical structure of bases, sugars, and / or phosphates, such as 5-position pyrimidine modification, 8-position purine modification, cytosine Substitution of exocyclic amines and 5 bromouracil; and 2 ′ sugar modifications include, but are not limited to, sugar modified ribonucleotides, where the 2′-OH of the ribonucleotide is H, OR, Substituted by R, halo, SH, SR, NH ₂ , NHR, NR ₂ , CN, or the like, where R is an alkyl moiety as defined herein. Nucleotide analogs are also intended to include bases (eg, inosine, cuocin, xanthine), sugars (eg, 2′-methyl ribose), unnatural phosphodiester linkages (eg, methyl phosphonate, phosphorothioate), and peptides. doing.
Modified base refers to a nucleotide base, such as adenine, guanine, cytosine, thymine, and uracil, xanthine, inosine, modified by substitution or addition of one or more atoms or groups, and Cueocin is mentioned. Some examples of the types of modifications that can be constructed from nucleotides modified with respect to the base moiety include, but are not limited to, various alkylated, halogenated, thiolated, aminated, amidated, or acetylated These combinations of bases can be mentioned. As more specific modified bases, for example, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N, N-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-amino Adenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides with modifications at position 5, 5- (2-amino) propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methyloxyuridine, deaza Nucleotides such as 7-deaza-adenosine, 6-azo Lysine, 6-azocytidine, 6-azothymidine, 5-methyl-2-thiouridine, other thiobases such as 2-thiouridine and 4-thiouridine, and 2-thiocytidine, dihydrouridine, pseudouridine, cuocin, alkaeosin, naphthyl and substitution Naphthyl groups, optional O- and N-alkylated purines and pyrimidines such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridin-4-one, pyridin-2-one, phenyl and modifications Phenyl groups such as aminophenol or 2,4,6-trimethoxybenzene, modified cytosine acting as G-clamp nucleotides, 8-substituted adenines, and guanines, 5-substituted uracils and thiamines, azapyrimidi , Carboxyalkyl hydroxyalkyl nucleotides, carboxyl alkyl nucleotides, and include alkylcarbonyl alkylated nucleotides. Modified nucleotides also include nucleotides that are modified with respect to the sugar moiety and nucleotides that have a sugar or analog that is not ribosyl. For example, sugar moieties include mannose, arabinose, glucopyranose, galactopyranose, 4′-thioribose, as well as other sugars, heterocycles, or carbocycles. The term nucleotide is also intended to include those known to those skilled in the art as a broad base. Examples include, as an example, a wide range of bases, including but not limited to 3-nitropyrrole, 5-nitroindole, or nebulaline.

  In addition, the term nucleotide also includes species having a detectable label (eg, a radioactive or fluorescent moiety) or mass label attached to the nucleotide.

(Nucleotide unit)
The phrase “nucleotide unit” refers to a single nucleotide residue, a modified or unmodified nitrogen base, a modified or unmodified sugar, and a conjugate that excludes two nucleotides or further linkages. It consists of a modified or unmodified part that allows ligation.

(Off target)
The terms “off-target” and the phrase “off-target effect” refer to an siRNA or shRNA directed to a given target and another mRNA sequence, DNA sequence, or cellular protein or other part. Any event that interacts directly or indirectly to produce an unintended effect. For example, an “off-target effect” is due to the partial homology or complementarity between the sense and / or antisense strand of the siRNA or shRNA and the other transcript, to the other transcript. It can occur when there is simultaneous degradation.

(Orthoester)
The term “orthoester protected” or “orthoester modified” refers to the modification of a sugar moiety within a nucleotide unit by an orthoester. Preferably the sugar moiety is a ribosyl moiety. Generally, it has an RC (OR ′) ₃ structure, where R ′ is the same or different, R is H, and underlined C is the orthoester central carbon. The orthoester of the present invention is composed of an orthoester in which the carbon of the sugar moiety of the nucleotide unit is bonded to oxygen and then to the central carbon of the orthoester. The next bond to the orthoester central carbon is two oxygen atoms, for a total of three oxygens to bond to the orthoester central carbon. These two oxygens bonded to the central carbon (none of which are bonded to the carbon of the sugar moiety) are then bonded to the carbons that make up the same or different two moieties. For example, one of the oxygens can be bonded to the ethyl moiety and the other can be bonded to the isopropyl moiety. In one example, R can be H, one R ′ can be a ribosyl moiety, and the other two R ′ can be two 2-ethyl-hydroxyl moieties. The orthoester can be placed at any position of the sugar moiety, for example, at the 2 ′, 3 ′, and / or 5 ′ positions. Preferred orthoesters and methods for producing orthoester protected polynucleotides are described in US Pat. Nos. 5,889,136 and 6,008,400.

(Overhang)
The term “overhang” refers to the formation of a terminal base pair resulting from a single strand or region where the first strand or region extends beyond the end of the complementary strand forming a duplex. One or more nucleotides that do not exist. One or both of two polynucleotides or polynucleotide regions capable of forming a duplex through base-paired hydrogen bonds are complementary 5 ′ and / or 3 shared by the two polynucleotides or regions. It is possible to have a 3 'and / or 5' end beyond the 'end. Single stranded regions extending beyond the 3 ′ and / or 5 ′ ends of the duplex are referred to as overhangs.

(Pharmaceutically acceptable carrier)
The phrase “pharmaceutically acceptable carrier” includes, but is not limited to, a composition that facilitates the introduction of dsRNA, dsDNA, or dsRNA / DNA hybrids into cells. Or dispersing agents, coating agents, anti-infective agents, isotonic agents, and agents that mediate the absorption time or release of the polynucleotides and siRNAs of the invention.

(Polynucleotide)
The term “polynucleotide” refers to a polymer of nucleotides, including but not limited to DNA, RNA, DNA / RNA hybrids, alternating regularly and irregularly. A polynucleotide chain consisting of a deoxyribosyl moiety and a ribosyl moiety (ie, the alternating nucleotide unit has -OH, then -H, then -OH, then H, etc. at the 2 'position of the sugar moiety); Such modifications of the polynucleotide in which the various components or moieties are attached to the nucleotide unit at any position. Unless otherwise stated or apparent from the context, the term “polynucleotide” includes unimolecular siRNAs and siRNAs composed of two different strands.

(Polyribonucleotide)
The term “polyribonucleotide” refers to a polynucleotide comprising two or more modified or unmodified ribonucleotides and / or analogs thereof.

(Ribonucleotides and ribonucleic acids)
The terms “ribonucleotide” and the phrase “ribonucleic acid” (RNA) refer to a modified or unmodified nucleotide or polynucleotide comprising at least one ribonucleotide unit. The ribonucleotide unit is located at the 1 'position of the ribosyl moiety with a nitrogen base attached to the N-glycoside linkage at position 1 of the ribosyl moiety, the 2' of the moiety that allows or prevents ligation to another nucleotide. Contains oxygen attached to the position.

(RNA interference and RNAi)
The phrases “RNA interference” and the term “RNAi” are synonymous and refer to a process in which a polynucleotide or siRNA comprising at least one ribonucleotide unit has an effect on a biological process. This process includes, but is not limited to, the degradation of mRNA, attenuation of translation, and interaction with tRNA, rRNA, hnRNA, cDNA, and genomic DNA, as well as by additional proteins. Includes gene silencing performed by methylation.

(Second 5 ′ terminal antisense nucleotide)
The phrase “second 5 ′ terminal antisense nucleotide” refers to a nucleotide directly adjacent to the first 5 ′ terminal antisense nucleotide and attached to the 3 ′ position of the first 5 ′ terminal antisense nucleotide. I mean. Thus, it is the second nucleotide that is the 5 ′ most end of the antisense strand or region within the set of nucleotides for which the corresponding sense nucleotide is present.

(Second 5 ′ terminal sense nucleotide)
The phrase “second 5 ′ terminal sense nucleotide” refers to a nucleotide directly adjacent to the first 5 ′ terminal sense nucleotide and attached to the 3 ′ position of the first 5 ′ terminal sense nucleotide. Say. Thus, it is the second nucleotide that is the 5'-most end of the sense strand or region within the set of nucleotides for which the corresponding antisense nucleotide is present.

(Sense strand)
The phrase “sense strand” refers to a polynucleotide or region having the same nucleotide sequence, partially or entirely, as a target nucleic acid such as messenger RNA or DNA sequence. The phrase “sense strand” includes both the sense region of a polynucleotide formed from two different strands and the sense region of a unimolecular siRNA capable of forming a hairpin structure. Where a sequence is provided, by convention it is the sense strand (or region) unless explicitly stated, and the presence of a complementary antisense strand (or region) is implied. The phrases “sense strand” and “sense region” are intended to be equivalent and are used interchangeably.

(SiRNA, ie short interfering RNA)
The terms “siRNA” and the phrase “short interfering RNA” refer to unimolecular nucleic acids, are capable of performing RNAi, and have a base pair length between 18 and 30 A nucleic acid composed of two different strands having a double strand. Also, the term “siRNA” and the phrase “short interfering RNA” include nucleic acids that also include moieties other than ribonucleotide moieties, including but not limited to modified nucleotides, modified nucleotide linkages, non-nucleotides, deoxy Examples include nucleotides, and analogs of the above nucleotides.

  siRNA can be double-stranded, short hairpin RNA, RNA having a loop length of about 4 to 23 nucleotides, RNA having a stem-loop-like protrusion, microRNA, and short transient RNA Is mentioned. Examples of RNA having a loop or hairpin loop include a structure in which the loop is bound to a stem part (stem) by a linker such as a flexible linker. Flexible linkers are composed of a wide variety of chemical structures and are of sufficient length and material to allow effective intermolecular hybridization of stem components. Generally, the span length is at least about 10-24 atoms.

  When the siRNA is a hairpin, the sense strand and the antisense strand are part of one long molecule.

(Stabilization)
The term “stabilized” refers to the ability of a dsRNA to resist degradation while remaining functional, and the ability is reduced by half in the presence of biological material such as serum, for example. Measured as a period. The half-life of siRNA refers to the time taken for 50% degradation of siRNA in serum, for example.

(Substantial complementarity)
Substantial complementarity refers to a polynucleotide chain that exhibits 90% or more complementarity.

(Track ability)
The term “trackability” refers to the ability to track the movement or placement of a molecule, such as after the molecule has been introduced into a cell. Molecules that are “trackable” are useful in observing successes and failures of cell transfection procedures and the like.

(Preferred embodiment)
The invention will now be described in connection with a preferred embodiment. These embodiments are provided to aid the understanding of the present invention and are not intended to limit the present invention in any way and should not be construed as having such intention. . All alternatives, modifications, and equivalents that can be understood by one of ordinary skill in the art upon reading this disclosure are within the spirit and scope of the present invention.

  According to a first embodiment, the present invention provides siRNA. The siRNA comprises a sense strand comprising a polynucleotide composed of at least one orthoester modified nucleotide and an antisense strand comprising at least one 2 'modified nucleotide unit. Preferably, the modified nucleotide is a ribonucleotide or an analogue thereof. Orthoesters can be placed at any position on the sugar moiety, for example at the 2 ', 3', and / or 5 'positions. Preferably, the orthoester moiety is at the 2 'position of the sugar moiety. Preferably, orthoesters and methods of making polynucleotide-protected orthoesters are described in US Pat. Nos. 5,889,136 and 6,008,400. Also preferably, the orthoester is attached to the 2 'position of the ribosyl moiety. Preferably, the orthoester contains two 2-ethyl-hydroxyl substituents. The most preferred orthoesters are shown below and are referred to herein as 2'-ACE or 2'-bis (hydroxyethyl) orthoester moieties. That is,

The advantages of including an orthoester group on the sense strand can be seen by reference to FIGS. 1A, 1B, 2A, and 2C.

  The generation of the data shown in FIG. 1 was performed in several variants by Dharmacon Inc. This was carried out using siRNA double-stranded target SEAP (human secreted alkaline phosphatase) synthesized by using the registered trademark ACE chemistry. These variants include naked or unmodified RNA, ie ACE-protected RNA in which each 2′-OH is modified with an orthoester, and fluorine bound to each 2 ′ carbon and to each C and U. 2 'fluoro modified variants.

  The siRNA duplex can be composed of a sense strand and an antisense strand. An array consisting of all possible combinations of sense and antisense strands was generated. Referring to FIG. 1, the following terms are used.

  Naked sense strand in S-siRNA duplex.

  AS-naked antisense strand within siRNA duplex.

  2'ACE protected sense strand in pS-siRNA duplex.

  2'ACE protected antisense strand within the pAS-siRNA duplex.

  2FS-sense strand in siRNA with all C and U, with the fluorine atom attached to the 2 'carbon of each C and / or U carrying nucleotide unit.

  2FAS—Antisense strand in an siRNA with all C and U, with the fluorine atom attached to the 2 'carbon, or each C and / or U carrying nucleotide unit.

  S-AS refers to a double-stranded siRNA formed from a naked sense strand and a naked antisense strand.

  pS-AS refers to a double-stranded siRNA formed from an ACE-modified sense strand and a naked antisense strand.

  2'-ACE modification of all nucleotides except Cs and Us, with P2'F-2 'modification.

  Double strands were co-transfected with pAAV6 plasmid (SEAP expression plasmid) and the desired siRNA, or siRNA was transfected into HEK293 cells that stably express SEAP. Under both conditions, standard transfection procedures were used, but the results did not change between Hela, MDA75, or 3TELi (mouse) cell lines.

  The level of siRNA-induced SEAP silencing was determined at different time points (24, 48, 72, or 144 hours) after transfection using the Clontech SEAP detection kit according to the manufacturer's protocol. Protein reduction levels are in good agreement with mRNA reduction levels (mRNA levels were measured using the QuantiGene kit (Bayer)). siRNA-induced toxicity was measured using the AlmaBlue toxicity assay or the housekeeping gene (cycloline) or both expression levels. Unless otherwise stated, no significant toxicity was observed.

  Cells were transfected with each duplex at varying concentrations between 1 and 100 nanomolar (Figure 1) and between 10 and 1 micromolar (Figure 2) (Figure 1). 1 and 2 show the effect of introducing ACE modifications on the sense and antisense strands of siRNA duplexes in combination with naked and 2 'fluoro modifications.

  The presence of ACE modifications to the antisense strand of the oligo significantly interferes with siRNA duplex functionality. ACE modified sense oligos affected SEAP silencing regardless of whether they were used with naked or 2'F modified AS oligos.

  The degree of silencing was compared at 24, 48, or 72 hours, and a detectable decrease in siRNA silencing was observed after 144 hours.

FIGS. 3 and 4 summarize siRNA functional screens in which the AS (FIG. 3) and sense (FIG. 4) strands are kept constant and the various strands on the opposite strand are screened in combination. In particular, the AS chain is (1) naked, (2) modified with a 2′F group at all C and U, (3) in positions 2, 4, 6, 14, and 18 (counting from the 5 ′ side) Modification with 2 ′ deoxy group, (4) Modification with 2′-O-methyl group on all of U and Cs, except those located at positions 67-10 (calculated from the 5 ′ side of the chain), (5) Either modification with phosphorothioate at all positions or (6) modification with phosphorothioate at positions 1-6 and 14-19 (calculated from the 5 'side of the chain). These AS chains are (1) naked, (2) 2′-ACE modified at all positions, (3) 2′F modified at all Cs and Us, and (4) modified at all Us. 2 ′ amine (NH ₂ ), (5) 2′-deoxy modifications to all odd nucleotides (eg, 1, 3, 5,...), (6) 2′- at all C and U O-methyl modification or (7) pairing with a sense strand carrying phosphorothioate at all positions. The results of these experiments show that while the described 2′-O-methyl modifications of the antisense strand are not tolerated, other modifications, including phosphorothioate, 2′F, and 2′deoxy modifications, are not In combination with sense strand modification, it shows what is possible on this strand. Similarly, in FIG. 4, amine modifications are generally not tolerated to the sense strand, while 2′-ACE, 2′F, 2′deoxy, 2′-O-methyl, and phosphorothioate additions vary. Has been shown to be possible without significant disruption of functionality.

  Figures 5, 6, 7, and 8 represent more detailed data grouped according to the type of modification used. In FIG. 5, a naked, 2′O-ACE modified, or 2′F modified sense strand starts with 2 phosphorothioates at each end of the 2′F-8T AS = strand, the second nucleotide at each end, Plus F groups in C and U; 8T AS = 4 phosphorothioates at each end of the chain, starting from the second nucleotide at each end; 4T-AS = each of the chains starting from the second nucleotide at each end Paired with either of the two phosphorothioates on the ends. In FIG. 6, the antisense strand containing the phosphorothioate modification at the even position, or position 12 (AS-Thio12, 6 phosphorothioates on either end of the chain), was bare 2 ′ at all positions. -Paired with a complementary sense strand that was ACE modified or phosphorothioate modified at all positions. Alternatively, the sense strand with phosphorothioate at all positions is bare, has a 2′F group at all C and U, has phosphorothioate at all positions, or has 12 phosphorothioates (6 at each end) Nucleotide modification) Paired with antisense strand. For FIG. 7, 2′-Ome = 2′-O-methyl modifications on U and C except those occurring between nucleotides 7-11 and S-Ome = sense 2 on C and U The antisense strand carrying the '-O-methyl modification is naked, S-2'ACE = 2'ACE modification on all nucleotides; S-deoxy hybrid = all odd nucleotides across the strand (1 , 3, 5 ...) 2'-deoxy modification; S-Ome = all C and U 2'-O-methyl modifications of the sense strand; AS-2'F = all C and C on the strand 2'F modification of U; AS-deoxy hybrid = 2'deoxy modification of nucleotides 2, 4, 6, 14, 16, 18; or As-Ome = something occurring between nucleotides 7-11 It matches any of the 2'-O- methyl modification of all U and C, excluding. With respect to FIG. 8, the AS chain carrying the deoxy modification (AS-deoxy hybrid = 2 'deoxy modification of nucleotides 2, 4, 6, 14, 16, 18) was combined with the sense strand. Where the sense strand is bare, S-2′ACE modification = 2′ACE modification on all nucleotides; S-Ome = 2′O-methyl modification on all U and C; or 2′deoxy (1 , 3rd, 5th, ... etc.). Similarly, sense-2 ′ deoxy hybrids (positions 1, 3, 5,... Other) are converted to naked 2′F modifications (C and U), 2′-O-methyl (positions 7˜ U and C except those between positions 11) and 2 ′ deoxy (2, 3, 6, 14, 14, 16, 18) were paired with the antisense strand.

  In particular, FIG. 5 shows that phosphorothioate modifications are well tolerated when placed on the antisense strand in combination with a naked 2'ACE modified and 2'F modified sense strand.

  FIG. 6 further shows that phosphorothioate backbone modifications are tolerated on both the sense and antisense strands with similar limitations for non-specifically induced toxicity.

  FIG. 7 shows that the presence of 2'-O-methyl modifications is well tolerated on the sense of the siRNA duplex, but not on the antisense strand of the siRNA duplex. It is worth mentioning that a functional siRNA duplex is formed by the combination of a 2'-O-methyl modified AS strand and a deoxy hybrid of the sense strand.

  FIG. 8 shows the suitability of deoxyhybrid modification with RNA interference. A deoxyribohybrid is an RNA / DNA hybrid oligo, in which both deoxy and ribo constituents are incorporated into an oligo in a sequence of alternating deoxyribonucleotides and ribonucleotides, for example. In designing these types of oligos, it is important to avoid introducing RNase H activity by keeping the size of the spread of the continuous DNA / RNA duplexes shorter than 5 nucleotides. The deoxyribohybrid was functional in both sense and antisense strands in combination with 2'fluoro and 2'ACE modified oligos. The deoxyhybrid sense strand was also the only modification (under those conditions) that supported siRNA activity when the antisense strand was modified with 2'-O-methyl.

  FIG. 9 shows the usefulness of a conjugate comprising cholesterol to improve the efficacy of ACE and 2'fluoro modified siRNA. By using a conjugate containing cholesterol on the sense strand, the ineffective effect due to the modification to the sense strand is reduced, but the negative effect due to the modification to the antisense strand is not improved.

  FIG. 10 shows equivalent data for PEG conjugates on the sense strand.

  FIG. 11 shows that the presence of a conjugate comprising cholesterol improves the efficacy of the modified siRNA oligo.

  Figure 12 shows the structure of protected RNA nucleotides used in Dharmacon's 2'-ACE RNA synthesis chemistry.

  FIG. 13 outlines the RNA synthesis cycle. Preferably, the cycle is performed in an automated fashion on a suitable synthesizer. In step (i), the incorporated phosphoramidite (here having uridine as the nitrogen base) can have any acceptable group on the phosphoramidite moiety at the 3 'position instead of the indicated methyl group. For example, an alkyl group or a cyanoethyl group can be used at that position. Depending on certain changes, when synthesizing polynucleotides with modified internucleotide linkages, and / or when synthesizing polynucleotides with other modifications (eg, at the 2 ′ position), Can be implemented as described.

  FIG. 14 shows the structure of the 2'-ACE protected RNA product immediately prior to 2 'deprotection. If it is necessary to retain the orthoester in the 2 'position, this 2' deprotection step is not performed.

  A 19-mer duplex with a di-dT overhang at the 3 ′ end, ie, having 2 ′ amine-modified nucleotide units at the second, fifth, seventh, ninth, eleventh and nineteenth positions of the sense strand For G2'-N-UGA2'-N-UG2'-N-UA2'-N-UG2'-N-UCAGAGAG2'-NUdTdT, the antisense strand is bare or 2'fluoro modified at all C and U Regardless of whether it is a deoxyhybrid that contains ribonucleotide units and deoxyribounits alternately or has a 2'-O-methyl modification, a significant loss of functionality occurred. Preferably, the sense strand does not contain 2 'amino modifications at the second, fifth, seventh, ninth, eleventh and nineteenth positions.

