# Patent

## Title
Oligonucleotides for RNA interference and biological applications thereof

## Abstract

The invention relates to compositions comprising double-stranded oligonucleotides of identical or different sequences and/or length, said oligonucleotides having sequences 3′N1N2 . . . Ni-1Ni . . . Nj5′ wherein—3′Ni . . . Nj5′ is half of a double-stranded 19-28 mer oligonucleotide of sequence complementary to a target nucleic acid sequence present in a living cell, and—3′N1 . . . Ni-15′ is a 3-50 mer overhang of sequence allowing oligomerization of said double-stranded oligonucleotide. Compositions of transfection comprising said oligonucleotide compositions and there used for therapeutical application.

## Background
This application is a divisional of application Ser. No. 11/921,089 (issued as U.S. Pat. No. 8,802,640), filed Jan. 30, 2008 (published as US 2008-0153772 A1), which is a U.S. national phase of international application PCT/EP2006/006340 filed 1 Jun. 2006, which designated the U.S. and claims benefit of U.S. Provisional No. 60/686,010, filed 1 Jun. 2005, the entire contents of each of which are incorporated herein by reference.


The invention relates to new double stranded oligonucleotides (dsONs) useful for RNA interference. It also relates to their use for oligonucleotides delivery to eukaryotic cells in culture or in animals for biological or therapeutic uses.
RNA interference (RNAi) is now a technology for gene silencing at the early gene function level, the mRNA (Fire et al, 1999; Tuschl et al., 1999). The technology provides sequence-specific mRNA degradation and inhibition of protein production (Yang et al, 2000, Zamore et al, 2000, Hammond et 2000, Parrish 2000). RNAi is highly effective due to a predictable design of active sequences of short dsRNA (siRNA, for small interfering RNA) and to the targeting of mRNA. When siRNA duplexes are introduced by transfection with a vector and delivered into the cytoplasm, RNAi has been shown to effectively silence exogenous and endogenous genes in a variety of mammalian cells (Elbashir et al, 2001).
Structural features of conventional dsRNA molecules required to mediate RNAi demonstrate that short dsRNAs having a length of preferably from 19-25 nucleotides (see patents WO 0244321, WO 01/075164 A3, EP20010985833), particularly 19-23 nucleotides, have RNAi activity in mammalian cell culture systems (Parrish et al., 2000; Elbashir et al., 2001; Tuschl, 2001). Short 19-25 nucleotides, when base-paired, with unpaired 3′ overhanging ends, act as the guide for sequence-specific mRNA degradation. It is possible to observe RNAi when both ends are blunt (0 nucleotide overhang) or when one strand is blunt-ended. Even if the sequence of the unpaired overhang of the siRNA is not critical for target RNA cleavage, the presence of 3′ overhang appears critical for optimized RNAi and stability of siRNA. Preferably, at least one strand has a 3′-overhang from 1 to 5 nucleotides, particularly from 1 to 3 nucleotides. The RNA strands preferably have 3′-hydroxyl groups and preferably comprise phosphate groups at the 5′-terminus, without 5′-overhangs. The most effective short dsRNAs are composed of two 21 nucleotides strands which are paired such that 1-3, particularly 2, nucleotides 3′-overhangs are present on the both ends of the dsRNA (Elbashir et al., 2001). The length of the RNA duplex was shown to be extendable to 27-28 mer (Siolas et al., 2005, Kim et al., 2005) and to tolerate various chemical and or backbone modifications (Kurreck, 2003).
The success of RNAi depends both on dsRNA length, sequence and chemical structure and on vector for cellular delivery. As compared to antisense or ribozyme technology, the secondary structure of the target mRNA is not a strong limiting factor for silencing with siRNA. Many sequences of siRNA may be effective for a given mRNA target. Thus, the stability and bioavailability of siRNA duplexes as well as the amount of dsRNA delivered to cells remains the limiting factors for efficient silencing rather than the target accessibility by the siRNA.
The inventors have found that dsONs with particular structural features that allow them to stick to each others have a high RNA interference activity in eukaryotic cells and provide higher gene silencing efficiencies than those obtained using conventional short dsRNAs, when introduced with as the same delivery system. Longer oligonucletoides than conventional short dsRNA exhibit a higher stability due to their better resistance to degradation.
It is then an object of the invention to provide new compositions comprising dsONs that are sequence-specific mediators of RNAi when introduced in mammalian cells. The invention thus describes the benefit for gene silencing of dsONs containing many copies of short dsONs mediating sequence-specific RNA interference of one or many targeted genes.
It also relates to various transfection delivery systems based on synthetic carriers and their use in biological applications.
The compositions of the invention comprise double-stranded oligonucleotides of identical or different sequences or length, said oligonucleotides having sequences 3′N1N2 . . . Ni-1Ni . . . Nj5′
wherein

    
    
        3′Ni . . . Nj5′ is half of a double-stranded 19-28 mer oligonucleotide of sequence complementary to a target nucleic acid sequence present in a living cell, and
        3′N1 . . . Ni-15′ is a 3-50 mer overhang of sequence allowing oligomerisation of said double-stranded oligonucleotide.
    