  On siRNA 19-mers with 3 ′ di-dT overhangs (see SEQ ID NOs: 171 to 314), replacement of any ribonucleotide unit with a deoxyribonucleotide unit may be performed on either the sense or antisense strand. , There is no significant effect on the functionality of the 19mer in RNAi (see FIG. 15A). Replacement of two adjacent ribonucleotide units by two adjacent deoxyribonucleotide units on the same siRNA 19mer does not significantly affect the functionality of the 19mer within RNAi. FIG. 15B shows that when 1 and 2 positions, 3 and 4 positions, 5 and 6 positions are individually modified to deoxyribonucleotides, the functionality is not significantly affected when the modification occurs in the sense strand. It shows that such modifications on the antisense strand have a slight negative impact on functionality. On the same siRNA 19-mer, replacement of three adjacent ribonucleotide units by three consecutive deoxyribonucleotide units does not significantly affect functionality if the modification is on the antisense. , When the modified unit is the first to third, or seventh to ninth nucleotides of the sense strand, it is significantly (in some cases) functionally affected. In this experiment, polyribonucleotide units 1-3, 4-6, 7-9 etc. were individually replaced by deoxyribonucleotide units (see FIG. 15C).

  On the same siRNA 19-mer polyribonucleotide with a 3 ′ di-dT overhang, modification of any individual unit with a 2′-O-methyl moiety, whether the modification is in the sense or antisense strand, RNAi It does not significantly affect the functionality of the 19-mer in (see FIG. 16A). Using the same siRNA 19-mer, substitution of two adjacent ribonucleotide units by two adjacent 2′-O-methyl modifications may occur at the first and second, or thirteenth and fourteenth positions of the antisense strand. Or, unless modification occurs at positions 17 and 18 of the sense strand, it does not significantly affect 19-mer functionality with RNAi (see FIG. 16B). Subsequent experiments confirmed that positions 13 and 14 of the antisense strand and positions 7 and 8 of the sense strand were not critical. Most notably, if no modification is added that corrects for the change in functionality, the first and second positions of the antisense strand should not have 2'-O-methyl modifications if functionality is maintained. Using the same siRNA 19-mer, substitution of three adjacent ribonucleotide units with successive 2′-O-methyl modifications is such that the modifications are on the antisense strand at positions other than positions 1 to 3. In some cases, it does not significantly affect functionality (see FIG. 16C). In this experiment, polyribonucleotides 1 to 3, 4 to 6, 7 to 9, etc. were individually modified with 2'O-methyl moieties.

  Modification of the same polyribonucleotide with either a single 2'-deoxy moiety or a single 2'-O-methyl moiety does not significantly affect functionality. Modification of the antisense strand at positions 1 and 2, or 1, 2, and 3 with two or more 2'-O-methyl moieties in tandem can significantly reduce functionality. Positions 7-9 on the sense strand and 13-15 on the antisense strand are sensitive to two or more 2'-O-methyl modifications in tandem. Thus, preferably the antisense strand is first and second, first, second, and third, thirteenth and fourteenth, and first, unless competing modifications occur at other positions in the duplex. It does not contain a 2'-O-methyl modification at positions 13, 14 and 15. Subsequent experiments show that 2'-O-methyl modifications at positions 1 and 2 (or 1 and 2, and 3) on the antisense strand are key to double-stranded functionality. Positions 7-9 on the sense strand and positions 13-15 on the antisense strand are less important.

  It is less wasted as a practical problem to synthesize a sense strand in which all nucleotides are modified with an orthoester group rather than a sense strand in which all of the nucleotides are modified only in the selected nucleotide. However, theoretically, since practical means for modifying only certain nucleotides have been developed to synthesize the sense strand, these polynucleotides could be used in the present invention.

Preferably, the 2 ′ modified nucleotide is selected from the group consisting of 2 ′ halogen modified nucleotides, 2 ′ amine modified nucleotides, 2′-O-alkyl modified nucleotides, and 2 ′ alkyl modified nucleotides. Where the modification is a halogen, the halogen is preferably fluorine. When the modification is fluorine, it is preferably attached to one or more nucleotides containing a cytosine or uracil base moiety. When the 2 ′ modified nucleotide is a 2 ′ amino modified nucleotide, the amine is preferably —NH ₂ . When the 2 ′ modified nucleotide is a 2′-O-alkyl modification, preferably the modification is a 2′-O-methyl, ethyl, propyl, isopropyl, butyl, or isobutyl moiety, most preferably 2′- The O-alkyl modification is a 2′-O-methyl moiety. Where the 2 ′ modified nucleotide is a 2′-alkyl modification, preferably the modification is a 2 ′ methyl modification, wherein the carbon of the methyl moiety is attached directly to the 2 ′ carbon of the sugar moiety.

  FIG. 2C shows that the siRNA effect begins to fade gradually 144 hours after transfection. The dose and potency of the modified oligo was comparable to the naked siRNA duplex.

  According to a second embodiment, the present invention provides siRNA comprising a sense strand. Wherein the sense region comprises at least one orthoester modified nucleotide provided according to the first embodiment, at least one 2 ′ modified nucleotide provided according to the first embodiment, and a conjugate. Including. Preferably, the modification is a 2 'methyl modification, and the carbon of the methyl moiety is attached directly to the 2' carbon of the sugar moiety.

  FIG. 2C shows that the siRNA effect begins to fade gradually 144 hours after transfection. The dose and potency of the modified oligo was comparable to the naked siRNA duplex.

  According to a second embodiment, the present invention provides siRNA comprising a sense strand. Wherein the sense region comprises at least one ortho ester modified nucleotide provided according to the first embodiment, at least one 2 ′ modified nucleotide provided according to the first embodiment, and a conjugate. Including.

  The conjugate within this embodiment is preferably selected from the group consisting of amino acids, peptides, polypeptides, proteins, sugars, carbohydrates, lipids, polymers, nucleotides, polynucleotides, and combinations thereof. More preferably, it is selected from the group consisting of cholesterol, polyethylene glycol, antigen, antibody, and receptor ligand. Even more preferably, the conjugate comprises cholesterol or polyethylene glycol. Most preferably, the conjugate comprises cholesterol and is linked to the 5 'terminal nucleotide unit of the sense strand at the 5' carbon position.

  Introduction of a cholesterol-modified conjugate at the 5 'end of the sense strand resulted in an increase in potency against orthoester modification and 2' antisense modified siRNA comparable to or better than naked or unmodified duplexes. See FIGS. 9 and 11. Introduction of a cholesterol-containing conjugate at the 5 'end of the sense strand results in a functionally effective amount for orthoester modification and 2' fluorine modified siRNA comparable to or better than the corresponding bare duplex. It was.

  FIG. 9 shows the usefulness of cholesterol to improve the efficacy of ACE and 2 'fluorine modified siRNA. A positive cholesterol effect was observed mainly due to modifications introduced on the sense chloride. For example, RNAi is produced in the absence of a transfection agent by attaching a cholesterol site to the 5 'end located at the 5' position of the sense strand of SEQ ID NOs: 1 to 16 (see Fig. 18).

  According to a third embodiment, the present invention provides an siRNA having a sense strand composed of at least one orthoester modified nucleotide, an antisense strand, and a conjugate. In this embodiment, the orthoester modification of the first embodiment may be used in combination with the conjugate of the second embodiment.

  According to a fourth embodiment, the present invention provides a siRNA having a sense strand, an antisense strand, and a conjugate, wherein the sense strand and / or the antisense strand has at least a 2 'modified nucleotide. The 2 'modified nucleotide of this embodiment is preferably selected based on the same parameters as the 2' modified nucleotide of the first embodiment. Similarly, the conjugate is selected based on the same parameters for which the conjugate was selected in the second embodiment.

  According to a fifth embodiment, the present invention comprises at least one orthoester modified nucleotide and 2 ′ halogen modified nucleotide, 2 ′ amine modified nucleotide, 2′-O-alkyl modified nucleotide, and 2 ′ alkyl modified nucleotide. An antisense strand composed of at least one 2′-modified nucleotide selected from the group consisting of amino acids, peptides, polypeptides, proteins, sugars, carbohydrates, lipids, polymers, nucleotides, polynucleotides, and combinations thereof A siRNA comprising a conjugate selected from the group, wherein the polynucleotide comprises 18-30 nucleotide base pairs.

The orthoester of this embodiment is selected based on the criteria for selecting the orthoester of the first embodiment. When the 2 ′ modification is a halogen, preferably the halogen is fluorine and at least one of the antisense strands is attached to a C and / or U containing nucleotide unit. When the 2 ′ modified nucleotide is a 2 ′ amino modified nucleotide, the amine is preferably NH ₂ . When the 2 ′ modified nucleotide is a 2′-O-alkyl modification, preferably the 2′-O-alkyl modification is a 2′-O-methyl, ethyl, propyl, isopropyl, butyl, or isobutyl moiety, most preferably Is a 2'-O-alkyl modification is a 2'-O-methyl moiety. When the 2 ′ modified nucleotide is a 2 ′ alkyl modification, preferably it is a 2 ′ methyl modification, and the carbon of the methyl moiety is attached directly to the 2 ′ carbon of the sugar moiety.

  According to a sixth embodiment, the present invention encompasses a composition comprising the following structure:

Wherein each of B ₁ and B ₂ is a nitrogen-containing base, heterocyclic, or carbocyclic compound, X is selected from the group consisting of O, S, C, and N, and W is OH, Selected from the group consisting of phosphates, phosphate esters, phosphate diesters, phosphotriesters, modified internucleotide linkages, conjugates, nucleotides, and polynucleotides, wherein R1 is an orthoester and R2 is a 2′-O-alkyl group , Alkyl groups, amines, and halogens, and Y is a nucleotide or polynucleotide. When R2 is halogen, this halogen is preferably fluorine. When R2 is fluorine, the fluorine is preferably attached to one or more C and / or U containing nucleotide units. If R2 is an amine, the amine is preferably -NH _2. When R2 is a 2'-O-alkyl modification, preferably it is a 2'-O-methyl, ethyl, propyl, isopropyl, butyl, or isobutyl moiety, more preferably a 2'-O-methyl moiety. It is. When R2 is a 2 ′ alkyl modification, preferably it is a 2 ′ methyl modification, and the carbon of the methyl modification is attached directly to the 2 ′ carbon of the sugar moiety.

  The R1, orthoester of this example is selected based on the parameters for selecting the orthoester of the first embodiment.

The broken line in a formula shows the interaction by the hydrogen bond between nitrogen containing chlorine. Preferably B ₁ and B ₂ are natural nitrogen-containing chlorines such as adenine, thymine, guanine, cytosine, uracil, xanthine, hypoxanthine, and queuosine, or analogs thereof. Preferably X is O.

  For each of the above embodiments, the siRNA is of any length, preferably 18-30 nucleotide base pairs, more preferably 18-19 base pairs, and any overhangs are removed. By using siRNA less than about 30 base pairs in length, non-specific processes (eg, interferon-related reactions that reduce the functionality of siRNA applications) can be avoided while functional in RNA interference applications. The reaction is maintained. Further preferably, the nucleotide is a ribonucleotide.

  In the above embodiments, overhangs can be present on one or both of the strands. Preferably, if the siRNA has an overhang, it is 1-6 nucleotide units in length, more preferably 2-3, even more preferably 2, and is located at the 3 'end of each strand of the siRNA. However, siRNAs with blunt ends are functional. A two nucleotide overhang is most preferred.

  Similarity in the above embodiments is that either strand or both strands of the siRNA can have one or more modified internucleotide linkages. Preferably, the modified internucleotide linkage is selected from the group consisting of phosphorothioate and phosphorodithioate. Furthermore, preferably the polynucleotide can have more than 4 modified nucleotide-related linkages. More preferably, the polynucleotide of the present invention comprises more than 8 modified internucleotide linkages. Most preferably, about 10 modified internucleotide linkages are used. Complete modification is preferred for maximum stability, but a number of factors affect the number of modification linkages that can actually be used. Such factors include the degree of stability afforded by the linkage, the extent to which the linkage affects functionality, the ability to chemically introduce linkages, and the toxicity of the linkage. Preferably, the modifications are placed at the 3 'and 5' ends for protection against exonuclease activity.

  The polynucleotide of the above embodiment of the present invention is stabilized. The half-life of these stabilized siRNAs is about 20 seconds to 100 hours or more. Preferably, the stabilized siRNA of the present invention has a half-life greater than 1 hour. More desirably, the stabilized siRNA of the invention exhibits a half-life greater than 10 hours. Most preferably, the stabilized siRNA of the invention exhibits a half-life of greater than 100 hours. Moreover, desirably, the effects of siRNA persist cell division for at least one generation.

  The polynucleotide of the present invention exhibits high stability in the presence of human serum. Preferably, for example, the half-life of the 19-mer duplex of the present invention in human serum is several minutes to 24 hours. More preferably, the half-life of 19-mer duplex in human serum is 24 hours to 3 days. Most preferably, the half-life of 19-mer duplex in serum is 3-20 days or longer.

  For a 19-mer polyribonucleotide duplex having an antisense strand with deoxyribonucleic acid modifications at positions 2, 4, 6, 14, 16, and 18, at 37 ° C for 1 hour 30 minutes, Exposure to fetal calf serum protected positions 4 and 6 from degradation, possibly by serum nucleases. Similarly, a 19-mer polyribonucleotide duplex comprising a 2'-O-methyl modification on the antisense strand in positions 2-6, 12, 14, 16, 16 and 17, and position 19. For these positions were protected from degradation by serum nucleases. Introduction of phosphorothioate modifications within the antisense strand to the 19-mer polyribonucleotide duplex between nucleotides 1-6 and 13-19 provides a modified internucleotide linkage that resists serum nuclease degradation. However, the 19-mer modified with the ACE orthoester moiety at each 2 ′ position of the antisense strand did not provide stability to human serum, but is probably caused by serum phosphodiesterase as well as serum ribonuclease. .

  Modifications at the 2 'position in the antisense strand of the polynucleotide duplex significantly increase the stability of the polyribonucleotide duplex in serum, in C and U nucleotide units. FIG. 17 shows 2′F (5′-2′GfUGAfUGfUAfUGfUfCAGAGAGfUdTdT-3 ′ for 2′-O-methyl (SEQ ID NO: 13) as a function of the type of modification at the 2 ′ position of both the sense and antisense strands. ) (SEQ ID NO: 17), phosphorothioate internucleotide linkage (SEQ ID NO: 10 and 11), and further ACE protection (SEQ ID NO: 3 and 4). The vertical axis shows the ratio of non-degradable polynucleotide and control. Thus, the higher the stability rate compared to the control, the less degradation is observed. From FIG. 17, modifying the sense strand is sufficient to achieve stability.

  Modification of each C and each U with either a 2'-O-methyl moiety or a 2 'fluoro moiety results in complete stabilization of the sense and antisense strands. Stable sense strands, such as those with 2'fluoro or 2'-O-methyl modifications, are annealed to the naked antisense strand to improve stability.

  The compositions of the present invention can be made based on Dharmacon's RNA synthesis chemistry according to a novel protecting group scheme. Silyl esters can be used to protect 5'-hydroxyl (5'-SIL) in combination with an acid labile orthoester protecting group (2'-ACE) on the 2'-hydroxyl. Such a set of protecting groups is then used in standard phosphoramidite solid phase synthesis techniques. The structure of several protected and functionalized ribonucleotide phosphoramidites are shown in FIG. As an alternative, the compositions of the present invention can be made using any other suitable RNA synthesis chemistry.

  According to a seventh embodiment, the present invention provides a method for performing RNA interference. This method consists of exposing the siRNA to the target nucleic acid to perform RNAi. In this method, the siRNA is composed of a sense strand and an antisense strand, and at least one of the sense strand and the antisense strand includes at least one orthoester-modified nucleotide.

  Preferably, the polynucleotide of the antisense strand exhibits 90% or more complementarity with the target nucleic acid of interest. More preferably, the antisense strand polynucleotide of the present invention exhibits 99% or more complementarity to the target nucleic acid of interest. Most preferably, the polynucleotide of the present invention has complete complementarity to the target nucleic acid of interest over at least 18 to 19 consecutive bases.

  Preferably, the at least one orthoester modified nucleotide is located on the sense strand and the composition of the orthoester is defined by the parameters described above for the first embodiment.

In addition to the orthoester modification, any of the other modifications described above can also be presented for use in this method. For example, the antisense strand preferably comprises at least one modified nucleotide selected from the group consisting of 2 ′ halogen modified nucleotides, 2 ′ amine modified nucleotides, 2′-O-alkyl modified nucleotides, and 2 ′ alkyl modified nucleotides, Including. When the modified nucleotide is a 2 ′ halogen modified nucleotide, the halogen is preferably fluorine. This halogen is preferably fluorine. When the halogen is fluorine, the fluorine is preferably attached to a C or U containing nucleotide unit. Where the 2 ′ modification is an amine, preferably the amine has a moiety of —NH ₂ . When the 2 ′ modification is a 2′-O-alkyl group, preferably the group has a moiety of mesooxy-OCH ₃ . When the 2 ′ modification is an alkyl group, the modification is preferably a methyl group (—CH ₃ ). Furthermore, preferably none of these modifications occur at nucleotides 8-11, more preferably none of these modifications occur at positions 7-12 of the antisense strand.

  The above method is also performed where the siRNA comprises a 5 'conjugate. This conjugate can be selected based on the aforementioned criteria for conjugate selection.

  When using these methods, the siRNA has an arbitrary number of base pairs, but is preferably 18 to 30 base pairs, more preferably 19 base pairs. More preferably, the polynucleotide comprises an antisense strand and a sense strand of ribonucleotides.

  One or more base pair overhangs of the 3 'and / or 5' terminal nucleotide units on one or both strands also exist based on the above parameters for overhangs.

  According to an eighth embodiment, the present invention provides a method for performing RNA interference, comprising exposing siRNA to a target nucleic acid. This siRNA is composed of a sense strand, an antisense strand, and a conjugate. Either the sense or antisense strand contains 2 'modified nucleotides. Preferably, the siRNA of this embodiment of the invention exhibits the same degree of complementarity as the previous embodiment.

According to this embodiment, the antisense strand is preferably at least one nucleotide selected from 2 ′ halogen modified nucleotides, 2 ′ amino modified nucleotides, 2′-O-alkyl modified nucleotides, and 2 ′ alkyl modified nucleotides including. Such modifications can be made to the antisense strand and / or the sense strand. When the modified nucleotide is a 2 ′ halogen modified nucleotide, the halogen is preferably fluorine. When the halogen is fluorine, the fluorine is preferably attached to at least two C or U containing nucleotides. Preferred 2 'amine modified is -NH _2. Preferred 2'-O- alkyl modification is -OCH _3. Preferably the 2 ′ alkyl modification is —CH ₃ .

  The above methods can also be performed with the siRNA comprising a conjugate. The conjugate is selected based on the parameters for selecting the conjugate already described. The siRNA may be any number of base pairs, but in the above-described embodiments, it is preferably 18-30 base pairs, most preferably 18-19 base pairs. Similarly, one or more base pair overhangs on the 3 'and / or 5' terminal nucleotides of either or both strands can be provided. Further, either sense or antisense can include at least one modified internucleotide linkage, preferably selected from phosphorothioate linkages and phosphorodiethate linkages. Preferably, the sense and antisense strands are polyribonucleotides.

  Each of the above-described embodiments can perform effective RNAi interference because the polynucleotide is more stable than the naked polynucleotide. Unlike naked polynucleotides, the polynucleotides of the invention are resistant to degradation by nucleases and other substances present in blood, serum, and other biological media.

  A further surprising advantage of the present invention is that it minimizes non-specific RNA interference, also called “off-targeting. Non-specific RNA interference is the function of unlabeled genes. The orthoester modifications and other modifications described herein can be used alone or in combination with each other to form the sense strand, the antisense strand, or both strands. Used to reduce or prevent such non-specific RNA interference.

  In order to reduce non-specific RNA interference, preferably the sense strand modification is performed at the 8th, 9th, 10th, or 11th nucleotide from the 5 ′ end, with the 5 ′ terminal nucleotide as the first. . More preferably, all of the 8th, 9th, 10th and 11th nucleotides are modified at the 2 'position. Most preferably, the 8th, 9th, 10th and 11th nucleotides are all modified at the 2 'position and the modification is an orthoester.

  According to a ninth embodiment, the present invention comprises exposing the siRNA to a target nucleic acid, wherein the siRNA is composed of a sense strand and an antisense strand, and the sense strand is substantially non-functional. A method for performing RNA interference is provided. What is meant by “substantially non-functional” is that the sense strand cannot inhibit the expression of any non-target gene by more than 50%. Thus, the expression of non-specific mRNA inhibited by the “substantially non-functional” sense strand is less than 50%. An additional advantage of the present invention is increased stability in serum-containing media and serum.

  According to this embodiment, the sense strand can comprise at least one 2′-O-alkyl modification, at least one cytosine-containing nucleotide base or uracil-containing nucleotide base, wherein the at least one cytosine-containing nucleotide base or Uracil-containing nucleotide bases have 2'-O-methyl modifications. Preferably, the 2'-O-alkyl modification is a 2'-O-methyl modification. More preferably, the 2'-O-alkyl modification is a 2'-O-methyl modification on the first, second, eighteenth and / or nineteenth nucleotide base.

  The sense strand can further include a conjugate. Preferably, the conjugate is cholesterol. Preferably, the cholesterol is attached to the 5 'and / or 3' end of the sense strand. Modification of siRNA duplexes with cholesterol significantly increases the affinity of the duplex for albumin and other serum proteins and alters the duplex biodistribution without causing any significant toxicity.

  The sense strand can include a cap at its 3 'end. Preferably, the cap is an inverted deoxythymidine or two consecutive 2'-O-methyl modified bases in the terminal positions (nucleotides 18 and 19).

  The antisense strand can include at least one modified nucleotide. Preferably, the at least one modified nucleotide is a 2'-halogen modified nucleotide. Most preferably, the modified nucleotide is a 2'-fluorine modified nucleotide.

  Where the sense strand comprises one or more cytosine-containing nucleotide bases and / or uracil-containing nucleotide bases, the one or more cytosine-containing nucleotide bases and / or uracil-containing nucleotide bases are 2′-fluorine modified. .

According to a tenth embodiment, the present invention provides a method of performing RNA interference, the method comprising exposing the siRNA to a target nucleic acid, wherein the siRNA comprises (a) a conjugate; b) a sense strand comprising at least one 2′-O-alkyl modification and being substantially non-functional; and (c) an antisense strand comprising at least one 2′-fluorine modification, wherein the sense strand The strand and the antisense strand form a 18-30 base pair duplex. Preferably, the at least one 2′-O-alkyl modification is the first, second, eighteenth and / or nineteenth nucleotide base. Preferably, the conjugate is cholesterol. Preferably, the cholesterol is attached to the 5 ′ and / or 3 ′ end of the sense strand.
The sense strand may further include a cap at its 3 ′ end. Preferably, the cap is an inverted deoxythymidine (idT) or two consecutive 2′-O-methyl modified bases in terminal positions (nucleotides 18 and 19).

  An advantage of the above-described embodiments of the present invention is that it allows modification of the sense strand of the siRNA duplex that promotes the orientation of the RISC complex assembly and prevents the sense strand from participating in gene silencing. The inventor systematically studied the effect of using siRNA with various modifications on the efficiency of siRNA-mediated silencing. The inventor has discovered that modifications at each position of the sense and antisense strands having duplexes with 2'-deoxy or 2'-O-methyl modifications do not interfere with siRNA function. When tandem inhibition is used for two or three such modifications, the pattern of well-tolerated modifications differs between the sense and antisense strands. The siRNA duplex with positions 1 and 2 of the sense strand modified with 2-O-methyl was fully functional. However, modification at similar positions in the antisense strand resulted in a completely non-functional siRNA. See Figures 19-31. Phosphorylation of the antisense strand at the 5 'end partially restored antisense strand functionality.

  The modifications described herein are inexpensive, reliable, non-toxic, and methods for modifying siRNA duplexes such that the sense strand cannot substantially function as an antisense strand is there. The net effect of this is an increase in siRNA specificity and potency. Recent microarray analysis suggests that the presence of 11 nucleotides is sufficient to induce non-specific silencing, as well as siRNA duplexes and sense strands of other mRNA transcripts. It has also been suggested that in some cases it is possible to produce at least half of the non-specific activity (Jackson, AL, et al. (2003) Expression profiling reviews off). -Target gene regulation by RNAi. Nature Biotechnology 21, 635-637). Therefore, if non-specific activity of the sense strand is inhibited, double strand specificity is increased. This also appears to have the effect of shifting the equilibrium towards functional RISC formation, as well as reducing the required siRNA concentration.

  The inventor provides modifications that are well tolerated and increase the stability of siRNA duplexes in the presence of serum (eg, human serum). Only stabilizing the sense strand modification of the siRNA duplex provides some degree of stability to the unmodified or naked antisense strand. Modification of the sense strand by C or U by 2'-O-alkyl modification (eg, 2'-O-methyl moiety) is quite effective for the stability of some sequences, but for other sequences Not effective. In addition, the inventors have discovered that 2'-O-methyl modification of the 5 'terminal nucleotide of the sense strand is important. Such modifications are also expected to result in a lesser degree of non-specific effects compared to fully modified siRNA. As the data herein describes, modifications at positions 1, 2, 18, and 19 of the sense strand do not interfere with duplex performance. An example of double-stranded stability caused by sense strand modification is shown in FIG. This figure shows that application of O-methyl modifications to all U and C to the double-stranded sense strand increased the half-life of anti-SEAP siRNA 2217 from 10 minutes to 4 hours. Since the idT version of the antisense strand is 2 times less stable, modification of the 3 'end with dTdT is important. Such a modification mode can be applied to any sequence since the antisense strand remains naked.

  A half-life of several hours in serum must be sufficient to enhance effective delivery of siRNA. This is because intracellular siRNA is stabilized by the RISC complex. FIG. 35 shows the stability of the siRNA duplex when C and U of the sense strand are modified with 2'-O-methyl and an idT cap is placed at the 3 'end. The functionality of this type of formulation is sequence dependent, but is significantly improved by the presence of cholesterol on the 5 'end of the sense strand. Further modifications (eg, 2′-O-methyl modifications of the sense strands C and U, plus 2′-O-methyl modifications at the 1 and 2 positions of the sense strand, 2′F of the antisense strands C and U) Modifications, even a phosphate group on the 5 ′ end of the antisense strand (see FIG. 37) further increase the serum stability of such molecules for 5 days without appreciably changing the silencing functionality. Can be made.

  The modification of siRNA with cholesterol conjugates has another unexpected feature. SiRNA modified by cholesterol exhibits a much higher affinity for albumin and other serum proteins. See FIG. Serum protein affinity has proven useful in previous studies on antisense biodegradation in mice. The presence of phosphothio modifications is responsible for most of the non-specific antisense binding activity, but has high affinity for serum proteins and thereby alters pharmacokinetic behavior, so in vivo antisense It proved beneficial for the application. Cholesterol modified siRNAs show the benefits of serum protein affinity without the disadvantage of increased non-specificity of phosphothio modifications.

  According to an eleventh embodiment, the present invention provides an siRNA composed of a sense strand and an antisense strand. The sense strand has a first 5 'terminal sense nucleotide and a second 5' terminal sense nucleotide, both of which have a 2 'carbon modification, preferably a 2'-O-alkyl modification. In some embodiments, the siRNA also includes a label. Where this label is present, it is preferably located on the first 5 'terminal sense nucleotide. Furthermore, at least one pyrimidine (eg, cytosine or uracil) on the sense strand, preferably each of the plurality of pyrimidines, has a 2 'carbon modification, preferably a 2'-O-alkyl modification. These modifications depend on the sequence of the siRNA in addition to any modification of the first or second 5 'terminal sense nucleotide, which may be a pyrimidine.

  The antisense strand of the eleventh embodiment comprises at least one 2'-halogen modified pyrimidine, preferably a fluorine modified pyrimidine, and a first 5 'terminal antisense nucleotide that is phosphorylated. Preferably, all pyrimidines on the antisense strand have a 2'-fluorine modification.

  Preferably, the siRNA has 18-30 base pairs, more preferably 19-25 base pairs, and most preferably 19-23 base pairs (over if present in a single molecule siRNA capable of forming a hairpin). Except hang or loop or stem structure). Preferably, the sense and antisense strands, except for overhangs, loops or stem structures, are at least 79% complementary, more preferably 90% complementary over a range of base pairs, and most preferably 100 over this range. % Complementarity. Similarly, preferably the antisense region is at least 79%, more preferably at least 90%, and most preferably at least 100% complementary to the target region. Preferably, the polynucleotide is RNA.

  The siRNA may include an overhang at either the 5 'or 3' end of either the sense strand or the antisense strand. However, in the case of a polynucleotide composed of two different strands, if any overhang is present, the overhang is present only at the 3 'end of the sense and / or antisense strand. Also, preferably any overhang has a length of 6 bases or less, preferably 2 bases or less. Most preferably, there is no overhang, or there is a 2 base overhang on the 3 'end of one or both of the sense and antisense strands. According to the eleventh embodiment, it is preferable not to have an overhang at the 5 'end of the antisense strand. Overhanging nucleotides are frequently removed by one or more intracellular enzymatic processes or events that leave non-phosphate 5'-nucleotides. Therefore, it is desirable not to have an overhang at the 5 'end of the antisense strand.

  Phosphorylation of the first 5 'terminal antisense nucleotide refers to the presence of one or more phosphate groups attached to the 5' carbon of the sugar moiety of the nucleotide. Preferably only one phosphate group is present.

The 2′-O-alkyl modification is preferably 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O—, regardless of which base the modification appears on. Isoprovir, 2′-O-butyl, 2′-O-isobutyl, 2′-O-ethyl-O-methyl (—CH ₂ CH ₂ OCH ₃ ), and 2′-O-ethyl-OH (—OCH ₂₎ Selected from the group consisting of (CH ₂ OH). Most preferably, the 2′-O-alkyl modification is a 2′-O-methyl moiety. Furthermore, the modification need not be identical for each of the first 5 'terminal sense nucleotide and the second 5' terminal sense nucleotide. However, from a practical point of view regarding the synthesis of the molecules of the present invention, it may be required to use the same modification as a whole.

  With respect to 2'-O-alkyl pyrimidine modifications, at least one pyrimidine other than the pyrimidine present in the first two 5 'terminal sense nucleotides is modified with a 2'-O-alkyl group. Preferably, all pyrimidines on the sense strand in the double stranded forming region are modified. Further preferably, the 2'-O-alkyl modification is a 2'-O-methyl group. Similar to the 2'-O-alkyl modification, the same alkyl modification need not be used on each nucleotide having a 2'-O-alkyl group. Where there are overhangs, loops or stems, the pyrimidines in those regions may or may not contain 2'-O-alkyl groups. However, at least one of the 2'-O-alkyl groups is desirably a sense region.

  With respect to 2 'halogen modified nucleotides, the modification occurs on at least one of the pyrimidines of the antisense strand, and more preferably occurs on all of the pyrimidines of the antisense strand. Further preferably, the halogen is fluorine. Where overhangs, stems, or loops are present, pyrimidines in those regions may or may not contain halogen groups, but preferably any stem or loop structure does not contain this modification, as described above. At least one halogen group is in the sense region. 2′-halogen modifications (including 2 ′ fluoro modifications) can be used to increase the stability of the siRNA without being affected by other modifications described herein. , You need to be careful. Thus, they can be used separately as well as using them with other modifications in siRNA applications.

  Other types of nucleotide modifications of the sense and / or antisense strands can be included unless they significantly negate the benefits of the present invention of stability and functionality in connection with the eleventh embodiment. Having a pyrimidine modified nucleotide (eg, halogen, preferably fluorine modified) and an additional 2'-O-alkyl modified pyrimidine provides a certain level of nuclease resistance.

  In addition to chemical modifications, base pair mismatches or bumps (bulges) can be added to the sense and / or antisense strand, which contribute to RISC-mediated attachment with targets that share less than 100% homology. Change the ability of those chains. Examples of such mismatches include (but are not limited to) purine-pyrimidine mismatches (eg, GU, CA) and purine-purine or pyrimidine-pyrimidine mismatches (eg, GA, U -C). Introducing these types of modifications may be combined with the aforementioned modifications to determine whether they change other attributes of functionality (eg, off-target effects) without compromising the benefits of the present invention. To be evaluated.

  In some applications, it may be required to use a label such as a fluorescent label. Where the label is fluorescent, the fluorescent label is Cy3 ™, Cy5 ™, or Alexa dye (Molecular Probes, Eugene, OR). These labels are preferably added to the 5 'end of the sense strand, more preferably to the 5' terminal sense nucleotide. These allow the user to visualize the distribution of labeled siRNA in the transfected cells, etc., and to evaluate the success of certain transfections. The use of labeled nucleotides is well known to those skilled in the art, and as a label other than a fluorescent label, for example, a mass or radioactive label can be used in applications where such a label would be beneficial.

  Fluorescence modification can be used to separate transfected cells from non-transfected cells. Specifically, after transfection of the molecule of the eleventh embodiment into the cell population, the transfected cells are separated from the non-transfected cells by sorting by FAGS. This makes it possible to obtain a more pure population (rather than a mixture) and subsequently improves the ability to clearly identify the phenotype obtained by gene silencing.

  The molecule of the eleventh embodiment has various uses, for example, it can be used as a transfection control reagent or a stable silencing reagent. If these molecules contain a label, transfection of the molecule into the cell allows the user to visualize and determine which fraction of the cell has been successfully transfected. Moreover, since these modifications do not change the siRNA function, the molecule of the eleventh embodiment can simultaneously serve as a transfection control and silencing reagent. Furthermore, since the above modifications do not limit the sequences that can be used, the modifications can be used in a variety of siRNA and RNAi applications and are not target sequence dependent.

  Thus, the molecule of the eleventh embodiment has its unique modification set, thus providing stability and “trackability” without changing functionality. These molecules can also be used to isolate a pure population of transfected cells. If a 2'-O-alkyl group is added to the first two or first three nucleotides of the antisense strand, an additional benefit of reducing off-target effects can be realized. Furthermore, the addition of 2'-O-alkyl groups to the first two or three nucleotides of the sense strand can have the added benefit of reducing the sense strand off-target effect.

According to a twelfth embodiment, the present invention provides another siRNA composed of a sense strand and an antisense strand. This sense strand is determined based on parameters similar to those of the sense strand of the eleventh embodiment, wherein the parameters include 2 ′ carbon modifications, preferably 2 ′, of the first and second 5 ′ terminal sense nucleotides. -O-alkyl modifications and at least one 2 'carbon modification of pyrimidines, preferably 2'-O-alkyl modifications. However, instead of the above modification to the antisense strand, the antisense of the twelfth embodiment consists of first and second, or first, second, and third 5 ′ terminal antisense nucleotides, Each of the nucleotides has a 2′-O-alkyl modification. In addition to the first and second 5 ′ terminal antisense nucleotides, there is at least one 2′-O-alkyl modified pyrimidine on the antisense strand. Furthermore, preferably there is no 2′Fl modification and there is no phosphorylation of the first 5 ′ terminal antisense nucleotide. The lack of phosphorylation gives a molecule with limited functionality compared to the corresponding functional polynucleotide. As another variation on the above description, the molecule of the twelfth embodiment includes a 5 ′ blocking group in the sense strand, antisense strand, or both the sense and antisense strands in addition to all of the above attributes. Such blocking groups further exclude that the molecule is phosphorylated and enter RISC, 5′-alkyl, 5′-O-alkyl, azide (N ₃ ), amines, and other It consists of parts.

  Other preferred parameters for size, overhang, and other modifications are the same as for the eleventh embodiment. However, since these compositions are not used for gene silencing, they can be 16-28 base pairs in length, preferably 18-30 base pairs in length.

  In addition to transfection control, the molecules of the twelfth embodiment can also act as potential “filters” or exaequo agents, which are negative in dose experiments or in microassay studies. It is particularly useful as a control. Many experiments test the effect of a given siRNA at different concentrations (eg, 100 nM, 50 nM, 25 nM, and 1 nM). If necessary, when performing transfection experiments, it is desirable to have a constant concentration of “total siRNA” to avoid any exceptions resulting from transfection at different nucleic acid levels. Unfortunately, adding any unmodified control siRNA (eg, a non-specific control) has a potential downtrend that competes with siRNA under studies on interactions with RISC. The molecule of the twelfth embodiment alleviates this problem. The addition of 2′-O-methyl modifications on positions 1 and 2 (or 1, 2 and 3) of both sense and antisense strands, in the absence of a phosphorylated 5 ′ terminal antisense nucleotide, Limit the ability of this molecule to interact with RISC. Thus, this molecule can be transfected but cannot compete with other siRNAs for RISC. As with the molecule of the eleventh embodiment, the addition of 2'-O-methyl groups to pyrimidines (C and U) minimizes the possibility of nuclease digestion.

  The molecules of the twelfth embodiment, unlike other “non-specific sequences or controls, do not interact well with RISC. Thus, these molecules are limited. Since it should only produce off-targeting side effects, it has the ability to be trackable without competing for sites on the RISC.

  The embodiments described above (both eleventh and twelfth) can be applied to siRNAs that do not have adjacent sense and antisense strands (ie, two separate strands), as well as single molecule siRNAs that can form hairpins ( shRNA). The term “siRNA” includes both types of RNA. For example, a hairpin comprises a loop structure (preferably containing 4-10 base pairs) and a sense region, wherein the sense region and the antisense region are each 19-23 base pairs long and are substantially Complementary. Preferred sequences of the loop structure are, for example, 5'-UUCG (SEQ ID NO: 319), 5'-UUUGUGUAG (SEQ ID NO: 320), and 5'-CUUCCUGUCA (SEQ ID NO: 321).

  The hairpin is desirably constructed with a loop basin downstream of the antisense region. This configuration is particularly desirable for the eleventh embodiment. This is because it is easier to phosphorylate the terminal antisense nucleotide. Thus, when designing unimolecular siRNAs, it is preferred that there is no overhang upstream of the 5 'terminal antisense nucleotide.

  When designing a unimolecular siRNA, particularly a left-handed unimolecular structure (eg, 5′-AS-Loop-S), according to the present invention, preferably a first 5 ′ terminal sense Nucleotides are counted from the base that is complementary to the first 5′-end antisense nucleotide (ie, counted from the 3 ′ end of the sense region) and the 18th, 19th, or 20th of the sense region. Defined as nucleotides that are bases of The first 5 'terminal sense nucleotide is thus defined. This is because when a single molecule siRNA capable of forming a hairpin enters a cell, Dicer converts a hairpin siRNA composed of two different strands (siRNA) into a hairpin composed of about 18 to 20 base pairs. This is because siRNA molecules are processed and these molecules are required to have a sense strand modification at the ends of the processed molecules. Most preferably, the first 5 'terminal sense nucleotide is defined as the nucleotide that is the 19th base of the sense region from the 3' end of the sense region. Furthermore, preferably the polynucleotide is capable of forming a counterclockwise hairpin.

  The shRNA can further comprise a stem region, the stem region comprising one or more nucleotides or modified nucleotides immediately adjacent to the 5 ′ end and the 3 ′ end of the loop structure, further comprising the stem region. One or more nucleotides or modified nucleotides are target specific or non-target specific. Preferably, the total length of the unimolecular siRNA is less than 100 base pairs, more preferably less than 85 base pairs.

  The unimolecular siRNA of the present invention is finally processed so that it is converted into two different strands depending on the cellular function. Alternatively, the molecule can be subjected to one or more steps of the RNAi pathway (eg, treatment with Dicer) and put into the RISC as a unimolecular hairpin molecule. In addition, these unimolecular siRNAs may be introduced into the cell with fewer than all modifications, and can be modified within the cell itself using the natural process or processing molecules introduced. (Eg, for the eleventh embodiment, intracellular phosphorylation by natural kinase). However, preferably all modifications already present are introduced into the polynucleotide (similarly, if the siRNA is composed of two different strands, preferably those strands are Including all modifications when introduced, even though the antisense strand can be modified, for example, by phosphorylating groups after introduction).

  Despite the fact that the above embodiments (11th and 12th) are aimed at increasing stability, their type or other types of modifications also affect other parameters, such as specificity. It is important to note.

  Furthermore, these stability and functional modifications may be combined (eg, an additional 2 ′ carbon modification (preferably —O-methyl modification) with at least one sense pyrimidine) and a 2 ′ carbon modification (preferably Are preferably all sense pyrimidines except for the first 2 (or 3) with —O-methyl modification) and the first 2 with 2 ′ carbon modification (preferably —O-methyl modification). Or first and second (or first) in combination with at least one, preferably all, 2′Fl modification of the antisense pyrimidine and phosphorylation of the 5 ′ antisense nucleotide except 3) , Second, and third) 5 'terminal sense and antisense nucleotide 2' carbon modifications (preferably -O-methyl modifications)). In addition, the above modifications of the present invention include sequences that are randomly selected or selected based on a logical design, eg, as described in US patent application Ser. No. 10 / 714,333. You may combine with siRNA.

  Stabilizing modifications to the phosphate backbone can be included in the polynucleotide for some uses of the invention. For example, at least one phosphorothioate and / or methylphosphonate can be present in any overhang, loop structure, or stem structure that may also be present in the sense and / or antisense strand of the oligonucleotide backbone. Or some or all of the phosphate groups in the 3 'position of all pyrimidines. Phosphorothioate (and methylphosphonate) analogs arise from modification of the phosphate groups of the oligonucleotide backbone. Within the phosphorothioate, phosphate O replaces the sulfur atom. In methylphosphonate, oxygen is replaced by a methyl group. In one embodiment, the phosphorothioate modification or methylphosphonate is located at the 3 'position of all antisense strand nucleotides, including also 2' fluorine (or other halogen) modified nucleotides. Furthermore, phosphorothioate 3 'modifications can be used instead of and independently of 2' fluorine substitution to increase the stability of the siRNA molecule. These modifications can be used in siRNA applications in conjunction with, or separately from, other modifications described herein.

  Nucleases generally mediate attacks on RNA using both the phosphate moiety and the oxygen group at the 2'OH position of the ribose ring. Substitution of the sulfur group for one of the oxygens removes the phosphate's ability to participate in this reaction, limiting its sensitivity to nuclease digestion. However, since it is necessary to keep in mind that phosphorothioates are typically toxic, it is primarily beneficial when any toxic effect is denied, eg, every other nucleotide, every third nucleotide, or every fourth nucleotide. It can be considered that this can be achieved by limiting the use of this modification for each nucleotide.

  The molecule of the twelfth embodiment, although of limited function, can be used in negative control studies as described above and can be used as an exaequo agent. The modification set associated with multiple molecules makes the molecule an inferior substrate for intracellular kinases that add an essential phosphate group to the 5 'end of the antisense strand of the siRNA. While modifications significantly inhibit phosphorylation, several small fractions of molecules carrying these modifications are still phosphorylated at C5 in the 5 ′ antisense position to produce small amounts of silencing. Is also possible. In such cases, it may be required to modify the 5 'carbon position of the 5' end of the sense and / or antisense strand of those molecules with a blocking group that prevents kinase phosphorylation. The blocking group can be, for example, any group that prevents phosphorylation of the 5 ′ carbon position of the terminal nucleoside, including but not limited to 5′-alkyl, 5′-O-methyl, and 5 ′. -Amine groups.

  Another example of a control or exaequio agent includes only the following 2 'carbon modification: (I) first and second, or first, second, and third 5 ′, each containing a 2 ′ modification, eg, a 2 ′ modification as described above (eg, a 2′-O-methyl modification). Terminal sense nucleotides; and (ii) first and second, or first, second, and first, each containing a 2 ′ modification, eg, a 2 ′ modification as described above (eg, a 2′-O-methyl modification). 3 '5' terminal antisense nucleotides, as well as the 5 'terminal antisense nucleotides are not phosphorylated.