    


Preferred dsONs of said compositions advantageously have a sequence 3′Ni . . . Nj5′ of 19-21 nucleotides and/or a sequence 3′N1 . . . Ni5′-1 comprising 5 to 8 nucleotides.
As demonstrated in the examples, short dsONs, when base-paired with unpaired 3′ overhanging ends, and oligomerized in long dsON, act as guides for sequence-specific mRNA degradation.
According to an embodiment of the invention, sequences 3′N1 . . . Ni-15′ may be stabilized against degradation, for example by nucleases, without significant loss of activity. Suitable stabilizing groups are selected in the group comprising purine nucleotides, pyrimidine nucleotides substituted by modified analogs such as deoxynucleotides, and/or modified nucleotide analogs such as sugar- or backbone modified ribonucleotides or deoxyribonucleotides.
In another embodiment, optionally in combination with anyone of the preceding features, the compositions of the invention comprise at least one dsON with a 5′ phosphate or hydroxyl group at one or both 5′ ends.
In the dsONs of the compositions according to the invention, the oligonucleotides sequences contain deoxyribonucleotides, ribonucleotides or nucleotide analogs (Verma and Eckstein, 1998), such as methylphosphonate (Miller, 1991), morpholino phosphorodiamidate, phosphorothioate (Zon and Geiser, 1991), PNA (Jepson and Wengel, 2004), LNA, 2′-alkyl nucleotide analogs (Kurreck, 2003).
Potent viral or non-viral vectors are useful for introducing oligonucleotides in cells. Viral delivery systems still suffer from their immunogenicity and potential risk in clinical situations. In contrast, the transfection of nucleic acids with synthetic systems is a versatile method showing flexibility and absence of immunogenicity. The transfection of oligonucleotides with non-viral vectors is useful for the delivery of dsONs in the cytoplasm. Currently non-viral vectors are mainly based on cationic lipids-mediated transfection, such as Oligofectamin, TRANSIT-TKO, LipofectAmine-2000, SiGuide, RNAiFect, or jetSi, or based on cationic polymer-mediated transfection, such as Superfect, jetPEI, or X-TREMGene.
The invention thus also relates to transfection compositions comprising at least an oligonucleotide composition such as above defined and a transfection agent or formulation.
The transfection agent or formulation is more particularly a non-viral delivery system suitable for introducing dsONs in living cells and liberating dsONs mediating RNAi in cells.
The non viral vector system advantageously comprises cationic lipid- or polymer- or peptide-based delivery reagents. The non-viral vector system is a formulation comprising at least a delivery reagent and others components stabilizing the formulation, targeting the cells, tissues or organs, or increasing the transfection efficiency.
When complexed with transfection reagents prior to introduction into the cells, the oligomerization of short dsONs is promoted by intermolecular interactions due to a 3′-overhang-3′-overhang interaction or by using a linker that interacts with 3′ overhangs of dsONs. Many linkers can be used such as oligonucleotides that comprise sequences of nucleotides complementary to the 3′-overhangs of dsONs that mediate RNAi. Others linkers can be: i) hairpin-like structure having terminal oligomerization domains that recognize the 3′ overhangs of dsONs mediating the RNAi, ii) short double stranded nucleic acid having 5′- or 3′-overhangs at each strand end which recognize the 3′-overhangs of dsONs mediating RNAi. The linker can also be one or several dsON (or many dsONs) that mediate sequence-specific RNAi or not and comprising overhangs that interact with 3′-overhangs of dsON mediating gene silencing by RNAi.
The invention also relates to a process for preparing a composition of oligonucleotides such as above defined, said process comprising

    
    
        synthetizing oligonucleotide strands having sequences 3′Ni . . . Nj5′ and 3′N1 . . . Ni-15′ such as above defined by a chemical or enzymatic way;
        annealing the synthetized oligonucleotides thus obtained
    
    