Any method by which one of ordinary skill in the art will conclude that the polynucleotides of the present invention are presently known or will be known, and further useful in connection with the present invention upon reading this disclosure, may be used in accordance with the present invention. It is useful in the synthesis of SiRNA duplexes containing specific modifications are described in Scaringe, S .; A. (2000) "Advanced 5'-silyl-2'-orthoatorapproach to RNA oligonucleotide synthesis," Methods Enzymol. 317, 3-18; Scaringe, S .; A. (2001) "RNA oligonucleotide synthesis via 5'-silyl-2'-orthoester chemistry," Methods 23, 206-217; Scaringe, S .; and Caruthers, M.A. H. (1999) US Pat. No. 5,889,136; Scaringe, S .; and Caruthers, M.A. H. (1999) US Pat. No. 6,008,400; Scaringe, S .; (2000) US Pat. No. 6,111,086; Scaringe, S .; (2003) Chemical synthesis is possible using the methods and methods described in US Pat. No. 6,590,093. The above synthesis method uses a nucleoside base protected 5′-O-silyl-2′-O-orthoester-3′-O-phosphoramidite to form a desired unmodified siRNA sequence on a solid support in the 3 ′ to 5 ′ direction. Assemble Briefly, the synthesis of the essential phosphoramidites begins with standard base-protected ribonucleosides (uridine, N ⁴ -acetylethine, N ² -isobutyryl guanosine, and N ⁶ -isobutyryl adenosine). Introduction of 5′-O-silyl and 2′-O-orthoester protecting groups, and a reactive 3′-O-phosphoramidite moiety is then accomplished by four steps. That is,
1. Simultaneous and temporary blocking of 5′- and 3′-hydroxyl groups of nucleoside sugars by Markiewicz reagent (1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane [TIPS-Cl ₂ ] -containing pyridine Solution {Markiewicz, WT (1979) (1979) "Tetraisopropyldisiloxane-1,3-diyl, a Group for Simulaneous Protection of 3'-and 5'-Hydroxyfunc. , 24-25} followed by purification by chromatography;
2. Regiospecific conversion of 2'-hydroxyl of TIPS-nucleoside sugar to bis (acetoxyethyl) orthoester [ACE derivative] using pyridinium p-toluenesulfonate as catalyst and using dichloromethane containing tris (acetoxyethyl) -orthoformate Followed by chromatographic purification;
3. Release of 5′- and 3′-hydroxyl groups of nucleoside sugars by specific removal of TIP protecting groups using hydrogen fluoride and acetonitrile containing N, N, N ″ N′-tetramethylethylenediamine followed by chromatography Purification by:
4). 4. Chromatographic purification after 5'-hydroxyl protection as 5'-O-silylethanol with benzhydroxyl-bis (trimethylsilyloxyl) chloride [BzH-Cl] containing dichloromethane; Purification by chromatography after conversion to 3′-O-phosphoramidite using bis (N, N-diisopropylamino) methoxyphosphine and 5-methylthio-1H-tetrazole-containing dichloromethane / acetonitrile.

  Phosphoramidite derivatives are generally thick, colorless to light yellow syrups. To adapt to an automated RNA synthesizer, each of the products is dissolved in a predetermined amount of anhydrous acetonitrile and this solution is added in equal portions to the appropriate number of serum vials, and the phosphoramidite in each vial is 1. The amount is 0 mmole. The vials are then placed in a suitable vacuum desiccator and the solvent removed by placing in a high vacuum overnight. The atmosphere is then replaced with dry argon, the vial is capped with a rubber septum, and the sealed phosphoramidite is stored at −20 ° C. until needed. Prior to loading into the synthesizer, each phosphoramidite is dissolved in sufficient anhydrous acetonitrile to the desired concentration.

  Synthesis of the desired oligoribonucleotide is performed using an automated synthesizer. It begins with the covalent attachment of the 3'-terminal nucleoside via a cleavable linkage to a solid beaded polystyrene support via the 3'-hydroxyl. An appropriate amount of carrier for the desired synthesis scale is measured and placed in a reaction cartridge which is then secured to the synthesizer. To protect the bound nucleoside with a 5′-O-dimethoxytrityl moiety and liberate the 5′-hydroxyl for chain assembly, the moiety is treated with acid anhydride (dichloromethane containing 3% [v / v] dichloroacetic acid). Remove.

Subsequent nucleosides of the assembled sequence are sequentially added to the chain growing on the solid support using a four step cycle consisting of the following general reaction.
1. Coupling: The appropriate phosphoramidite is activated with 5-ethylthio-1H-tetrazole and reacted with the carrier-bound nucleoside or the free 5 ′ hydroxyl of the oligonucleotide. Optimizing the concentration and molar excess of these two reagents, as well as their reaction times, generally result in coupling yields exceeding 98% per cycle.
2. Oxidation: The intermolecular linkage formed in the coupling step leaves the phosphorus atom in its P (III) [phosphite] oxidation state. The biologically relevant oxidation state is P (V) [phosphate]. Therefore, phosphorous acid is oxidized from P (III) to P (V) using a toluene solution containing tert-butyl hydroperoxide.
3. Capping: A small amount of residual unreacted 5′-hydroxyl group must be blocked from participating in subsequent binding cycles to prevent formation of sequences containing deletions. This is accomplished by treating the support with acetonitrile containing excess acetic anhydride and 1-methylimidazole, effectively blocking the residual 5′-hydroxyl group as the acetate ester.
4). Desilylation: The silyl protected 5'-hydroxyl must be deprotected prior to the next coupling reaction. This is achieved through treatment with N, N-dimethylformamide containing triethylamine trihydrogen fluoride, where other protecting groups (2'-O-ACE, N-acyl base protecting groups or methylphosphonates) are simultaneously Release 5'-hydroxyl quickly and specifically without being removed.

  It should be noted that there are several washes with acetonitrile between the above four reaction steps, and using the wash removes excess reagents and solvent prior to the next reaction step. The above cycle is repeated as many times as necessary until the unmodified part of the oligoribonucleotide is assembled. The above synthesis methods are exemplary only and are not intended to limit the means by which molecules are made. Any method currently or will be known for synthesizing siRNAs, and any method that one of ordinary skill in the art would conclude by reading this disclosure, useful in connection with the present invention may be used. Is possible.

The siRNA of some embodiments of the eleventh and twelfth embodiments includes two modified nucleosides (eg, 2′-O-methyl derivatives) at the 5 ′ end of each strand. With these modified nucleosides, 5′-O-silyl-2′-O-methyl-3′-O-phosphoramidite derivatives are prepared using procedures similar to those already described (steps 4 and 5 above) and Protected 2′-O-methyl nucleosides (2′-O-methyl-uridine, 2′-O-methyl-N ⁴ -acetylcytidine, 2′-O-methyl-N ² -isobutyryl guanosine, and 2′- O-methyl-N ⁶ -isobutyryl adenosine). The absence of 2'-hydroxyl in these modified nucleosides eliminates the need for ACE protection of these compounds. Thus, derivatization of the 5′-O-silyl and reactive 3′-O-phosphoramidite moieties is accomplished by the following two steps.

1. Protecting 5'-hydroxyl as 5'-O-silyl ether using N, N-dimethylformamide containing benzhydroxy-bis (trimethylsilyloxy) silyl chloride (BzH-Cl), followed by purification by chromatography, And 2. Purification by chromatography after conversion of 3′-O-phosphoramidite using bis (N, N-diisopropylamino) methoxyphosphine and 5-ethylthio-1H-tetrazole-containing dichloromethane / acetonitrile.

  Post-purification packaging of phosphoramidites is performed using the procedure already described for standard nucleoside phosphoramidites. Similarly, the incorporation of the 5′-O-silyl-2′-O-methyl nucleoside through two 5′-O-silyl-2′-O-methyl nucleoside phosphoramidite derivatives is a standard nucleoside. This is achieved by applying the same 4 steps / cycle already described via phosphoramidite twice.

  The siRNA duplexes of some embodiments of the eleventh embodiment of the invention include a phosphate moiety at the 5 'end of the antisense strand. This phosphate is chemically introduced as the final coupling to the antisense strand. In short, the desired phosphoramidite derivative (bis (cyanoethyl) -N, N-diisopropylaminophosphoramidite) is synthesized as follows. That is, phosphorus trichloride is reacted with anhydrous tetrahydrofuran containing 1 equivalent of N, N-diisopropylamine in the presence of excess triethylamine. Next, 2 equivalents of 3-hydroxypropionitrile are added to complete the reaction. Finally, the product is purified by chromatography. Post-purification packaging of phosphoramidites is carried out using the procedure already described for standard nucleoside phosphoramidites. Similarly, incorporation of phosphoramidites at the 5 'end of the antisense strand is accomplished by applying the same four-step cycle already described for standard nucleoside phosphoramidites.

The modified and protected oligonucleotide remains linked to the solid support at the end of the chain assembly. A two-step rapid cleavage / deprotection procedure is used to remove the methylphosphonate protecting group, cleave the oligonucleotide from the solid support and remove the N-acyl base protecting group. It should be noted that this procedure removes the cyanoethyl protecting group from the 5'-phosphate on the antisense strand. Furthermore, this procedure removes the acyl functionality from the ACE orthoester and converts the 2'-O-ACE protecting group to the bis (2-hydroxyethyl) orthoester. This new orthoester is extremely unstable to weak acids and is also more hydrophilic than the parent ACE group. The two-step procedure is briefly described below.
1. The carrier bound oligoribonucleotide is treated with disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate containing N, N-dimethylformamide. This reagent removes methyl protecting groups from internucleotide phosphate linkages quickly and efficiently. At this time, the oligonucleotide is not separated from the solid support. The carrier is then washed with water to remove excess dithiolate.
2. Oligoribonucleotides are cleaved from the solid support with 40% (w / v) water soluble methylamine at room temperature. The crude oligoribonucleotide-containing methylamine solution is then heated to 55 ° C. to remove protecting groups from the nucleoside base. Crude orthoester protected oligonucleotides are obtained in vacuo from the following solvents:

  Removal of the 2'-orthoester is the final step in the synthesis process. This is accomplished by treating the crude oligonucleotide with acetic acid and an aqueous solution of N, N, N ′, N′-tetramethylethylenediamine, pH 3.8, 55 ° C. for 35 minutes. The fully deprotected oligonucleotide is then desalted by ethanol precipitation and further isolated by centrifugation.

  Incorporation of a fluorescent label at the 5 'end of the polynucleotide is a common and well-known procedure for those skilled in the art. In general, there are two methods used to achieve this uptake, and the necessary materials are available from several commercial sources (eg Glen Research Inc., Sterling, Virginia, USA; Molecular Probes Inc., Eugene, Oregon, USA). Available from TriLink BioTechnologies Inc., San Diego, California, USA; and others). The first method utilizes a fluorescent molecule, which was derivatized with a phosphoramidite moiety similar to the previously described nucleoside phosphoramidite derivatives. In such cases, this fluorescent dye is added to the carrier-bound polynucleotide in the final cycle of chain assembly. The fluorophore modified polynucleotide was then cleaved from the solid support and deprotected using the standard procedure described above. This method is referred to as “direct labeling”. Alternatively, the second method utilizes a linker molecule that is derivatized with a phosphoramidite moiety. The moiety includes a protected reactive functional group (eg, amino, sulfhydryl, carbonyl, carboxyl, etc.). This linker molecule is added to the carrier-bound polynucleotide in the last cycle of chain assembly. The linker-modified polynucleotide is then cleaved from the solid support and deprotected using the standard procedures described above. Linker functional groups are also deprotected while performing standard deprotection procedures or by utilizing subsequent group specific procedures. The crude linker-modified polynucleotide is then reacted with a fluorophore derivative. The derivative results in the formation of a covalent bond between the fluorophore site and the linker functional group. This method is called “indirect labeling”.

Once synthesized, the polynucleotides of the invention may be used immediately or stored for future use. Preferably, the polynucleotide of the present invention is stored as a double strand in an appropriate buffer. Many buffers are known in the art to be suitable for storing siRNA. For example, the buffer may include a 100mM KCl, 30mM HEPES-pH 7.5 , and 1 mM MgCl _2. Preferably, the siRNA of the invention retains 30% to 100% of its activity when stored in such a buffer at 4 ° C. for 1 year. More preferably, the siRNA of the present invention retains 80-100% biological activity when stored in such a buffer at 4 ° C. for 1 year. Alternatively, the composition can be stored in such a buffer at −20 ° C. for at least one year. Preferably, storage for more than one year at −20 ° C. results in less than 50% reduction in biological activity. Preferably, the decrease in biological activity due to storage at −20 ° C. is less than 20% after 1 year or more. Most preferably, the decrease in biological activity due to storage for more than one year at −20 ° C. is less than 10%.

  To ensure the stability of the siRNA pool prior to use, it can be kept dry at -20 ° C until they are ready for use. Prior to use, it must be resuspended, but once resuspended, it must be kept at -20 ° C until use, for example with the buffer described above. The buffer described above may be stored at approximately 4 ° C. or room temperature prior to use. The effective temperature at which transfection is performed is well known to those skilled in the art and includes, for example, room temperature.

  In developing the present invention, two or more modifications were added to the duplex to increase stability. Applicants understand that combinations with other modifications that will help improve stability can be discovered in the future. Moreover, the modifications of the present invention could be combined with modifications that are desired for other purposes. For example, in some examples, one modification can stabilize a molecule against a specific set of conditions (eg, a single nuclease), while a second modification is a condition (eg, The molecule can be stabilized against a second set of different families of nucleases). Alternatively, two separate modifications could act additively or synergistically to stabilize the molecule towards a certain set of conditions. In yet another example, one modification could stabilize the molecule, but it has detrimental consequences on other desired properties (eg, siRNA potency or toxicity). In cases like these, additional modifications could be made that would repair these aspects of molecular functionality.

  Various approaches can be used to identify the type of molecules and key positions required to enhance stability. In one non-limiting example, a modification-function walk is performed. In this procedure, one type of modification is added to one or more nucleotides across the sense and / or antisense strand. In effect, modified and unmodified were tested for (1) functionality and (2) stability by one of several methods. Thus, for example, the 1 and 2 positions, 3 and 4 positions, 5 and 6 positions, 7 and 8 positions, 9 and 10 positions, 11 and 12 positions, 13 and 14 positions, 15 and 16 of the sense and / or antisense strand 2'-O-Me groups can be added to positions 17, 17 or 18 and 19 and also for functionality (eg their ability to silence specific targets) Test and stabilize the molecule against action (eg by nuclease). If key positions that enhance stability are identified but lead to loss of double-stranded functionality, then a second round of modified walks may result in mismatches that may increase double-stranded functionality or Additional chemical groups where bulges occur (eg, 5′-terminal 5 ′ phosphate on the antisense strand) are added to molecules that already contain modifications that enhance stability.

The thirteenth embodiment of the present invention relates to siRNA, wherein the siRNA is
(A) an antisense strand, the antisense strand being composed of a first 5 'terminal antisense nucleotide, the first 5' terminal antisense nucleotide being a first 5 'terminal antisense An antisense strand phosphorylated at the 5 ′ carbon position of the nucleotide;
(B) a sense strand, the sense strand being composed of a first 5 'terminal sense nucleotide and a second 5' terminal sense nucleotide, wherein the first 5 'terminal sense nucleotide comprises a first 5' terminal sense nucleotide; A sense strand comprising a 2 ′ carbon sense modification, wherein the second 5 ′ terminal sense nucleotide comprises a second 2 ′ carbon sense modification.

  Preferably, the siRNA contains 18-30 base pairs, more preferably 19-25 base pairs, most preferably 19-23 base pairs, excluding overhangs. Preferably, the sense and antisense strands are at least substantially complementary over a range of base pairs, more preferably 100% complementarity over this range. Preferably, the polynucleotide is RNA.

  The siRNA may have an overhang at either the 5 'or 3' end of either the sense strand or the antisense strand. Preferably, however, if any overhang is present, the overhang is at the 3 'end of the sense and / or antisense strand. Furthermore, the arbitrary overhang is preferably 6 or less base length, more preferably 2 or less base length. Most preferably, there is no overhang, or there are two base overhangs on one or both of the sense and antisense strands at the 3 'end. Overhanging nucleotides are frequently removed by one or more intracellular enzymatic processes or events, leaving unphosphorylated 5'-nucleotides and overhangs on the 5 'end of the antisense strand Preferably not.

  Phosphorylation of the first 5 'terminal antisense nucleotide refers to the presence of one or more phosphate groups attached to the 5' carbon of the sugar moiety of the nucleotide. Preferably only one phosphate group is present.

  A 2 'carbon modification refers to a modification on the 2' carbon of the sugar moiety of the nucleotide. "First 2 'carbon sense modification" refers to a modification on the first 5' terminal sense nucleotide. "Second 2 'carbon sense modification" refers to a modification on the second 5' terminal sense nucleotide.

It is known that the addition of chemically modified RNA-RNA, RNA-DNA, or key positions along the DNA-DNA duplex can significantly alter the chemical and functional properties of these molecules. Yes. Applicants understand that adding a modification to the sense strand of the siRNA can prevent the strand from entering the RISC and induce sense strand-specific off-target effects. Such modifications include the addition of any number of chemical moieties. Preferably, the moiety is attached to the 2 ′ position (ie, 2 ′ carbon) of the ribose ring of nucleotides. According to the invention, preferably the modification is a 2′-O-alkyl group. However, other modifications minimize off-target effects with this strand when used in the context of the present invention. Where such modifications are included under conditions that minimize off-target effects, for example, the group consisting of 2 ′ modified nucleotides, 2 ′ halogen modified nucleotides, 2 ′ amine modified nucleotides, and 2 ′ alkyl modified nucleotides You may choose from. When the modification is halogen, this halogen is preferably fluorine. When the 2 ′ modified nucleotide is a 2 ′ amine modified nucleotide, the amine is preferably —NH ₂ . When the 2 ′ modified nucleotide is a 2′-alkyl modification, preferably the modification is a 2 ′ methyl modification, and the carbon of the methyl moiety is attached directly to the 2 ′ carbon of the sugar moiety.

As noted above, preferably the modification is a 2′-O-alkyl group. More preferably, the modification is 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-isopropyl, 2′-O-butyl, 2′-O-isobutyl, 2'-O- methyl -O- methyl _{(-CH 2 CH 2 OCH 3)} , and 2'-O- ethyl -OH is selected from the group consisting of _(-OCH 2 _CH 2 OH). Most preferably, the 2′-O-alkyl modification is a 2′-O-methyl moiety. Furthermore, the modification need not be identical for each of the first 5 'terminal sense nucleotide and the second 5' terminal sense nucleotide. However, from a practical point of view regarding the synthesis of the molecules of the present invention, it may be required to use the same modification as a whole.

  The above modifications should not be construed as suggesting that any other part will be modified. Other types of modifications are allowed as long as the off-target effect is not increased. For example, in some embodiments, additional modifications can be made to 1, 2, 3, or more consecutive nucleotides or every other nucleotide in the sense strand. Alternatively, additional modifications can be limited to specific positions identified as keys for introducing the sense strand into the RISC complex. In addition, additional 2′-O-methyl groups (or other 2 ′ modifications) can be added to one or more of the sense strands, preferably all pyrimidines (eg C and / or U nucleotides). And / or 2'Fl modifications (or other halogen modifications) can be added to one or more of the antisense strands, preferably all pyrimidines (eg C and / or U nucleotides) When used in conjunction with the following sixteenth and seventeenth embodiments, preferably all pyrimidines other than those that fall within the first two 5 ′ terminal antisense nucleotides are halogen-modified, or If three contain other modifications, the first three 5 ′ terminal antisense nucleotides).

  In addition to chemical modification, base pair mismatches or bulges (bulges) that alter the ability of these strands to participate in RISC-mediated binding to targets that share less than 100% homology are added to the sense and / or antisense strand It is assumed that it can. Examples of such mismatches include (but are not limited to) purine-pyrimidine mismatches (eg, GU, CA) and purine-purine or pyrimidine-pyrimidine mismatches (eg, GA, U-). C). The introduction of this type of modification can be combined with the above modifications, and they can be evaluated to determine whether to reduce off-target effects.

  In order to determine what modifications are allowed, several non-limiting assays can be performed to identify modifications that limit off-target effects. In one non-limiting example, the sense strand (carrying the modification being tested) can be labeled with one of many labeled nucleotides. A binding assay can then be performed to compare the affinity of the RISC for the modified sense strand with that of the unmodified form.

According to a fourteenth embodiment, the present invention relates to a unimolecular siRNA capable of forming a siRNA with a hairpin, wherein the unimolecular siRNA comprises:
(A) an antisense region, the antisense region comprising a first 5 ′ terminal antisense nucleotide, wherein the first 5 ′ terminal antisense nucleotide comprises the first 5 ′ terminal antisense nucleotide; An antisense region phosphorylated at the 5 ′ carbon position of the sense nucleotide;
(B) a sense region, wherein the sense strand is comprised of a first 5 'terminal sense nucleotide and a second 5' terminal sense nucleotide, wherein the first 5 'terminal sense nucleotide comprises: A sense strand comprising a 2 ′ carbon sense modification, wherein the second 5 ′ terminal sense nucleotide comprises a second 2 ′ carbon sense modification;
(C) a loop region, wherein the loop region is located between the sense region and the antisense region;
including.

  According to this embodiment, the scope of modification is the same as that of the thirteenth embodiment. However, since a polynucleotide is a single molecule and can form a hairpin, it is not two different strands, so there is a single continuous molecule that contains both a sense region and an antisense region. . Preferably, the sense region and antisense region are at least substantially complementary, more preferably 100% complementary. Similar to the thirteenth embodiment, preferably the sense and antisense regions comprise 18-30 base pairs, more preferably 19-25 base pairs, even more preferably 19-25 base pairs, most preferably Contains 19-23 base pairs. Preferably, the nucleotide is RNA.

  When designing a unimolecular siRNA, particularly a left-handed unimolecular structure (eg, 5′-AS-Loop-S), according to the present invention, preferably a first 5 ′ terminal sense Nucleotides are counted from the base that is complementary to the first 5′-end antisense nucleotide (ie, counted from the 3 ′ end of the sense region) and the 18th, 19th, or 20th of the sense region. Defined as nucleotides that are bases of The first 5 'terminal sense nucleotide is thus defined. Because, when a unimolecular siRNA capable of forming a hairpin enters a cell, typically a dicer becomes a molecule composed of two different strands (siRNA) of about 18-20 base pairs. This is because it is required that these molecules retain the sense strand modification that is processed in the picture hairpin siRNA containing a long double-stranded region and bound to the 5 ′ end of the processed molecule. Most preferably, the first 5 'terminal sense nucleotide is defined as the nucleotide that is the 19th base of the sense region from the 3' end of the sense region. Furthermore, preferably the polynucleotide is capable of forming a counterclockwise hairpin.