According to an embodiment, said process further comprises adding linker(s) after the annealing step of said oligonucleotides, said linker(s) having nucleotidic sequences ends complementary to sequence 3′N1 . . . Ni-15′.
Said linker(s) is (are) advantageously selected in the group comprising oligonucleotides, single-stranded oligonucleotides, hairpin-like structures, short double-stranded nucleic acids having 3′ or 5′ overhangs, double stranded oligonucleotides.
The linkers are selected in the group comprising deoxyribonucleotides, ribonucleotides or nucleotide analogs.
The invention also relates to a method for in vitro and in vivo inhibition of gene expression, comprising the use of an oligonucleotide composition or a transfection composition such as above defined.
Said compositions and method are particular useful for therapeutical applications such as treatment of cancers, such as bladder (Urban-Klein et al., 2004), prostate (Pal et al., 2005) or leukaemia (Guan et al., 2005) cancers, or viral infections, such as HIV, Hepatitis virus, or influenza virus infections (Ge et al., 2004).
Other characteristics and advantages of the invention will be given in the following, with reference to FIGS. 1 to 7, which represent, respectively:



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1A: Luciferase gene expression of A549-GL3Luc cells, as described herein, measured after 24 h. Experiments are made in triplicate and the GL3 luciferase silencing efficiency was calculated from the endogenous luciferase level of nontransfected A549-GL3Luc cells normalized by the content of protein in cell lysates.
FIG. 1B: Luciferase gene expression of A549-GL3Luc cells, as described herein, measured after 48 h. Experiments are made in triplicate and the GL3 luciferase silencing efficiency was calculated from the endogenous luciferase level of nontransfected A549-GL3Luc cells normalized by the content of protein in cell lysates.
FIG. 2A: Luciferase gene expression of A549-GL3Luc cells, as described herein, measured after 24 h. Experiments are made in triplicate and the GL3 luciferase silencing efficiency was calculated from the endogenous luciferase level of nontransfected A549-GL3Luc cells normalized by the content of protein in cell lysates.
FIG. 2B: Luciferase gene expression of A549-GL3Luc cells, as described herein, measured after 48 h. Experiments are made in triplicate and the GL3 luciferase silencing efficiency was calculated from the endogenous luciferase level of nontransfected A549-GL3Luc cells normalized by the content of protein in cell lysates.
FIG. 3: RNA interference by (dA)5-GL3Luc-(dT)5 dsRNA (SEQ ID NOs: 5 and 6) duplexes mediates sequence-specific RNA interference. Experiments are made in triplicate and the GL3 luciferase silencing efficiency was calculated from the endogenous luciferase level of nontransfected A549-GL3Luc cells normalized by the content of protein in cell lysates.
FIG. 4: Double-stranded RNA having 3′-overhang that induce their intermolecular oligomerization when complexed with jetPEITM mediates high GL3Luciferase silencing. Experiments are made in triplicate and the GL3 luciferase silencing efficiency was calculated from the endogenous luciferase level of nontransfected A549-GL3Luc cells normalized by the content of protein in cell lysates.
FIG. 5: Double-stranded RNA having 3′-overhang that induce their intermolecular oligomerization when complexed with jetPEITM mediates a sequence-specific GL3Luciferase silencing. Experiments are made in triplicate and the GL3 luciferase silencing efficiency was calculated from the endogenous luciferase level of nontransfected A549-GL3Luc cells normalized by the content of protein in cell lysates.
FIG. 6: Oligomerization of dsRNA promoted by intermolecular interactions using a linker interacting with symmetric 3′ overhangs of dsRNAs duplexes mediates efficient GL3Luciferase silencing when complexed with jetPEITM. Experiments are made in triplicate and the GL3 luciferase silencing efficiency was calculated from the endogenous luciferase level of nontransfected A549-GL3Luc cells normalized by the content of protein in cell lysates.
FIG. 7A: Oligomerization of dsRNA promoted by intermolecular interactions using a linker interacting with symmetric 3′ overhangs of dsRNAs duplexes mediates efficient GL3Luciferase silencing when complexed with a cationic lipid formulations such as jetSi-ENDOTM. Experiments are made in triplicate and the GL3 luciferase silencing efficiency was calculated from the endogenous luciferase level of nontransfected A549-GL3Luc cells normalized by the content of protein in cell lysates.
FIG. 7B: Oligomerization of dsRNA promoted by intermolecular interactions using a linker interacting with symmetric 3′ overhangs of dsRNAs duplexes mediates efficient GL3Luciferase silencing when complexed with a cationic lipid formulations such as RNAiFect. Experiments are made in triplicate and the GL3 luciferase silencing efficiency was calculated from the endogenous luciferase level of nontransfected A549-GL3Luc cells normalized by the content of protein in cell lysates.