  The hairpin includes a loop structure composed of 4 to 10 bases and a sense region, and each of the sense region and the antisense region has a length of 19 to 23 base pairs and is substantially complementary to each other. Preferred sequences of the loop structure include, for example, 5'-UUCG (SEQ ID NO: 323), 5'-UUUGGUGUAG (SEQ ID NO: 324), and 5'-CUUCCUGUCA (SEQ ID NO: 325).

  The hairpin is preferably constructed by a loop region downstream of the antisense region. This configuration is desirable because it is easier to phosphorylate the terminal antisense nucleotide. Thus, when designing a unimolecular siRNA, it is preferred that there is no overhang upstream of the 5'-end antisense nucleotide. It may be desirable to have an overhang of, for example, 1, 2, or 3 nucleotides at the sense region 3 'end of some embodiments. Furthermore, preferably, the unimolecular siRNA can form a counterclockwise hairpin.

  The shRNA can further comprise a stem region, the stem region comprising one or more nucleotides or modified nucleotides immediately adjacent to the 5 ′ end and the 3 ′ end of the loop structure, further comprising the stem region. One or more nucleotides or modified nucleotides are target specific (or non-target specific). Preferably, the total length of the unimolecular siRNA is less than 100 base pairs, more preferably less than 85 base pairs.

  The unimolecular siRNA of the present invention is finally processed so that it is converted into two different strands depending on the cellular function. In addition, these unimolecular siRNAs may be introduced into the cell with fewer than all modifications, and can be modified within the cell itself using the natural process or processing molecules introduced. (Eg, intracellular phosphorylation). However, preferably, all the existing modifications are introduced into the siRNA (similarly, if the siRNA is composed of two different strands, preferably the strands of the first embodiment are all (For example, it can be modified by a phosphorylation group after introduction).

According to a fifteenth embodiment, the present invention relates to a method for minimizing off-target effects, wherein the method expresses a modified siRNA as a target nucleic acid, or expresses the target nucleic acid, or expresses the target nucleic acid. The siRNA has an antisense strand and a sense strand, comprising exposing to a cell or tissue capable of
(A) the sense strand is comprised of a first 5 'terminal sense nucleotide and a second 5' terminal sense nucleotide, wherein the first 5 'terminal sense nucleotide comprises a first 2' carbon sense modification; A sense strand, wherein the second 5 'terminal sense nucleotide comprises a second 2' carbon sense modification;
(B) the antisense strand is composed of a first 5 ′ terminal antisense nucleotide, and the first 5 ′ terminal antisense nucleotide is at the 5 ′ position of the first 5 ′ terminal antisense nucleotide; An antisense strand that is phosphorylated, and
including.

  The modified siRNA of the fifteenth embodiment is preferably the siRNA of the thirteenth embodiment.

According to a sixteenth embodiment, the invention relates to a method for minimizing off-target effects, said method expressing a modified siRNA as a target nucleic acid, or expressing said target nucleic acid, or expressing said target nucleic acid. Exposing the cell or tissue to a single siRNA capable of forming a hairpin, wherein the siRNA has an antisense strand and a sense strand;
(A) the sense region is comprised of a first 5 'terminal sense nucleotide and a second 5' terminal sense nucleotide, wherein the first 5 'terminal sense nucleotide comprises a first 2' carbon sense modification; A sense region, wherein the second 5 ′ terminal sense nucleotide comprises a second 2 ′ carbon sense modification;
(B) the antisense region is composed of a first 5 ′ terminal antisense nucleotide, and the first 5 ′ terminal antisense nucleotide is the 5 ′ carbon position of the first 5 ′ terminal antisense nucleotide; An antisense region that is phosphorylated at
(C) a loop region, wherein the loop region is located between the sense region and the antisense region;
including.

  The modified siRNA of the sixteenth embodiment is preferably the siRNA of the fourteenth embodiment.

  In other embodiments, additional modifications may still be present, as combinations of modifications are described as follows. (1) 1 and 2 positions (or 1, 2 and 3) of the sense region (or sense region of shRNA) counting from 5 ′ of the strand modified by the 2 ′ modifying group (carbon 2 on ribose) Position), (2) 1 and 2 positions (or 1, 2) of the sense region (or antisense region of shRNA) counting from the 5 'end of the strand modified by the 2' modifying group (carbon 2 on ribose) And 3), and (3) the 5 ′ end of the antisense strand is phosphorylated (carbon 5 of ribose). The combination of all three modifications appears to be beneficial in that it substantially reduces off-target effects induced by the sense and antisense strands.

According to a seventeenth embodiment, the present invention provides siRNA, wherein the siRNA is
(A) an antisense strand, wherein the antisense strand is composed of a first 5'-end antisense nucleotide and a second 5'-end antisense nucleotide, and the first 5'-end antisense The nucleotide comprises a first 2 'carbon antisense modification and the second 5' terminal antisense nucleotide comprises a second 2 'carbon antisense modification, the first 5' terminal antisense nucleotide An antisense strand that is phosphorylated at the 5 ′ carbon position of the first 5 ′ terminal antisense nucleotide;
(B) a sense strand, wherein the sense strand is composed of a first 5 'terminal sense nucleotide and a second 5' terminal sense nucleotide, wherein the first 5 'terminal sense nucleotide is a first A sense strand comprising a 2 ′ carbon sense modification, wherein the second 5 ′ terminal sense nucleotide comprises a second 2 ′ carbon sense modification;
including.

  According to this seventeenth embodiment, the modification is achieved by adding a 2 ′ carbon modification to the first 5 ′ terminal antisense nucleotide and the second 5 ′ terminal antisense nucleotide to form the thirteenth embodiment. Defined in the same way as Alternatively, the modification is defined as in the thirteenth embodiment by the addition of a 2 'carbon modification on the second terminal antisense nucleotide. These 2 'carbon modifications are defined in the same way as modifications to the first 5' terminal sense nucleotide and the second 5 'terminal sense nucleotide. In addition, there may be additional 2 'carbon modifications on the third 5' terminal antisense nucleotide.

According to an eighteenth embodiment, the present invention provides a unimolecular siRNA molecule capable of forming a hairpin. This siRNA
(A) an antisense region, the antisense region comprising a first 5'-end antisense nucleotide and a second 5'-end antisense nucleotide, wherein the first 5'-end antisense The nucleotide comprises a first 2 ′ carbon antisense modification, the second 5 ′ terminal antisense nucleotide comprises a second 2 ′ carbon antisense modification, and further comprises a first 5 ′ terminal antisense nucleotide An antisense region that is phosphorylated at the 5 ′ carbon position of the first 5 ′ terminal antisense nucleotide;
(B) a sense region, wherein the sense strand is comprised of a first 5 'terminal sense nucleotide and a second 5' terminal sense nucleotide, wherein the first 5 'terminal sense nucleotide is a first A sense region comprising a 2 ′ carbon sense modification, wherein the second 5 ′ terminal sense nucleotide comprises a second 2 ′ carbon sense modification;
(C) a loop region, wherein the loop region is located between the sense region and the antisense region;
Consists of

  According to this eighteenth embodiment, the modification is the same as that of the seventeenth embodiment by adding a 2 ′ carbon modification to the first 5 ′ terminal antisense nucleotide and the second 5 ′ terminal antisense nucleotide. Defined in the same way as These 2 'carbon modifications are defined in the same way as modifications to the first 5' terminal sense nucleotide and the second 5 'terminal sense nucleotide. In addition, there may be additional 2 'carbon modifications on the third 5' terminal antisense nucleotide.

  According to a nineteenth embodiment, the present invention provides a method for reducing off-target effects during gene silencing. This method comprises administering the siRNA of the seventeenth embodiment to a target nucleic acid, or a cell or tissue that expresses or can express the target nucleic acid.

  According to a twentieth embodiment, the present invention provides a method for reducing off-target effects during gene silencing. This method comprises administering the siRNA of the eighteenth embodiment to a target nucleic acid, or a cell or tissue that expresses or is capable of expressing the target nucleic acid.

  Although the above embodiments are aimed at increased specificity, other types of modifications that can be designed to affect other parameters (eg, those described above that affect stability) It is important to note that their combination modifies the siRNA.

  However, some stability-directed modifications give siRNAs with limited functionality and are not used except for use as, eg, a control or exaequo agent (eg, the sense strand, where the sense strand is the first Composed of a 5 ′ terminal sense nucleotide and a second 5 ′ terminal sense nucleotide, wherein the first 5 ′ terminal sense nucleotide comprises a first 2′-O-alkyl (preferably methyl) sense modification; The second 5 ′ terminal sense nucleotide comprises a second 2′-O-alkyl (preferably methyl) sense modification; at least one (preferably all) 2′-O-alkyl (preferably Methyl) pyrimidine modified sense nucleotide, wherein said at least one, preferably all 2'-O-alkyl (preferably methyl) The pyrimidine-modified sense nucleotide is a nucleotide other than the first 5 'terminal sense nucleotide or the second 5' terminal sense nucleotide; an antisense strand, wherein the antisense strand is the first 5 'terminal nucleotide. A terminal antisense nucleotide and a second 5 'terminal antisense nucleotide, wherein the first 5' terminal antisense nucleotide is a first 2'-O-alkyl (preferably methyl) antisense Including a modification, wherein the second 5 ′ terminal sense nucleotide comprises a second 2′-O-alkyl (preferably methyl) antisense modification; a 2′-labeled pyrimidine, preferably at least 1, on the antisense strand Two 2′-O-alkyl (preferably methyl) pyrimidine modified antisense nucleotides, The at least one 2′-O-alkyl (preferably methyl) pyrimidine modified antisense nucleotide is a nucleotide other than the first 5 ′ terminal antisense nucleotide or the second 5 ′ terminal antisense nucleotide. And, if necessary, the 5 ′ terminal sense nucleotide has a label such as a fluorescent label).

  In addition, several other related combinations of modifications can be used to develop siRNA that can be used as a control group or an exaequo agent. For example, siRNA or shRNA can be developed similar to the 17th and 18th embodiments described in this disclosure without phosphorylating the first 5 'terminal antisense nucleotide. Thus, under one embodiment, (i) first and second, or first, second, each comprising a 2 ′ modification, eg, each of the 2 ′ modifications described above (for example, 2′-O-methyl modification). , And a third 5 ′ carbon-terminal sense nucleotide, and (ii) first and second, each comprising a 2 ′ modification, eg, a 2 ′ modification as described above (eg, a 2′-O-methyl modification), respectively, Or there are 2 'modifications limited to the first, second, and third 5' carbon-terminal antisense nucleotides.

  When using an exaequo agent or a control group, it may be required to modify the 5 'carbon position of the 5' end of the sense strand and / or the antisense strand with a blocking group. This blocking group may be, for example, an alkyl group or any other group that prevents phosphorylation of the 5 'carbon position of the nucleotide. Phosphorylation appears to occur inside the cell by the action of a kinase present in the cell. Exemplary blocking groups include, but are not limited to, methyl, O-methyl, and amine groups.

  These molecules are not functional but are used in negative control studies and can be used as exaequo agents. In addition, if the labeling method described above is used, these agents are useful as tracking agents, which may help detect transfection and also help detect where the molecule is present in the cell. I think that the. Since these agents are not used for silencing, they are preferably 18-30 base pairs, but they can contain 16-28 base pairs.

Furthermore, stabilizing modifications to the phosphate backbone can be included in the siRNA for some uses of the invention. For example, at least one phosphorothioate and / or methylphosphonate can be present in any overhang, loop structure, or stem structure that may also be present in the sense and / or antisense strand of the oligonucleotide backbone. Or some or all of the phosphate groups in the 3 'position of all pyrimidines. Phosphorothioate (and methylphosphonate) analogs arise from modification of the phosphate groups of the oligonucleotide backbone. Within the phosphorothioate, the phosphate O ⁻ is replaced by a sulfur atom. In methylphosphonate, oxygen is replaced by a methyl group. In one embodiment, the phosphorothioate modification or methylphosphonate is placed at the 3 ′ position of all antisense strand nucleotides, including also 2 ′ fluorine (or other halogen) modified nucleotides. Furthermore, phosphorothioate 3 ′ modifications can be used instead of and independently of 2 ′ fluorine substitution to increase the stability of the siRNA molecule. These modifications can be used in siRNA applications in conjunction with, or separately from, other modifications described herein.

  Nucleases generally mediate attacks on RNA using both the phosphate moiety and the oxygen group at the 2'OH position of the ribose ring. Substitution of the sulfur group for one of the oxygens removes the phosphate's ability to participate in this reaction, limiting its sensitivity to nuclease digestion. However, since it is necessary to keep in mind that phosphorothioates are typically toxic, it is primarily beneficial when any toxic effect is denied, eg, every other nucleotide, every third nucleotide, or every fourth nucleotide. It can be considered that this can be achieved by restricting the use of this modification per leotide.

  Further, in some applications it may be desirable to incorporate a label (eg, a fluorescent label, a radioactive label, or a mass label) into the nucleotides of the invention.

  The above modifications of the invention may be combined with siRNA, which is selected at random, or based on a logical design, eg, as described in US patent application Ser. No. 10 / 714,333. Containing the selected sequence. In addition, it may be desirable to select a sequence based on overall or partial internal thermal stability that can facilitate treatment by cellular machinery.

  In developing the thirteenth through twentieth embodiments of the present invention, two or more types of modifications were added to the duplex to minimize the off-target effect. As noted above, Applicants understand that other modifications and combinations that will help in minimizing off-target effects may be discovered in the future. Moreover, the modifications of the present invention could be combined with modifications desired for other purposes. For example, in some examples, one modification can affect one particular step of off-target silencing (eg, the sense strand bound to RISC) while the second modification Could completely affect the different steps without changing, for example, the ability of the sense / antisense strand to bind to targets with less than 100% homology. Alternatively, two separate modifications could affect the same step. In some examples, two or more modifications act additively or synergistically to limit off-target effects by minimizing unnecessary interactions or processes in one or more steps did. In yet another example, one modification could remove off-target effects, but it has detrimental consequences on other desired properties, such as siRNA potency or stability. In cases like these, additional modifications could be made that would repair these aspects of molecular functionality.

  Various approaches can be used to identify the type of molecule and key position required to eliminate sense and / or antisense strand off-targeting effects. In one non-limiting example, a modification-function walk is performed. In this procedure, one type of modification is added to one or more nucleotides across the sense and / or antisense strand. In effect, modified and unmodified were tested for (1) functionality and (2) off-target effects by one of several methods. Thus, for example, the 1 and 2 positions, 3 and 4 positions, 5 and 6 positions, 7 and 8 positions, 9 and 10 positions, 11 and 12 positions, 13 and 14 positions, 15 and 16 of the sense and / or antisense strand 2'-O-Me groups can be added to positions 17, 17 or 18 and 19 and further functionality (eg, the ability of those molecules to silence specific targets) and Test for off / target effects (eg, by microarray analysis). If key positions that eliminate some or all off-target effects are identified but result in loss of double-stranded functionality, then the second round of the modified walk will result in double-stranded function Additional chemical groups that cause mismatches or bumps that appear to enhance sex (eg, 5 'phosphate at the 5' end on the antisense strand) are added to molecules that already contain modifications that remove off-target effects. The

  It should be noted that the modifications of the thirteenth to twentieth embodiments of the present invention can have different effects depending on the functionality of the siRNA used. Thus, in highly functional siRNA, the modifications of the present invention may be responsible for the loss of the functionality of the molecule, but are nevertheless desirable because off-target effects are reduced. In contrast, when moderately or poorly functional siRNA is used, there is a slight decrease in functionality, and in some instances, functionality can be enhanced.

  Since the stability of the dsRNA of the present invention that retains functionality and resists degradation does not depend on the nucleotide sequence, cell type, or introduced species, the present invention is applicable to a wide range of organisms. Yes, such organisms include, but are not limited to, plants, animals, protozoa, bacteria, viruses, and fungi. The invention is particularly advantageous for use with mammals such as cows, horses, goats, pigs, sheep, canines, rodents (eg hamsters), mice and rats, as well as primates (eg gorillas, chimpanzees and humans). It is.

  The present invention can be advantageously used with a variety of cell types (eg, but not limited to, primary cells, germline, and somatic cells). The cell is, for example, a stem cell or a differentiated cell. For example, germ cells, oocytes, sperm cells, adipocytes, fibroblasts, muscle cells, cardiomyocytes, endothelial cells, neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils Examples include spheres, eosinophils, basophils, mast cells, leukocytes, granulocytes (keratinocytes), chondrocytes, osteoblasts, osteoclasts, hepatocytes, and endocrine or exocrine gland cells.

  The present invention is applicable to applications that use RNA interference against a wide range of genes (and / or used as controls), including but not limited to diseases such as diabetes, Alzheimer and cancer. 45,000 genes of the human genome, as well as humans, mice, hamsters, chimpanzees, goats, sheep, horses, camels, pigs, dogs, cats, linear animals (eg C. elegans), flies (eg Drosophila melanogaster D. melanogaster) ), And all genes contained in the genomes of other vertebrates and invertebrates.

  The siRNAs of the present invention can be administered to cells by any method that is presently known or will be known and further related to the present invention by reading this disclosure. Any method that would be concluded by one skilled in the art to be useful. For example, siRNA can be passively delivered to cells.

  Passive incorporation of the modified siRNA can be achieved, for example, by the presence of a conjugate, eg, a polyethylene glycol or cholesterol moiety at the 5 ′ end of the sense strand, and / or, where appropriate, a pharmaceutically acceptable carrier. Can be adjusted.

  Other methods for delivery include, but are not limited to, DEAE-dextran, calcium phosphate, cationic lipid / liposomes, microinjection, electroporation, immunoprecipitation, and siRNA and certain conjugates or ligands (For example, coupling with an antibody, an antigen, or a receptor).

  Preferably, the siRNA comprises a double strand when administered.

  Furthermore, the method for assessing the level of gene silencing is not limited. Thus, the silencing capacity of any given siRNA is by any one of a number of art tested procedures including, but not limited to, Northern analysis, Western analysis, PTPCR, expression profiling, etc. Can be studied.

  Furthermore, the siRNA of the present invention can be used in various combinations of applications, and includes, but is not limited to, basic research, drug discovery and development, diagnosis, and drug therapy. . For example, the present invention can be used to determine whether a gene product is a target for drug discovery or development. In such applications, mRNA corresponding to the target nucleic acid sequence of interest is identified for targeted degradation. An siRNA of the invention that is specific for targeting a particular gene is introduced into a cell or organ, preferably in a double stranded form. By maintaining the cell or organism under conditions that allow degradation of the target mRNA, the activity or expression of the gene is reduced. Measure the degree of decrease in gene expression or activity, along with the effect of such decreased expression or activity, if the expression or activity is decreased, the target nucleic acid sequence acts for drug discovery or development Is a factor. Thus, a phenotypically desirable effect can be accompanied by RNA interference of a specific target nucleic acid of interest, and in a suitable example, toxicity and pharmacokinetic studies can be performed, Developed.

  The present invention relates to RNA that induces a transient or permanent state of a disease or disorder in a living body, for example, by attenuating the activity of the target nucleic acid of interest that is considered to be the cause or factor of the target disease or disorder It can also be used for interference applications. Increased activity of the target nucleic acid of interest tends to exacerbate the disease or disorder or, in some cases, ameliorate or treat the target disease or disorder. Similarly, a decrease in the activity of the target nucleic acid of interest may result in a disease or disorder that tends to worsen or in some cases improve or heal. Target nucleic acids of interest can include genomic or chromosomal nucleic acids or extrachromosomal nucleic acids (eg, viral nucleic acids).

  Furthermore, the present invention can be used in RNA interference applications for determining the function of a target nucleic acid or target nucleic acid sequence. For example, knockdown experiments that exclude or reduce the activity of certain target nucleic acids of interest. This can be achieved by performing RNA interference with one or more siRNAs that target a specific target nucleic acid of interest. Observing the effect of such knockdown can lead to inferences regarding the function of the target nucleic acid of interest. RNA interference attenuates the activity of a target nucleic acid of interest having either one of the alleles and observes its effect on a living organism or system, thereby causing polymorphic effects (eg, biallelic polymorphism). It can also be used to study (formality). Therapeutically, one or the other of the alleles, or both, can be selectively allelic silenced using RNA interference where selective allelic silencing is desired.

  Furthermore, the present invention relates to RNA interference such as diagnosis, prevention, and treatment, including the use of compositions in the manufacture of pharmaceuticals for animals, preferably mammals, more preferably humans, in the treatment of diseases or in the over or under expression of targets. It can be used for various purposes. Preferably, the disease or disorder is due to an abnormal function of one or more proteins, and the disease or disorder is related to the expression of a gene product comprising one or more proteins. For example, it is widely accepted that certain human breast cancers are associated with dysfunction of proteins expressed from a gene commonly known as the “bcl-2” gene. using one or more siRNAs against the bcl-2 gene, and optionally combining the drug with a pharmaceutically acceptable carrier, diluent and / or adjuvant in which the drug is used for the treatment of breast cancer, It can be prepared according to the compositions and teachings of the present invention. Applicants have established the utility of the methods and compositions in cell models. Methods for delivery of polynucleotides (eg, siRNA) to animal cells, including humans, are well known. Any delivery vehicle currently known or known in the art, which has utility for introducing polynucleotides such as siRNA into animals such as humans, is in accordance with the present invention. Unless this delivery vehicle is incompatible with any modification that may be present in the manufactured composition, it is expected to be useful in the manufacture of a medicament according to the present invention. A delivery vehicle that is not compatible with a composition made according to the present invention is one that reduces the efficacy of the composition by more than 95% as measured for efficacy in cell culture.

  Animal models exist for many, many diseases, including, for example, cancer, vascular system, birth defects or metabolic diseases. Persons skilled in the art will be able to administer nucleic acids to animals in a dosage regime to reach the optimal dosage regimen for a particular disease or disorder in an animal such as a mammal (eg, a mouse, rat or non-human primate). Is well known. Once efficacy has been established by a person skilled in the art by a tedious experiment in a mammal, a dosing regimen during the start of a human trial can be achieved based on data reached in such studies.