FIG. 1: RNA interference by conventional siRNA duplexes complexed with representatives of the two major classes of transfection reagents, i.e., a cationic lipid-based and a polymer-based transfection reagent (jetSi-ENDOTM and jetPEITM, respectively).
A549-GL3Luc cells, stably expressing the GL3 luciferase gene, were transfected with GL3Luc siRNA complexed with jetSi-ENDOTM and jetPEITM to evaluate the potency of transfection reagents. Luciferase gene expression was measured after 24 h (a) and 48 h (b) incubation period. Cell lysates were assessed for firefly luciferase expression using a commercial kit (Promega). As nonspecific control, siRNA matching the GL2 luciferase sequence are transfected in the same conditions. Experiments are made in triplicate and the GL3 luciferase silencing efficiency was calculated from the endogenous luciferase level of nontransfected A549-GL3Luc cells normalized by the content of protein in cell lysates.









GL3Luc siRNA (SEQ ID NO: 1/SEQ ID NO: 2):



SEQ ID NO: 1:



5′-CUUACGCUGAGUACUUCGA(dT)2-3′;



 



SEQ ID NO: 2:



3′-(dT)2GAAUGCGACUCAUGAAGCU-5′;



 



GL2Luc siRNA (SEQ ID NO: °3/SEQ ID NO: 4):



SEQ ID NO: 3:



5′-CGUACGCGGAAUACUUCGA(dT)2-3′;



 



SEQ ID NO: 4:



3′-(dT)2GCAUGCGCCUUAUGAAGCU-5′.






FIG. 2: RNA interference by (dA)5-GL3Luc-(dT)5 dsRNA (SEQ ID NOs: 5 and 6) duplexes complexed with a cationic polymer delivery reagent, jetPEITM.
A549-GL3Luc cells were transfected and luciferase gene expression was measured after 24 h (a) and 48 h (b) incubation period. Standard GL3Luc siRNA (SEQ ID NOs: 1 and 2) was used for comparison. Experiments were made in triplicates and the luciferase silencing efficiency was calculated from the endogenous luciferase level of nontransfected A549-GL3Luc cells normalized by the content of protein in cell lysates.









GL3Luc siRNA (SEQ ID NO: 1/SEQ ID NO: 2):



SEQ ID NO: 1:



5′-CUUACGCUGAGUACUUCGA(dT)2-3′;



 



SEQ ID NO: 2:



3′-(dT)2GAAUGCGACUCAUGAAGCU-5′;



 



(dA)5-GL3Luc-(dT)5 dsRNA (SEQ ID NO: 5/SEQ 



ID NO: 6):



SEQ ID NO: 5:



5′-CUUACGCUGAGUACUUCGA(dT)5-3′;



 



SEQ ID NO: 6:



3′-(dA)5GAAUGCGACUCAUGAAGCU-5′.






FIG. 3: RNA interference by (dA)5-GL3Luc-(dT)5 dsRNA (SEQ ID NOs: 5 and 6) duplexes mediates sequence-specific RNA interference.
A549-GL3Luc cells were transfected with (dA)5-GL3Luc-(dT)5 dsRNA (SEQ ID NOs: 5 and 6), a sequence mutated at position 9 (dA)5-GL3Luc-(dT)5 Mut dsRNA (SEQ ID NOs: 7 and 8), and (dA)5-GL2Luc-(dT)5 dsRNA (SEQ ID NOs: 9 and 10) duplexes complexed with jetPEITM. Luciferase gene expression was measured after 48 h incubation period. Experiments were made in triplicate and the luciferase silencing efficiency was calculated from the endogenous luciferase level of nontransfected A549-GL3Luc cells normalized by the content of protein in cell lysates.







(dA)5-GL3Luc-(dT)5 dsRNA (SEQ ID NO: 5/SEQ ID 


NO: 6):


SEQ ID NO: 5:


5′-CUUACGCUGAGUACUUCGA(dT)5-3′;


 


SEQ ID NO: 6:


3′-(dA)5GAAUGCGACUCAUGAAGCU-5′;


 


(dA)5-GL3Luc-(dT)5 Mut dsRNA(SEQ ID NO: 7/SEQ ID 


NO: 8):


SEQ ID NO: 7:


5′-CUUACGCUAAGUACUUCGA(dT)5-3′;


 


SEQ ID NO: 8:


3′-(dA)5GAAUGCGAUUCAUGAAGCU-5′;


 


(dA)5-GL2Luc-(dT)5 dsRNA(SEQ ID NO: 9/SEQ ID 


NO: 10):


SEQ ID NO: 9:


5′-CGUACGCGGAAUACUUCGA(dT)5-3′;


 


SEQ ID NO: 10:


3′-(dA)5GCAUGCGCCUUAUGAAGCU-5′.