  The dosage of the drug produced according to the present invention varies from several micrograms to several milligrams per kilogram of subject. As is known in the art, dosing is based on the mass of the mammal being administered, the nature of the mammal receiving the dose, the disease or severity of the disease, as well as other known to those skilled in the art. Among factors, it depends on the drug stability of the subject's serum.

  For these applications, organisms suspected of having a disease or diseases that are subject to modulation by manipulation of a specific target nucleic acid are treated by administering siRNA. As a result of siRNA treatment, remission, temporary relief, prevention, and / or diagnosis for a particular disease is obtained. Preferably, the siRNA is administered in a pharmaceutically acceptable manner with a pharmaceutically acceptable carrier or diluent.

The therapeutic application of the present invention can be carried out by various therapeutic compositions and dosing methods. Pharmaceutically acceptable carriers and diluents are well known to those skilled in the art. Methods of administration to cells and organisms are also well known to those skilled in the art. It is known that the dosage regimen will depend on, for example, the severity and extent of the response of the disease or disorder being treated, and the course of treatment may range from days to months or Until the desired effect is achieved. Chronic administration of siRNA may be necessary for the desired effect that persists in some diseases or disorders. For example, an appropriate dosing regimen can be determined by administering varying amounts of one or more siRNAs in a pharmaceutically acceptable carrier or diluent near the pharmaceutically acceptable delivery route, The amount of the drug accumulated in the body of the ent organism can be measured at various times after administration. Similarly, the desired effect (eg, the degree of suppression of expression of a gene product or gene activity) can be measured at various times after administration of siRNA, and this data can be compared to other pharmacokinetics Data (eg, accumulation in living organisms or organs). Persons of ordinary skill in the art can determine optimum dosages, dosing schedules and the like. One skilled in the art can use EC ₅₀ from in vivo and in vitro as _{per the} research guide for humans.

  Still further, the present invention can be used in RNA interference applications (eg, diagnostics, prophylactics, and pharmacotherapy). For these applications, a living organism suspected of having a disease or disorder that is subject to modulation by manipulation of a specific target nucleic acid is treated by administering siRNA. The result of siRNA treatment is ameliorating, temporary relief, prophylactic and / or diagnosis of a specific disease or disorder. Preferably, siRNA is administered in a pharmaceutically acceptable manner with a pharmaceutically acceptable carrier or diluent.

  Furthermore, the siRNA can be applied topically as a cream or ointment, oral formulation (eg, capsule or tablet or suspension or solution). The route of administration is intravenous, intramuscular, transdermal, dermal, cutaneous, subcutaneous, intranasal, oral, rectal, instillation, or even in an advantageous location, such as a location close to an organ or tissue, with the target nucleic acid of interest. A device for releasing siRNA into a cell type may be transplanted into a tissue.

  According to another embodiment, the invention comprises siRNA, a sense strand and an antisense strand, each of the sense strand and the antisense strand having at least one orthoester modification at the 2 'position.

  According to another aspect of the present invention, the present invention provides an siRNA comprising a sense strand and an antisense strand, the antisense strand comprising at least one orthoester modification and / or 2′-halogen modification, 2 It includes at least one modification selected from the group consisting of a '-alkyl modification, a 2'-O-alkyl modification, a 2'-amine modification, and a 2'-deoxy modification.

  According to another embodiment, the present invention comprises siRNA, said siRNA comprising a sense strand and an antisense strand, said sense strand and / or antisense strand comprising at least one orthoester modification, and At least the sense strand and / or the antisense strand is selected from the group consisting of 2′-halogen modification, 2′-alkyl modification, 2′-O-alkyl modification, 2′-amine modification, and 2′-deoxy modification Contains one 2 'modification.

  In yet another embodiment, the invention comprises an siRNA, wherein the siRNA is (a) a sense strand, wherein the sense strand is a first 5 ′ terminal sense nucleotide and a second 5 ′ terminal sense nucleotide. A sense comprised of nucleotides, wherein the first 5 'terminal sense nucleotide comprises a first 2' carbon sense modification and the second 5 'terminal sense nucleotide comprises a second 2' carbon sense modification A strand; and (b) an antisense strand, the antisense strand comprising a first 5 'terminal antisense nucleotide and a second 5' terminal antisense nucleotide, wherein the first 5 ' The terminal antisense nucleotide comprises a first 2 ′ carbon antisense modification and a 5 ′ modification, and the second 5 ′ terminal antisense nucleotide is a second 2 ′ carbon antisense modification. Including a scan modification, including the anti-sense strand, the.

  In yet another embodiment, the invention comprises a siRNA, the siRNA comprising a sense strand and an antisense strand, the antisense strand comprising at least one 2′-orthoester modification, and the antisense strand Contains at least one 2′-orthoester modification, and the antisense strand is further divided into 2′-orthoester modification, 2′-alkyl modification, 2′-halogen modification, 2′-O-alkyl modification, 2 ′ Modified by one or more modifications selected from the group consisting of a '-amine modification and a 2'-deoxy modification, wherein the 2'-alkyl modification, 2'-O-methyl modification, and 2'-halogen modification are anti Located on one or more pyrimidines of the sense strand, the siRNA further includes a 3 ′ cap. This 2 'carbon modification is for example a 2'-O-alkyl modification such as a 2'-O-methyl modification. The first 5 'terminal sense nucleotide and / or the first 5' terminal antisense nucleotide is further modified with a 5 'blocking group. The 5 'blocking group can be selected from the group consisting of 5'-methyl modification, 5'-O-methyl modification, and 5'-azide modification.

  In another embodiment, the first and second 2′-O-alkyl modifications of the 5 ′ terminal sense nucleotide and / or 5 ′ terminal antisense nucleotide are 2′-O-methyl modifications, The first 5 ′ terminal sense and / or antisense nucleotide can further comprise a 5 ′ blocking group. The 5 'blocking group is selected from the group consisting of 5'-methyl modification, 5'-O-methyl modification, or 5' azide modification. In another embodiment, the present invention provides an siRNA consisting of an antisense strand and a sense strand, the sense strand comprising a first 5 'terminal sense nucleotide and a second 5' terminal sense nucleotide. Constructed, the first 5 'terminal sense nucleotide comprises a first 2' carbon sense modification and the second 5 'terminal sense nucleotide comprises a second 2' carbon sense modification.

  In another embodiment, the present invention provides an siRNA, wherein the siRNA is (a) a sense strand, wherein the sense strand is a first 5 'terminal sense nucleotide and a second 5' terminal. The first 5 'terminal sense nucleotide comprises a first 2' carbon sense modification, and the second 5 'terminal sense nucleotide comprises a second 2' carbon sense modification. A sense strand comprising (b) an antisense strand, the antisense strand comprising a first 5'-end antisense nucleotide and a second 5'-end antisense nucleotide; One 5 ′ terminal antisense nucleotide contains a 5 ′ phosphate modification and the second 5 ′ terminal antisense nucleotide contains a 2 ′ carbon antisense modification; It includes a chain, a.

  In other embodiments, any of the compositions of the present invention further comprises a 3 'cap. This 3 'cap is, for example, an inverted deoxythymidine.

  In other embodiments, the compositions of the invention can include at least one 2'-deoxy modification and / or a methylphosphonate internucleotide linkage. The compositions of the present invention also comprise a modification selected on one or more pyrimidines from 2'-alkyl modifications, 2'-O-alkyl modifications, and 2'-halogen modifications. The 2'-alkyl modification can be a 2'-methyl modification, such as a 2'-O-methyl modification. The 2'-halogen modification can be, for example, 2'-fluorine modification.

In other embodiments, the compositions of the present invention can include at least one 2'-amine modification and / or 2'-halogen modification. This 2′-amine modification is, for example, a 2′-NH ₂ modification. The 2′-halogen modification is, for example, 2′-fluorine modification. The compositions of the present invention can include one or more modified internucleotide linkages, such as one or more phosphorothioate linkages, phosphorodithioate linkages, methylphosphonate linkages, and combinations thereof.

  In other embodiments of the invention, any of the above compositions can include a conjugate. The conjugate can be selected from the group consisting of amino acids, peptides, polypeptides, proteins, sugars, carbohydrates, lipids, polymers, nucleotides, polynucleotides, and combinations thereof. The conjugate may be cholesterol or PEG. The conjugate further includes a label such as a fluorescent label. The fluorescent label is selected from the group consisting of TAMRA, BODIPY, Cy3, Cy5, fluorescein, and Dabsyl. Alternatively, any fluorescent label known in the art may be used as the fluorescent label.

  In other embodiments, the compositions of the present invention comprise at least one 2'-orthoester modification, which is preferably a 2'-bis (hydroxyethyl) orthoester modification.

  In other embodiments, any of the compositions of the invention can be used in a method of performing RNA interference, the method comprising administering to a cell one or more compositions of the invention. Preferably, an ortho ester modification is present in the siRNA and the ortho ester modification is a 2'-bis (hydroxyethyl) ortho ester.

  In order to describe the invention with some specificity, examples are now provided. These examples are not intended to limit or limit the scope of the claims in any way. Although the present invention is readily understood through reference to the following examples, they are intended to be illustrative of the invention and are not intended to limit the invention unless otherwise stated. .

Example 1
(Synthesis of siRNA)
RNA oligonucleotides were synthesized in a stepwise fashion using the nucleotide addition reaction cycle illustrated in FIG. The synthesis is preferably performed as an automated process on a suitable machine. Some such synthesis machines are well known to those skilled in the art. Each nucleotide is added sequentially (3 ′ to 5 ′ orientation) to the oligonucleotide bound to the solid support. A polystyrene carrier is preferred, but any suitable carrier may be used. The first nucleoside at the 3 ′ end of the chain is covalently attached to the solid support (attachment to the solid support). Activators such as nucleotide precursors, activated ribonucleotides such as phosphoramidites and H-phosphonates, and tetrazole, for example S ethyl tetrazole (although any other suitable activator may be used) (Step i in FIG. 13) to couple the second base onto the 5 ′ end of the first nucleoside. The support is washed and any unreacted 5 ′ hydroxyl group is capped with an acetylating agent such as, but not limited to, acetic anhydride or phenoxyacetic anhydride to yield unreacted 5 ′ acetyl moieties (steps) ii). The P (III) bond is then oxidized to the more stable and ultimately desired P (V) bond using, for example, a suitable oxidizing agent such as t-butyl hydroperoxide or iodine and water (step iii). ). At the end of the nucleotide addition cycle, the 5 ′ silyl group is cleaved with fluoride ions (eg, triethylammonium fluoride or t-butylammonium fluoride) (step iv). This cycle is repeated for each subsequent nucleotide. Although FIG. 13 illustrates a phosphoramidite with a methyl protecting group, it should be emphasized that any other suitable group may be used to protect or displace the oxygen of the phosphoramidite moiety. For example, an alkyl group, a cyanoethyl group, or a thio derivative may be used at this position. Furthermore, the activated nucleoside entering step (i) may be a different type of activated nucleoside (eg H-phosphonate, methyl phosphoramidite, or thiophosphoramidite). It should be noted that the initial or 3 ′ nucleoside attached to the support can have different 5 ′ protecting groups such as dimethoxytrityl groups rather than silyl groups. When used in standard DNA synthesis chemistry, cleavage of the dimethoxytrityl group requires acid hydrolysis. That is, acids such as dichloroacetic acid (DCA) and trichloroacetic acid (TCA) are used alone for this step. Apart from the DCA cleavage step, this cycle is repeated as many times as necessary to synthesize the desired polynucleotide.

  Following synthesis, a protecting group on the phosphate (shown as a methyl group in FIG. 13 but not necessarily limited to a methyl group) was converted to 1 molar disodium-2-carbamoyl-2-in DMF (dimethylformamide). Cleavage with cyanoethylene-1,1-dithiolate trihydrate (dithiolate) in 30 minutes. This deprotection solution is washed with oligonucleotides bound to a solid support using water. The carrier is then treated with 40% methylamine at 55 ° C. for 20 minutes. This releases the RNA oligonucleotide into solution, deprotects the exocyclic amine, and removes the acetyl protection on the 2'ACE group. Oligonucleotides can be analyzed at this stage by anion exchange HPLC (high performance liquid chromatography).

  If removal is desired, the 2'orthoester group is the last protecting group to be removed. The structure of RNA protected with 2'ACE immediately before 2'deprotection is as shown in FIG.

  In the case of automated processing, the solid support with the first nucleoside is attached to the synthesizer. This equipment contains all the necessary auxiliaries and monomers required for the synthesis. Reagents are maintained under inert gas. This is because some monomers may hydrolyze if not maintained under an inert gas. Prepare the apparatus to fill all lines with reagents. A synthesis cycle is defined that defines the correct order of reagent distribution according to the synthesis cycle, and the reagents are distributed in the order specified in FIG. Once the cycle is defined, the amount of each reagent to be added is defined, the time between steps is defined, the wash step is defined, and the synthesis proceeds once the solid support with the first nucleoside is added. Ready to do.

For the RNA analogs described herein, modification is performed in three different general ways. The first is for carbohydrate and base modifications, as well as for the introduction of certain linkers and conjugates, using modified phosphoramidites where the modifications already exist. Examples of such modifications are modified species at the carbohydrate 2 ′ position (2′-F, 2′-NH ₂ , 2′-O-alkyl, etc.) where the ortho ester at the 2 ′ position is the desired modification. Has been replaced by 3 'or 5' terminal modifications such as fluorescein derivatives, dabsyl, cholesterol, cyanine derivatives, or polyethylene glycol can also be introduced. Certain internucleotide linkage modifications are also introduced via incoming reactive nucleoside inclusions. Examples of resulting internucleotide linkage modifications include, but are not limited to, methylphosphonate, phosphoramidate, phosphorothioate, or phosphorodithioate.

  Many modifiers can be used, using the same or similar cycles. Examples of this class are, for example, 2-aminopurine, 5-methylcytidine, 5-aminoallyluridine, diaminopurine, 2-O-alkyl, multi-atom spacers, single monomer spacers, 2 ′ aminonucleosides 2′-fluorinated nucleoside, 5-iodouridine, 4-thiouridine, acridine, 5-bromouridine, 5-fluorocytidine, 5-fluorouridine, 5-iodouridine, 5-iodocytidine, 5-biotin thymidine, 5- Fluoroceintomidine, inosine, pseudouridine, abasic monomer, nebularane, deazanucleoside, pyrene nucleoside, azanucleoside and the like. Often, the remaining steps in the synthesis remain the same except for modifications that introduce substituents that are subject to standard deprotection conditions. Here, modification conditions that do not affect the substituent are used. The second, specific internucleotide linkage modification requires a change in oxidation step that allows their introduction. Examples of this class include phosphorothioates and phosphorodithioates that require oxidants with elemental sulfur or another suitable sulfur transfer agent. A third, specific conjugate and modification is introduced by a “post-synthesis step” where the desired molecule is added to the biopolymer after the solid phase synthesis is complete. An example of this is the addition of polyethylene glycol to a pre-synthesized oligonucleotide containing a primary amine attached to a hydrocarbon linker. Attachment in this case can be achieved using N-hydroxysuccinimidyl ester of polyethylene glycol in a solution phase reaction.

  Although the most preferred methods for the synthesis of synthetic RNA and its analogs have been outlined here, any nucleic acid synthesis method that can assemble these molecules should be able to be used in their assembly. Examples of alternative methods include the synthesis of 5'DMT-2'TBDMS and 5'DMT-2'TOM. Some 2'-O-methyl, 2'-F, and backbone modifications can be introduced into transcription reactions using, for example, modified and wild type T7, and SP6 polymerase.

(Synthesize modified RNA)
The following guidelines are provided for the synthesis of modified RNA and can be readily adapted to any automated synthesizer known in the art.

(3 'terminal modification)
There are several ways to incorporate the 3 ′ modification. The 3 ′ modification can be anchored or “loaded” onto a selected solid support using methods well known in the art. Alternatively, a 3 ′ modification can be used as the phosphoramidite. This phosphoramidite is coupled to a common carrier using standard synthetic methods, which is a hydroxyl that produces a 3 ′ terminal modification by introducing an activated phosphoramidite of the desired terminal modification. I will provide a. Alternatively, the 3 ′ modification can be introduced post-synthetically after the polynucleotide is removed from the solid support. The free polynucleotide initially has a hydroxylamino, thiol, or halogen at the 3 ′ end that reacts with an appropriately activated form of the selected modification. Examples include, but are not limited to, reactions of N-hydroxysuccinimidyl esters, thioethers, disulfides, maleimides or haloalkyls. This modification now becomes the 3 ′ end of the polynucleotide. Examples of modifications that can be post-synthetically conjugated include, but are not limited to, fluoresceins, acridines, TAMRA, dabsyl, cholesterol, polyethylene glycols, multi-atom spacers ( multi-atom spacer), cyanines, lipids, carbohydrates, fatty acids, steroids, peptides, or polypeptides.

(5 'terminal modification)
There are several ways to introduce a 5 ′ modification into a polynucleotide. For example, nucleotides with 5 ′ modifications can be purchased and then activated with phosphoramidites. This phosphoramidite having a 5 'modification is also commercially available. The activated nucleoside with the 5 'modification is then used in the cycle just as any other activated nucleoside can be used. However, not all 5 'modifications are used as phosphoramidites. In such cases, the 5 ′ modification can be introduced in a similar manner as described above for the 3 ′ modification.

(Thioate)
Polynucleotides having one or more thioate moieties, such as phosphorothioate linkages, were made according to the synthesis cycle described above and illustrated in FIG. However, instead of the t-butyl hydroperoxide oxidation step, elemental sulfur or another sulfurizing agent was used.

(5'-position thio modification)
Monomers having a 5 'thiol can be purchased as phosphoramidites from commercial sources such as Glen Research. These 5'-position thiol-modified monomers generally carry a trityl protecting group. After synthesis, the trityl group can be removed by any method known in the art.

(Other modifications)
Due to specific modifications, the steps of this synthesis cycle vary somewhat. For example, if the 3 ′ end has an inverted thymidine (dT) (the first base is attached to the solid support by the 5 ′ hydroxyl group and the first coupling is a 3′-3 ′ bond): Since detritylation and coupling occur more slowly, excess detritylating agent such as dichloroacetic acid (DCA) should be used and the coupling time should be increased to 300 seconds. Some 5 'modifications may require long coupling times. Examples include cholesterol and fluorophores such as Cy3 or Cy5 biotin, dabsyl, amino linkers, thiolinkers, spacers, polyethylene glycol, phosphorylating agents, BODIPY or photocleavable linkers and the like.

  If the polynucleotide has only one modification, this modification removes the carrier with the partially constructed polynucleotide on it, manually couples the monomer with the modification, and then an automated synthesizer It should be noted that it can be performed most efficiently manually by repositioning the support therein and resuming automatic synthesis.

(Example 2)
(Deprotection and cleavage of synthetic oligo from carrier)
Cutting was done manually or by an automated process on the machine. Cleavage of the protecting moiety from the internucleotide linkage, such as a methyl group, can be accomplished by using any suitable cleavage reagent known in the art, such as dithiolate or thiophenol. One mole of dithiolate in DMF is added to the solid support over 10-20 minutes at room temperature. The support is then washed thoroughly, for example with DMF, then with water and with acetonitrile. Alternatively, a water wash followed by a complete acetonitrile wash is sufficient to remove any residual dithiolate.

  Cleavage of the polynucleotide from the support and removal of the exocyclic base protection can be accomplished using a 40% aqueous N-methylamine (NMA) solution followed by heating to 55 ° C. for 20 minutes. Once the polynucleotide is in solution, NMA is carefully removed from the solid support. The solution containing the polynucleotide is then dried under vacuum until the NMA is removed. Further processing, including duplexing, desalting, gel purification, quality control, etc., can be performed by any method known in the art.

  NMA steps may be changed for some modifications. For example, for 3 'amino modification, treatment with NMA should be at 55 ° C for 40 minutes. Puromycin, 5 'terminal amino linkage modification, and 2' amino nucleoside modification are heated for 1 hour after adding 40% NMA. Cy5-modified oligonucleotides are treated with ammonium hydroxide for 24 hours while protected from light.

(Preparation of cleavage reagent)
Use HPLC grade water and synthetic grade acetonitrile. Dithiolate is prepared in advance as crystals. Add 4.5 grams of dithiolate crystals to 90 mL of DMF. Forty percent NMA can be purchased from a supplier such as Sigma Aldrich and is ready for use.

(Annealing of single-stranded polynucleotide)
Single-stranded polynucleotides can be annealed using any suitable buffer by any method known in the art. For example, equal amounts of each strand can be mixed with a suitable buffer, for example, 50 mM HEPES pH 7.5, 100 mM potassium chloride, 1 mM magnesium chloride. The mixture is heated at 90 ° C. for 1 minute and cooled to room temperature. In another example, each polynucleotide is prepared separately, each at 50 micromolar concentration. 30 microliters of each polynucleotide solution is then added to the tube along with 15 microliters of annealing buffer (x5). Here, the final concentration of annealing buffer is 100 mM potassium chloride, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium chloride. The final volume is 75 microliters. The solution is then incubated at 90 ° C. for 1 minute, spun in for 15 seconds, incubated at 37 ° C. for 1 hour, and then allowed to return to room temperature. The solution can then be stored frozen at −20 ° C. and freeze-thawed up to 5 times. The final concentration of duplex is 20 micromolar. An example of a suitable buffer for polynucleotide storage is 20 mM KCl, 6 mM HEPES pH 7.5, 0.2 mM MgCl ₂ . All buffers used must be free of RNase.

(Removal of ortho ester moiety)
If desired, the orthoester moiety or portions thereof can be removed from the polynucleotide by any suitable method known in the art. Such a method uses a volatile tetramethylene diamine acetate (TEMED) pH 3.8 buffer system. The buffer system can be removed by lyophilization after removal of the orthoester moiety or portions thereof. Deprotection at a pH above 3.0 helps minimize the possibility of acid-catalyzed cleavage of the phosphodiester backbone. For example, deprotection is accomplished using 100 mM acetic acid adjusted to pH 3.8 with TEMED by suspending the orthoester protected polynucleotide and incubating at 60 ° C. for 30 minutes. be able to. The solution is then lyophilized or quickly placed under vacuum (SpeedVac) to dry before use. If desired, desalting after deprotection can be performed by any method known in the art, such as ethanol precipitation or desalting of a reverse phase cartridge.

(Example 3)
(SiRNA synthesized for use in RNA interference)
The following is a list of 19-mer siRNAs with di-dT overhangs synthesized and designed using Dharmacon's registered ACE reagent and used in accordance with the invention described herein. “SEAP” means human secreted alkaline phosphatase. “Human cyclo” means human cyclophilin. An asterisk between nucleotide units means a modified internucleotide linkage that is a phosphorothioate bond. The structure 2'-FC or 2'-FU refers to a nucleotide unit having a fluorine attached to the 2 'carbon of the ribosyl moiety. The structure 2′-N—C or 2′-N—U refers to a nucleotide unit having a —NH ₂ group attached to the 2 ′ carbon of the ribosyl moiety. Structure 2′-OME-C or 2′-OME-U is a 2′-carbon of either the Cs or Us ribosyl moiety, each with a nucleotide unit exposed to 2′-O-methyl modification. means. dG, dU, dA, dC, and dT refer to a nucleotide unit that is deoxy with respect to the 2 ′ position and instead has a hydrogen attached to the 2 ′ carbon of the ribosyl moiety. Unless otherwise stated, all nucleotide units in the list below are ribosyl with —OH at the 2 ′ carbon.

Example 4
(Implementation of RNA interference)
(Transfection)
siRNA duplexes were annealed using standard buffer (50 mM HEPES pH 7.5, 100 mM KCl, 1 mM MgCl ₂ ). Transfection is performed according to the standard protocol described below.

(Standard transfection protocol for 96-well and 6-well plates: siRNA)
Protocols for 1.293 and Calu6, HeLa, MDA 75 are identical.
2. Cells are plated to be 95% confluent on the day of transfection.
3. SuperRNAsin (Ambion) is added to the transfection mixture for protection against RNAses.
4). All solutions and handling must be performed under RNAse-free conditions.
Plate 1 0.5-1 ml of 25 ml medium in a small flask or 1 ml of 50 ml medium in a large flask.

(96 hole plate)
Add 1.3 ml 0.05% trypsin-EDTA to culture flask (6 inches large) and incubate for 5 minutes at 37 ° C.
2. Resuspend cells by adding 2.7 ml (14 ml large) normal medium and pipetting 10 times.
3. Take 25 microliters of the cell suspension from step 2 and place 10 microliters in a 75 microliter trypan blue stain (1: 4) and cell counter.
4). Count cells with a standard hemocytometer.
5. The average cell number x 4 x 10,000 is the number of cells per ml.
6). Dilute to 350,000 / ml with normal medium.
7. Plate 100 microliters (35,000 cells for HEK293) into a 96-well plate.

(Transfection to 2 x 96-well plate (60-well format))
1. OPTI-MEM 2 ml + 80 microliters Lipofectamine 2000 (1:25) + 15 microliters SuperRNAsin (AMBION).
2. Aliquots (aliquots) of siRNA (100 micromol for screening, 0.8 microliter (total dilution ratio is 1: 750, 0.8 microliter of 100 micromolar solution to obtain a final concentration of 100 nanomoles) Transfer to a pan in order (usually 3 columns x 6 for 60-well format, or 4 columns x 8 for 96-well).
3. Transfer 100 microliters of OPT1-MEM.
4). Transfer 100 microliters of OPT1-MEM with Lipofectamine 2000 and SuperRNAsin to each well.
5. Leave at room temperature (RT) for 20-30 minutes.
6). Add 0.55 ml of normal medium to each well. Cover the plate with film and mix.
7. Array out 100x3x2 directly to cells (enough for two plates).

(Transfection for 2 x 6-well plates)
8.8 ml OPTI-MEM +160 microliter Lipofectamine 2000 (1:25), 30 microliter SuperRNAsin (AMBION).
9. Transfer aliquots of siRNA (total dilution ratio is 1: 750, 5 microliters of 100 micromolar solution to give a final concentration of 100 nanomolar) to a polystyrene tube.
10. Transfer 1,300 microliters of OPT1-MEM with Lipofectamine 2000 and SuperRNAsin (AMBION).
11. Leave at room temperature (RT) for 20-30 minutes.
12 Add 0.55 ml of normal medium to each well. Cover the plate with film and mix.
13. Transfer 2 ml to each well (sufficient for 2 wells).

  mRNA or protein concentration is measured at 24, 48, 72, and 96 hours after transfection using standard kits or Custom B-DNA set and Quantigen kit (Bayer).

(Example 5)
(Activity measurement / detection)
The level of siRNA-induced RNA interference, or gene silencing, was assessed by target mRNA level or corresponding protein level. The mRNA level assay was performed using B-DNA ™ technology (Quantage Corp.). Protein levels for fLUC and rLUC were assayed with the STEADY GLO ™ kit (Promega Corp.). Human alkaline phosphatase levels were measured using Great EscAPe SEAP Fluorescence Detection Kits (# K2043-1), BD Biosciences, Clontech. Was used to assay.

(Example 6)
(2'-deoxy modification / firefly luciferase gene)
Functional effects of siRNAs with two tandem 2'-deoxy modifications on the sense and antisense strands, 21-mer with 19-mer region complementation and di-dT overhangs at the 3 'end of the duplex Systematically tested by introducing into siRNA. siRNA was directed to the firefly luciferase gene (fLUC5) introduced into HEK293 cells. siRNA functionality was measured as described above. Toxicity, when measured with ALMAR blue, appeared unaffected. Functionality was evaluated at three different concentrations. That is, 1, 10, and 100 nM final. The sequence of the siRNA used and the 2′-deoxy modification substitutions are shown in Table 1. The results of these experiments are shown in FIGS. 19-23.

(Example 7)
(2'-O-methyl modification / firefly luciferase gene)
The functional effect of siRNA having a single 2′-O-methyl modification, two tandem 2′-O-methyl modifications, and three tandem 2′-O-methyl modifications on the sense and antisense strands is 19 Systematic testing was performed by introducing complementation of the body region and di-dT overhangs into 21-mer siRNAs with double-stranded 3 ′ ends. siRNA was directed to the firefly luciferase gene (fLUC5) transfected into HEK293 cells. siRNA functionality was measured as follows. Functionality was evaluated at three different concentrations. That is, 1, 10, and 100 nM final. Toxicity, when measured with ALMAR blue, appeared unaffected. The sequence of siRNA used and the arrangement of 2′-O-methyl modifications are shown in Table 5. The results of these experiments are shown in FIGS.

(Example 8)
(2'-deoxy and 2'-O-methyl modifications / sense vs. antisense strand)
Fifteen duplexes (of which 5 are directed to the human cyclophilin gene and 10 are directed to firefly / luciferase) were tested with various modifications (see Figures 29-31). For human cyclophilin B, the duplexes tested were (1) unmodified, (2) 2'-O-methyl modifications at the first and second positions of the sense strand, and (3) the first strand of the antisense strand. It contains 2′-O-methyl modifications at the 1 and second positions, and (4) 2′-O-methyl modifications on the sense and antisense strands. For luciferase, in addition to all of the modifications described for human / cyclophilin B, (1) 2′-O-methyl modifications on the AS chain in combination with 5 ′ phosphorylation of the AS chain, and (2) the antisense strand The 2'-O-methyl modification of the sense and antisense strands in combination with the 5 'phosphorylation of was tested. For all 15 duplexes, modifications at positions 1 and 2 of the sense strand with a 2′-O-methyl moiety did not interfere with functionality. The same modification of the antisense strand limited duplex functionality. This decrease in functionality was partially reduced when the antisense strand was phosphorylated at its 5 ′ end (FIGS. 30 and 31). Considering the above data, 2′-O-methylation at the 1st and 2nd positions of the sense strand in combination with 5 ′ phosphorylation of the antisense strand can turn off the sense strand without changing the functionality of the duplex. An inexpensive, reliable and non-toxic method that modifies siRNA duplexes to limit targeting. This information is of commercial value. This is because it increases the specificity and efficacy of siRNA. Recent microarray data indicate that the presence of exactly 11 nucleotides is sufficient to induce non-specific silencing. The homology present within the sense strand of the siRNA duplex generally constitutes at least half non-specific functionality. If inherent non-specificity is blocked, the sense strand cannot contribute to off-targeting and siRNA specificity should increase. Transfer of equilibrium to a functional RISC antisense strand complex also reduces the effective concentration of siRNA.

Example 9
(Modified siRNA with 5 ′ conjugate)
The effect of various modifications on duplex stability, functionality, and passive uptake was assayed. FIG. 33 shows cholesterol conjugation for duplex stability and (2′-O-methyl modifications on positions 1 and 2 of the sense and antisense strands, plus 2 ′ of all C and U of the sense strand. -O-methyl modification, all C and U 2'F modifications of the antisense strand, plus, 5 'phosphorylation of the antisense strand). Naked or modified siRNAs were ³² P-labeled (AS chain, T4 kinase, promega), incubated with serum (or serum albumin), and further run on gel shift chromatography (denaturation, 15% PAGE). The results of these experiments show that while unmodified duplexes degraded rapidly, cholesterol and siRNA with the modifications already described are stable in the presence of serum or serum albumin. Similarly, increased stability is also shown for siRNA carrying C and U 2′-O-methyl modifications in combination with 3′idT capping and CHO (5 ′ sense) conjugation (FIG. 35).

  Studies on similarly modified molecules (eg, cholesterol modification of the 5 ′ end of the sense strand in combination with 2′Fl modification of the sense strand) show that such modification is functional (FIG. 34) and passive uptake (FIG. 36, modification = 2′F) for both sense and antisense C and U with cholesterol is shown to be enhanced.

(Example 10)
(Deoxy and 2'-O-methyl modified walk at 2 'on SEAP2217 target)
The constructs used for 2′-deoxy and 2′-O-methyl walks using siRNA targeting the SEAP construct (see FIGS. 31 and 32) are shown in Table 6.

(Example 11)
(Molecular 1 modification and stability)
For the purposes of Examples 11-18, the term “molecule 1 modification” means 2′-O-methyl modifications at positions 1 and 2 on the sense strand, all C and on the sense strand. Of molecules containing 2'-O-methyl modifications on U, all C and U 2'-fluorine (Fl) modifications on the antisense strand, and phosphate modifications on the 5 'end of the antisense strand That means. Similarly, the term “molecule 2 modification” includes 2′-O-methyl modifications on positions 1 and 2 of the sense strand, 2′-O-methyl modifications on all Cs and Us of the sense strand, Molecules that contain a Cy3 label on the 5 'end of the sense strand, 2'-fluorine (Fl) modifications on all C and U of the antisense strand, and phosphate modifications on the 5' end of the antisense strand Say.

  To evaluate the effect of molecule 1 modification on siRNA stability, four unique siRNAs were synthesized in modified and unmodified forms. Subsequently, these molecules were incubated in 100% serum at 37 ° C. at different times, and double-stranded intactness was evaluated by analysis by PAGE. Visualization of the sequence was performed by ethidium bromide staining.

  These experimental results shown in FIG. 37 show that the duplex with molecule 1 modification is significantly more stable than the unmodified equivalent. Unmodified molecules were degraded by more than 50% within 2 minutes when exposed to serum at room temperature. In comparison, the half-life of the sequence with molecule 1 modification was approximately 125-135 hours. Thus, molecule 1 modification significantly increased stability by about 500 times.

(Example 12)
(Molecular 1 modification and siRNA silencing drag)
To evaluate the effect of molecule 1 modification on siRNA drag, two unique siRNAs directed against human cyclophilin B (U1 and U2) were modified and unmodified using 2'-O-ACE chemistry It was synthesized in form and its functionality was examined in a whole cell assay. Briefly, modified and unmodified siRNA were transfected (Lipofectamine 2000) into HeLa cells (10,000 cells / well, 96-well plate) at a concentration of 0.01-200 nM and cultured for 24-48 hours. Subsequently, the expression level of designated targets was assessed using a branched DNA assay (Genospectra, Femont, CA).

  The results of these experiments are shown in FIG. These experimental results also indicate that the double-stranded with molecule 1 modification functions equivalently to unmodified siRNA at all concentrations tested. Thus, molecule 1 modification does not appear to alter siRNA drag.

(Example 13)
(Molecular 1 modification and silencing life)
In order to determine the effect of molecule 1 modification on siRNA silencing longevity, siRNA targeting the human cyclophilin B gene was synthesized in modified and unmodified forms, as described above, in HeLa cells (100 nM). Transfected. Subsequently, the level of silencing was monitored over 7 days using a branched DNA assay.

  An example of the results of these experiments shown in FIG. 39 shows that the level of silencing induced by unmodified molecules decreases from about 70% to 0% over 7 days, and the modified duplex is in the course of the experiment. It is demonstrated to induce> 80% functionality throughout. Thus, siRNA modified with a molecule 1 modification increases the lifetime of silencing induced by these duplexes.

(Example 14)
(Molecular 1 modification and toxicity)
To determine the effect of molecule 1 modification on siRNA toxicity, four siRNAs (U1-U4) targeted to human cyclophilin B were synthesized in modified and unmodified forms and in a concentration range of 0.01-200 nM. HeLa cells were transfected. At time t = 48 hours after transfection, an Almer Blue viability assay was performed to assess the level of cell death within the cell population. In a side-by-side comparison of modified and unmodified duplexes, there was no difference in the level of cell death induced at any of the concentrations tested (FIG. 40).

(Example 15)
(Molecular 1 modification and off-target effect)
To evaluate the effect of molecule 1 modification on off-target effects, four siRNA target human cyclophilins B (U1-U4) were synthesized in modified and unmodified forms and transfected into HeLa cells at a concentration of 100 nM. . Subsequently, (1) mock-transfected, (2) transfected (unmodified), and (3) total RNA obtained from transfected (modified) cells was purified (Qiagen), converted to cDNA, and further transformed into Agilent ' Labeling with Cy3 (mock transfection) or Cy5 (transfection-unmodified, transfection-modified) was performed using the s Low RNA Input Linear Amp Kit. Labeled cDNAs obtained from mock transfected and non-transfected cells were mixed and hybridized to Agilent Human 1A (V2) Oligo Microarray containing more than 21,000 probes. Using software (Agilent's Feature Extraction, Sptfire, and Sptfire Functional Genomics) (versions 7.2, 7, 2, and 7.1, respectively), the number of off targets bound to the modified and unmodified samples was determined. evaluated.

  A summary of these off-target studies is shown in FIG. Also, according to this summary, the modified and unmodified siRNAs showed similar functions with respect to the number of off-targeted genes. Specifically, if the off-target gene is isolated based on the level of induction or suppression (compared to wild-type gene expression), the modified siRNA functions similarly (or better) than the unmodified one. did.

(Example 16)
(Molecular 2 modification and silencing efficacy)
To test the functionality of siRNA containing molecule 2 modification, cyclo14 (5′GOCCTTAGCTACAGGAGAG, sense strand SEQ ID NO: 322), a siRNA targeting human cyclophilin B, was synthesized by molecule 2 modification and further specified. The ability to silence selected targets was tested. Briefly, cyclo14 was synthesized by appropriate modification using 2'-O-ACE chemistry. Subsequently, T482HeLa cells (10,000 cells / well, 96-well plate) were plated and cultured overnight, and further transfected with cyclo14 duplex at a concentration of 100 nM (Lipofectamine 2000). Three days after transfection, the level of mRNA silencing was assessed using a branched DNA assay (Genospectra, Fremont, CA).

  The results of these studies are shown in FIG. Unmodified cycl14 duplex induces approximately 80-95% silencing. Cyclo14 duplex modified with Cy3 label alone induces about 70-80% silencing, and cycl14 siRNA with molecular 2 modification induces silencing with 80% or better and addition of molecular 2 modification Has little effect on duplex functionality.

(Example 17)
(Effect of molecule 2 modification on track ability)
In order to test the usefulness of the molecule 2 modification, as a means of evaluating transfection efficiency, cyclo14 siRNA was prepared by the above-mentioned modification and visualized with a fluorescence microscope. Specifically, cyclo14 was synthesized by appropriate modification using 2′-O-ACE chemistry. Subsequently, T482HeLa cells (10,000 cells / well, 96-well plate) were plated and cultured overnight, and further transfected with appropriate double strands (Lipofectamine 2000) at a concentration of 100 nM. Forty-eight hours after transfection, the medium was incubated with Hoechst 33342 (2 μg / ml, 20 minutes, 37 ° C.) and then visualized with a Leica DMIL fluorescence microscope using Dapi and Rhodamine filters.

  Fluorescence micrographs of HeLa cells transfected with cycl14 siRNA with molecular 2 modification (not attached) show strongly stained nuclei and their surroundings. In cells transfected with unmodified cyclo14 siRNA, no comparable staining is seen (data not shown), so molecular 2 modification is an excellent means of assessing the intracellular location of a given siRNA and the success of the transfection. I understand that.

(Example 18)
(Effect of molecule 2 modification on stability)
To test whether molecular 2 modification increased the intracellular stability of the double strand, cycl14 siRNA with molecular 2 modification was transfected into HeLa cells and compared to Cy3-labeled Cyclo14 transfected cells for 7 days. It shows significantly enhanced siRNA stability over duplexes modified with Cy3 alone by the addition of the aforementioned modifications. Both samples show a strong staining pattern on day 2 (Day2), while Cy3-Cyclo14 transfected cells are lost by day 7 (Day7). In contrast, cells containing cycl14 siRNA with a molecular 2 modification retain a strong staining pattern on day 7 (Day 7). Furthermore, unlike Cy3-labeled duplexes, siRNAs with molecular 2 modifications also facilitate nuclear access to the duplex.

(Example 19)
(Identification of chemical modifications that modify silencing activity)
A modified walk consisting of 1, 2 or 3 consecutively modified nucleotides in the sense (S) and antisense (AS) strands using 2'-O-ACE chemistry as a platform for RNA synthesis Was performed on SEAP-2217, human secreted alkaline phosphatase (SEAP, SEAP-2217-sense strand 5'-GUUGAAUGUAUAGUCACAGAGAGAUdTdT (SEQ ID NO: 326)). . Subsequently, the silencing efficiency of these modified siRNAs was determined by co-transfecting each duplex with SEAP expression vector (Clontech) into HEK293 cells (100 nM siRNA, 50 ng / well SEP expression vector, Lipofectamine 2000). Assessed by assaying for a decrease in target protein activity 24 hours later. Figures 43A and 43B show the correlation between modification and function for 2'-O-methylated SEAP-2217 siRNA. Unmodified duplexes that target SEAP induce> 90% silencing of the SEAP gene. Single base modifications of the S and AS strands induced little effect on siRNA activity. This suggests that there is no single 2′-hydrosyl group on either strand that plays an essential role in RNAi. In contrast, double side-by-side modified walks identified several key positions where the introduction of modified bases significantly interfered with silencing activity.

  The most significant interference with function is observed when modifying two consecutive bases (positions 1 and 2) or three consecutive bases (positions 1, 2 and 3) at the 5 ′ end of the AS strand Therefore, the cooperation effect between adjacent modification groups is implied. Since similar modifications of the S chain have failed to alter duplex functionality, the paired 2'-O-methyl modified bases allow the S and AS chains to be distinguished.

  The same set of experiments was performed with 2'-deoxy modification. The studies shown in FIGS. 44A and 44B show that adding three consecutive 2′-deoxy groups at different positions (eg, 1 + 2 + 3, 4 + 5 + 6, etc.) on either strand of the duplex , Indicating no effect on double-stranded silencing. Similar results were observed for walks testing the effect of 2 and 1 consecutive 2'-deoxy groups (data not shown).

To further test the effect of 2'-O-methyl modification on double-stranded functionality, a series of siRNAs directed to the luciferase gene (luc 8, 18, 56, 58, 63, and 81) were It is synthesized using -O-ACE chemistry and modified to include an O-methyl group at the 2 'position of the ribose ring.
Luc 8 5′-GAAAAAUCAGAGAGAUCUCU (SEQ ID NO: 327)
Luc 18 5'-UACCGGGAAAACUCGACGCA (SEQ ID NO: 328)
Luc 56 5′-ACGUCGCCAGUCAAGUAAC (SEQ ID NO: 329)
Luc 58 5'-GAUUAACGUCGCCAGUCAAG (SEQ ID NO: 330)
Luc 63 5'-AGAGALTCGUGGALIJACGUC (SEQ ID NO: 331)
Luc 81 5′-UGUUGUUUGGAGCACCGGA (SEQ ID NO: 332)
(The sequence listed above is the sense strand).

  Specifically, siRNAs containing 2′-O-methyl modifications on the two 5 ′ extreme nucleotides of (1) the sense strand, (2) the antisense strand, or (3) both strands are HEK283 cells are transfected via the expression plasmid (pCMVLuc, 50 ng / well). Subsequently, a side-by-side comparison of the silencing capacity of each duplex was performed to determine the effect of this modification on target transcription degradation.

  The results of these studies showed that only the addition of 2′-O-methyl groups to the AS strand significantly reduced the ability of the duplex to silence the target mRNA (see FIGS. 45A-F). ). In contrast, duplexes with this modification on the sense strand functioned well (luc 58, 63, 81) or better (luc 56, 8, 18) than the equivalent unmodified siRNA. . This suggests that modification of the sense strand biased strand selection by RISC and (in some cases) increased the effective antisense strand concentration. Enhanced silencing reduces the binding affinity of RISC to the 5 'sense end of the molecule (thus increasing the availability of free RISC to bind to the opposite end), a natural kinase to phosphorylate the sense strand May be the result of a reduced ability of RISC (thus reducing sense and antisense strand competition for access to RISC), or the ability of RISC to release the duplex from the 5 'sense end. SiRNA containing 2'-O-methyl modifications on both strands showed a reduced silencing capacity between the values observed for molecules containing the modifications on either single strand. One interpretation to these results is that the binding affinity of the 2'-O-methyl modification with respect to the modified strand is lower than that of RISC. If both strands are modified, neither strand will benefit over its complement and a new equilibrium will be established that represents the average functionality of both modified molecules.

  Test whether the reduced level of silencing observed in cells containing 2'-O-methylated S / AS siRNA is a result of the weakened ability of cellular kinases to phosphorylate duplexes Therefore, siRNA with 2′-O-methyl modification was modified to have a phosphate group on the 5 ′ end of the AS strand. Specifically, 2′-O at either (1) positions 1 and 2 at the 5 ′ end of the antisense strand, or (2) positions 1 and 2 at both 5 ′ ends of the antisense and sense strands. -Luc siRNA having a methyl group was 5'-phosphorylated on the AS strand during the synthesis. These duplexes were then introduced into HEK293 cells using the procedure previously described and tested for the ability to silence the desired target. The results show that in 83% (10/12) of the tested examples, 5 ′ phosphorylation of the antisense strand improved duplex silencing efficiency over the equivalent non-phosphorylated molecule. (FIGS. 45A-F). In the remaining two examples, silencing remains unchanged or only slightly improved.

  More detailed experiments on the effects of chemical modifications on silencing using dose response curves support previous data. Five different siRNAs directed to the luciferase gene (luc 8, 56, 58, 63, and 81) were synthesized using 2-O-ACE chemistry in 2'-O-methyl modified and unmodified forms. These duplexes were then transfected into HeLa cells (with a luciferase expression vector) at varying concentrations between 0.01 and 200 nM and assayed for the level of silencing of luciferase protein (t = 24-48). The results of these experiments are summarized in FIG. 46 and the following conclusions are drawn. (1) Silencing activity is consistently disrupted by the addition of 2'-O-methyl groups at positions 1 and 2 of the 5 'end of the antisense strand. (2) Addition of 2′-O-methyl group to the 1st and 2nd positions of the 5 ′ end of the sense strand of siRNA improves the effect of target-specific silencing or against this silencing One of them has little effect. (3) an unmodified duplex on the 5 ′ end of the antisense strand by addition of a 2′-O-methyl group to positions 1 and 2 at the 5 ′ end of both strands (sense and antisense); An intermediate silencing effect occurs between that observed with the duplex with the '-O-methyl modification. (4) by adding a phosphate group to the 5 ′ end of the antisense strand of the molecule having 2′-O-methyl modification at the 1 and 2 positions of the 5 ′ end of both the sense and antisense strands, The silencing function of the molecule is improved over duplexes with 2'-O-methyl modifications on the 5 'ends of both strands.

  These results indicate that in addition to 5 ′ phosphorylation of the first terminal nucleotide of the antisense strand, addition of 2′-O-methyl modifications at the 1 and 2 positions of the sense and antisense strands of siRNA Suggests increasing the potency of the duplex at concentration. Furthermore, the effects of various modifications lead to the following conclusions. (1) Addition of a 2'-O-methyl group to the 5 'end of the sense or antisense strand results in a significant decrease in silencing by that strand. (2) Addition of 5 'phosphate group almost completely restores silencing even in the presence of 2'-O-methyl group. Thus, some fractions of the inhibitory effect of As-2′-O-methylation are probably the result of inhibition of double-stranded phosphorylation, while some part of the effect is in other parts of the RNAi pathway. There appears to be considerable probability that this is due to the effects of this form of modification on these steps (eg, RISC binding, siRN unwinding, RISC-mediated siRNA target binding, or RISC-mediated target cleavage). Importantly, the potential effect of 2′-O-methylation at these other steps suggests to the authors that the modification described above is intended with 100% homology with the antisense strand, It made me think that RISC's ability to distinguish between off-targets with poor homology may also change.

(Example 20)
(Effects of 2'-O-methylation and 5 'phosphorylation on off-targeting)
The off-target produced by the sense strand by the observation that 2'-O-methylation of the antisense strand disrupts silencing while the equivalent sense strand label has no effect on target-specific silencing • Suggest strategies to eliminate silencing. Furthermore, the observation that the steps downstream of double-stranded phosphorylation may be affected by 2′-O-methylation is the combination (in sense 1 and antisense strand 5 ′ positions 1 and 2). The possibility that siRNA containing 2′-O-methylation, plus, 5 ′ end phosphorylation of the AS strand) has high potency and / or low sense / antisense off-targeting effects Lead.

  Off-targeting of 5′-phosphorylation of the antisense strand in combination with (1) sense strand, (2) antisense strand, and (3) 2′-O-methylation of sense and antisense strand To test the effects, two siRNAs targeting four different genes (total 8 siRNAs; MAPK14-193 and -153, MPHOSPH1-202 and -203, IGF1R-73, and -74, and PTEN- 213, and further -214) were synthesized using 2'-O-ACE chemistry and further modified at the appropriate site. Extensive off-targeting is observed when experiments are performed under conditions where MAPK14-153 contains only a phosphate group on the 5 'end of the antisense strand. If the sense strand is further modified with 2'-O-methyl groups at positions 1 and 2, off-targeting by that strand is removed. However, a new set of antisense strand offset targets occurs. The increase in antisense strand off-targeting is likely due to increased AS strand-RISC interaction in the absence of competition by the sense strand. When the antisense strand has a phosphate group on the 5 ′ end and is modified with 2′-O-methyl groups at positions 1 and 2, off-target by the antisense strand is lost, but the sense strand Off-target is observed again. Finally, both the sense and antisense strands are modified by 2'-O-methyl groups (on the 1 and 2 positions of each strand) and a phosphate group is attached on the 5 'end of the antisense strand When doing so, the degree of off-targeting by both strands decreases rapidly.

  Results similar to those described above were obtained for the remaining seven siRNAs corresponding to IGF1R, PTEN, MAPK1, and MPHOSPHI. From these experiments it is therefore possible to conclude that (1) another siRNA directed at the same target induces varying levels of off-target effects. (2) The 2′-O-methyl modification at the 1 and 2 positions of the 5 ′ end of the antisense strand (in combination with 5′-phosphorylation of the antisense strand) removes the off-target effect by that strand, However, in many cases, this results in an additional sense strand off target. (3) 2′-O-methyl modifications at the 1 and 2 positions of the 5 ′ end of the sense strand (in combination with 5′-phosphorylation of the antisense strand) remove off-target effects based on that strand, Often results in additional antisense strand off-target. (4) The 2'-O-methyl modification at positions 1 and 2 on both the sense and antisense strands (in combination with 5 'phosphorylation of the antisense strand) dramatically removes the target effect from both strands. Thus, a combination of three modifications (5′-phosphorylation of the antisense strand, 2′-O-methylation of positions 1 and 2 of the sense strand, and 2′-O-methylation of 1 and 2 of the antisense strand) ) Leads to a sharp decrease in the off-target effect commonly caused by both chains. In addition, in three cases where specific silencing activity was tested (MAPK14, PTEN, and MPHOSPH1), fully modified molecules with minimal off-target effects (eg, 5 ′ phosphate on the antisense strand) 2′-O-methylation at positions 1 and 2 of the sense strand, and 2′-O-methylation at positions 1 and 2 of the antisense strand) are 5 ′ on the 5 ′ end of the antisense strand. The given target was silenced to the same or better than siRNA containing only phosphate groups.

(Effect of 2'-O-methylation)
The above data clearly show that siRNA activity is interrupted by adding 2'-O-methyl groups to the 1- and 2-positions of the 5 'end of the AS strand. The way this modification disrupts double-stranded function can be broken down into two distinct steps in the RNAi pathway. In the first step, 2′-O-methylation clearly interrupts the activity of resident kinases that phosphorylate the duplex. By adding a 5 ′ phosphate group, the need for cellular enzymes is totally reduced, thereby satisfying this function and further improving the double-stranded functionality. Thus, one of the effects of this chemical modification is well defined. While increasing the activity by synthetic addition of a phosphate group to the 5 ′ end of the antisense strand of siRNA modified with 2′-O-methyl groups at the 1 and 2 nucleotides of both strands, Full functionality is not restored. This result indicates that steps downstream of the phosphorylation event (eg, RISC binding, RISC-mediated siRNA target / off-target interaction, and / or RISC-mediated siRNA target / off-target cleavage) are also 2′-O— This suggests that it is affected by methylation.

(2'-O-methylation and enhanced duplex functionality)
An important parameter for functionality is siRNA binding to RISC followed by double-stranded unwinding. The studies presented here, in some cases, alter the dose response curve by 2'-O-Me modification of the sense strand in a manner that is inversely proportional to the original functionality of the molecule. One interpretation of these findings is that the sense and antisense strand and RISC linkages exist in equilibrium, which is an “on” and “off” factor (eg, k ₁ , k ₁ ′, k ₂ , and k _{2 ′} , FIG. 47). Highly functional molecules exhibit K _overall (k ₁ k ₂ / k ₁ 'k ₂ '), reflecting a slope in the direction of binding to the antisense strand (eg, K _overall = 100). In contrast, a non-functional or semi-functional molecule (<F70) represents a K _overall that exhibits a more even and balanced strand interaction (eg, K _overall is about 1 for <70 siRNA) is there). Since chemical modification of the sense strand of a non-functional or semi-functional molecule drives the equilibrium state for RISC-AS interaction, RISC-AS: RISC-S ( _Koverall is about 5) and overall dual function Significantly changes gender. In contrast, for highly functional molecules, RISC-AS interaction is already preferred, so that chemical modification is less likely to further enhance RISC-AS association and functionality.

(2'-O-methylation and off-target silencing)
Part of the silencing phenotype by synthetic addition of a phosphate group to the 5 'end of the double stranded antisense strand containing the 2'-O-methyl modifications at the 1st and 2nd positions of both strands While reaching rescue but lacking full recovery, additional steps where this modification is downstream of double-stranded phosphorylation (eg, RISC binding, RISC-mediated siRNA target / off-target interactions, and (Or / and RISC-mediated siRNA target / off-target cleavage). This fact opens up the possibility that these same modifications may place greater demands on the degree of siRNA-target complementarity required for silencing. If the 2'-O-methyl position overlaps with the off-target and the homologous part of a given siRNA (sense or antisense strand), the addition of a chemical modification destabilizes the siRNA off-target interaction. By doing so, there is a possibility of suppressing an important step in RNAi-mediated down-regulation. Alternatively, if the position of the 2′-O-methyl group overlaps with the portion of homology between the off-target and a given siRNA, additional chemical modifications will change the flexibility of the duplex in that region, Can inhibit the ability of RISC to degrade target molecules. In yet another scenario, the site of 2'-O-methyl modification cannot overlap with the site of homology with the off target. In this case, the destabilizing effect of the modification is transferred downstream of the molecule, thereby removing the RISC's ability to pair and / or degrade with a target having less than 100% homology to the siRNA. There is a possibility. Therefore, 2′-O-methyl modifications at positions other than the 5 ′ end of the S or AS chain can be used to remove off-target effects.

(Example 21)
(Modified double strand as an exaequo agent)
During continuous dosing with siRNA or microarray experiments, it is desirable to keep the total siRNA concentration constant during transfection. Unfortunately, the addition of a second siRNA (even if it is non-specific) can reduce the degree of silencing induced by the target-specific siRNA, which is probably due to RISC. Due to competition between the two sequences for access. For that reason, it is important to develop a duplex that can act as an exaequo agent that does not disrupt gene targeting.

  To evaluate the ability of the modified duplex to act as a non-competitive exaequo agent, such as in a dose experiment, the effect of modified and unmodified nonspecific siRNA on specific siRNA silencing was examined. To accomplish this, siRNA duplexes directed against the human cyclophilin B gene (eg, cycl4) were first transfected into cells at various concentrations (eg, dose response). Cyclo4 alone induced target specific silencing is then followed by silencing induced by this molecule in the presence of (a) unmodified and non-specific sequence # 4 (NS4) or (b) modified NS4. Compared. In this case, the modified version of NS4 is either 19 or 17 base pairs long (SEQ ID NO: 333: UAGCGACUAAACACAUCAA and SEQ ID NO: 334: UAGCGACUAAACACAUCUC, respectively) and is in positions 1 and 2 on the sense and antisense strands -O-methyl group was included, and also included 2'-O-methyl group 2'-O-methyl group on C and U, and 2'Fl on C and U of the antisense strand. No phosphate group is attached to the 5 'end of the sense and antisense strands.

Specifically, HeLa cells were plated on 96-well plates and allowed to adhere overnight. Subsequently, the cells were transfected with one of the following using Lipofectamine 2000. That is,
a) cyclo4 siRNA (1-100 nM), (SEQ ID NO: 318: GGA AAG ACU GUU CCA AAA A)
b) cyclo4 siRNA (1-100 nM), plus, NS4 (19 base pairs),
c) cyclo4 siRNA (I-100 nM), plus, modified NS4 (17 or 19 base pairs),
d) Cyclo4 siRNA (1-100 nM) plus, modified NS4 (17 or 19 base pairs), which also contains a Cy3 label on the 5 ′ end of the sense strand. The concentration of the second siRNA was matched to the concentration of cyclo4 so that the total siRNA concentration was kept at 100 nM during transfection. Next, target-specific silencing was measured using a branched DNA assay (Genospectra, Fremont, CA) (t = 24 hours).

The results of these experiments are shown in FIG. 43 and described below.
1. Cyclo4 is a powerful double strand. Decreasing the concentration of cyclo4 from 100 nM to 1 nM does not change this double-stranded silencing ability (F95, see FIGS. 48A-I).
2. Addition of 19 base pair unmodified NS4 siRNA to cycl4 has a favorable concentration-dependent decrease for cycl4 induced gene silencing and NS4 competes with cycl4 for access to, for example, the RISC complex (See FIGS. 48B-I).
3. 2'-O-methyl groups on positions 1 and 2 of the sense and antisense strands, 2'-O-methyl groups on C and U of the sense strand, and 2'Fl on C and U of the antisense strand NS4 duplexes containing groups (17 or 19 base pairs) do not reduce target-specific silencing by cycl4, suggesting that these duplexes do not compete with cycl4, eg, for RISC ( (See FIGS. 43BII and BIII).
4). 2'-O-methyl groups on positions 1 and 2 of the sense and antisense strands, 2'-O-methyl groups on C and U of the sense strand, and 2'Fl on C and U of the antisense strand NS4 duplexes (17 or 19 base pairs) containing a group and Cy3 on the 5 ′ end of the sense strand do not reduce target-specific silencing by cycl4, which duplexes are for example against RISC Suggesting that it does not compete with cyclo4 (see FIGS. 48AII and AIII).

  These results indicate that siRNAs containing the above modifications do not compete with functional siRNA and can be used as exaequo agents in siRNA studies such as microarray studies.

  The reason why the siRNA having the modification does not compete with the target-specific siRNA is thought to be because the modified duplex cannot enter the cell. To test this, the 2'-O-methyl group at positions 1 and 2 of the sense and antisense strands, the 2'-O-methyl group on C and U of the sense strand, and the C of the antisense strand And siRNA carrying a 2 ′ Fl group on U (no 5 ′ phosphate group on the antisense strand) is labeled with the Cy3 moiety on the 5 ′ end of the sense strand and transfected into HeLa cells did. Forty-eight hours after transfection, cells were analyzed with a fluorescence microscope. The results of these experiments indicate that Lipofectamine 2000 transfection does not alter the above modifications to their duplex ability to enter cells. (A) 2'-O-methyl group on positions 1 and 2 of the sense and antisense strands, 2'-O-methyl group on C and U of the sense strand, added to C and U of the antisense strand HeLa cells stained with a cycl14 siRNA duplex modified by a 2'Fl group, as well as a Cy3 group on the 5 'end of the sense strand, and stained as in (B) (A) and the location of the nucleus Photomicrographs (not replicated here) with counterstained with Hoechst 33342 to identify the siRNA nuclei and perinuclear containing these modifications.

Test whether the addition of 2'-O-methyl groups on positions 1 and 2 of the sense and antisense strands is sufficient to remove the ability of the duplex to compete with other co-transfected siRNAs In order to do this, siRNA directed against GAPDH (GAPDH4) was (1) non-specific control # 2 (NS2-SEQ ID 334: UAAGGCUAUGAGAGAAUAC), (2) positions 1 and 2 of both sense and antisense strands With non-specific control # 2 carrying 2'-O-methyl modification at (3) cyclo14 (SEQ ID NO: 335: cyclophilin siRNA that does not compete with GAPDU4), at various concentrations (0.781-100 nM) HeLa cells were transfected. The concentration of the second siRNA was matched to the concentration of GAPDH4 so that the total siRNA concentration during transfection was 100 nM. The cells were then cultured for an additional 24 hours before performing a branched DNA assay to assess the level of GAPDH transcript. The results of these experiments are illustrated in FIG. 49 and shown below.
1. GAPDH4 induces approximately 90% silencing of the wild type transcript in the presence of Cyclo14 (or by itself, data not shown).
2. The addition of unmodified NS2 resulted in a dramatic decrease in GAPDH silencing.
3. Modification of NS2 with 2′-O-methyl groups at both the 1 and 2 positions of both the sense and antisense strands eliminated the interference effect observed by unmodified molecules.

  These results demonstrate that siRNAs containing the previously described modifications do not compete with functional siRNAs and can be used as exaequo agents in a variety of siRNA applications including microarray studies.

  While the present invention has been described and illustrated in combination with certain specific or preferred inventive embodiments, it will be understood by those skilled in the art that the present invention can be subject to many further modifications. This application is generally known in the art to cover any and all variations, uses, or applications consistent with the principles of the invention and is applicable to the essential features recited in the application and scope of the appended claims. Or it includes deviations from the disclosure in accordance with convention.

Claims (10)

siRNA,
(A) An antisense strand composed of a plurality of nucleotides, wherein the 5′-most nucleotide is the first 5′-end antisense nucleotide, and 5 of the first 5′-end antisense nucleotide 'Antisense strand phosphorylated at the carbon position;
(B) a sense strand composed of a plurality of nucleotides, the 5′-most nucleotide being the first 5′-end sense nucleotide , and immediately downstream of the first 5′-end sense nucleotide, 'it is a terminal sense nucleotide, wherein said first 5' second 5 terminal sense nucleotide 'includes a carbon sense modification and the second 5' first 2 terminal sense nucleotide and the second 2 'carbon A sense strand containing a sense modification;
Hints, the 2 'each nucleotide other than the nucleotide having the carbon modified to have a 2'-OH,
The siRNA is composed of base pairs between 18 and 30;
Each of the first 2 ′ carbon sense modification and the second 2 ′ carbon sense modification is a 2′-O-methyl modification;
siRNA. The siRNA of claim 1 , wherein the siRNA consists of between 19 and 25 base pairs. The siRNA of claim 1 , wherein the antisense strand and the sense strand are 100% complementary and form a duplex that does not contain an overhang. A method for reducing an off-target effect induced in RNAi, comprising introducing the siRNA according to claim 1 into a cell in vitro . A method of reducing off-target effects in RNAi, comprising exposing siRNA to a target nucleic acid in vitro , wherein the siRNA comprises an antisense strand and a sense strand,
(A) The sense strand is composed of a plurality of nucleotides, the 5′-most nucleotide is the first 5′-terminal sense nucleotide, and the first 5′-terminal sense nucleotide is immediately downstream of the second The first 5 ′ terminal sense nucleotide comprises a first 2 ′ carbon modification, and the second 5 ′ terminal sense nucleotide comprises a second 2 ′ carbon modification. And (b) the antisense strand is composed of a plurality of nucleotides, and the 5′-most nucleotide is the first 5′-end antisense nucleotide, and the first 5′-end antisense nucleotide Is phosphorylated at the 5 'carbon position,
The 2 'each nucleotide other than the nucleotide having carbon modifications have a 2'-OH,
The siRNA is composed of base pairs between 18 and 30;
Each of the first 2 ′ carbon sense modification and the second 2 ′ carbon sense modification is a 2′-O-methyl modification ;
Off-target effect reduction method. 6. The method of claim 5 , wherein the siRNA consists of between 19 and 25 base pairs. 6. The method of claim 5 , wherein the antisense strand and the sense strand are 100% complementary and form a duplex that does not contain an overhang. A unimolecular siRNA capable of forming a hairpin, comprising:
(A) An antisense region composed of a plurality of nucleotides, the 5′-most nucleotide being the first 5′-terminal antisense nucleotide, and 5 of the first 5′-terminal antisense nucleotide 'Antisense region where the carbon position is phosphorylated, and
(B) a sense region composed of a plurality of nucleotides, wherein the 5′-most nucleotide is the first 5′-end sense nucleotide, and immediately downstream of the first 5′-end sense nucleotide is the second Wherein the first 5 'terminal sense nucleotide comprises a first 2' carbon sense modification and the second 5 'terminal sense nucleotide comprises a second 2' carbon sense modification. Including a sense region;
(C) a loop region located between the sense region and the antisense region;
Hints, the 2 'each nucleotide other than the nucleotide having the carbon modified to have a 2'-OH,
The siRNA is composed of base pairs between 18 and 30;
Each of the first 2 'carbon sense modification and said second 2' carbon sense modification is Ru der 2'-O- methyl modified, siRNA. 9. The siRNA of claim 8 , wherein the siRNA is composed of between 19 and 25 base pairs. A method for reducing off-target effects in RNAi comprising:
Exposing the target nucleic acid to a single molecule siRNA capable of forming a hairpin in vitro, the single molecule siRNA comprising an antisense region and a sense region;
(A) The sense region is composed of a plurality of nucleotides, the 5′-most nucleotide is the first 5′-terminal sense nucleotide, and the downstream immediately after the first 5′-terminal sense nucleotide is the second A 5 ′ terminal sense nucleotide, wherein the first 5 ′ terminal sense nucleotide comprises a first 2 ′ carbon sense modification and the second 5 ′ terminal sense nucleotide comprises a second 2 ′ carbon sense modification ,
(B) The antisense region is composed of a plurality of nucleotides, and the 5′-most nucleotide is the first 5′-end antisense nucleotide, and the 5 ′ of the first 5′-end antisense nucleotide The carbon position is phosphorylated,
(C) including a loop region located between the sense region and the antisense region;
The 2 'each nucleotide other than the nucleotide having carbon modifications have a 2'-OH,
The siRNA is composed of base pairs between 18 and 30;
Each of the first 2 'carbon sense modification and said second 2' carbon sense modification is Ru der 2'-O- methyl modification, off-target effects reduction method.