FIG. 4: Double-stranded RNA having 3′-overhang that induce their intermolecular oligomerization when complexed with jetPEITM mediates high GL3Luciferase silencing.
A549-GL3Luc cells were transfected with (dA)5-GL3Luc-(dT)5 dsRNA (SEQ ID NOs: 5 and 6), and (dA)8-GL3Luc-(dT)8 dsRNA (SEQ ID NOs: 12 and 13) duplexes complexed with jetPEITM. Luciferase gene expression was measured after 48 h incubation period. As dsRNAs that are unable to promote their intermolecular oligomerization by their 3′ overhang, (dT)5-GL3Luc-(dT)5 dsRNA (SEQ ID NOs: 5 and 11) and (dT)8-GL3Luc-(dT)8 dsRNA (SEQ ID NOs: 12 and 14) duplexes were transfected in the same conditions with jetPEITM. Experiments were made in triplicate and the luciferase silencing efficiency was calculated from the endogenous luciferase level of nontransfected A549-GL3Luc cells normalized by the content of protein in cell lysates.







(dA)5-GL3Luc-(dT)5 dsRNA(SEQ ID NO: 5/SEQ ID NO: 6):


SEQ ID NO: 5:


5′-CUUACGCUGAGUACUUCGA(dT)5-3′;


 


SEQ ID NO:6: 


3′-(dA)5GAAUGCGACUCAUGAAGCU-5′;


 


(dT)5-GL3Luc-(dT)5 dsRNA(SEQ ID NO: 5/SEQ ID 


NO: 11):


SEQ ID NO: 5:


5′-CUUACGCUGAGUACUUCGA(dT)5-3′;


 


SEQ ID NO: 11:


3′-(dT)5GAAUGCGACUCAUGAAGCU-5′;


 


(dA)8-GL3Luc-(dT)8 dsRNA(SEQ ID NO: 12/SEQ ID 


NO: 13):


SEQ ID NO: 12:


5′-CUUACGCUGAGUACUUCGA(dT)5-3′;


 


SEQ ID NO: 13:


3′-(dA)5GAAUGCGACUCAUGAAGCU-5′;


 


(dT)8-GL3Luc-(dT)8 dsRNA(SEQ ID NO: 12/SEQ ID 


NO: 14):


SEQ ID NO: 12:


5′-CUUACGCUGAGUACUUCGA(dT)8-3′:


 


SEQ ID NO: 14:


3′-(dT)8GAAUGCGACUCAUGAAGCU-5′.






FIG. 5: Double-stranded RNA having 3′-overhang that induce their intermolecular oligomerization when complexed with jetPEITM mediates a sequence-specific GL3Luciferase silencing.
A549-GL3Luc cells were transfected with (dA)5-GL3Luc-(dT)5 dsRNA (SEQ N° 3), and (dA)8-GL3Luc-(dT)8 dsRNA (SEQ ID NOs: 12 and 13) duplexes complexed with jetPEITM. Luciferase gene expression was measured after 48 h incubation period. As nonspecific control, (dA)5-GL2Luc-(dT)5 dsRNA (SEQ ID NOs: 9 and 10) and (dA)8-GL2Luc-(dT)8 dsRNA (SEQ ID NOs: 15 and 16) were transfected in the same conditions with jetPEITM. Experiments were made in triplicate and the luciferase silencing efficiency was calculated from the endogenous luciferase level of nontransfected A549-GL3Luc cells normalized by the content of protein in cell lysates.







(dA)5-GL3Luc-(dT)5 dsRNA(SEQ ID NO: 5/SEQ ID NO: 6):


SEQ ID NO: 5:


5′-CUUACGCUGAGUACUUCGA(dT)5-3′;


 


SEQ ID NO: 6:


3′-(dA)5GAAUGCGACUCAUGAAGCU-5′;


 


(dA)5-GL2Luc-(dT)5 dsRNA(SEQ ID NO: 9/SEQ ID 


NO: 11):


SEQ ID NO: 9:


5′-CGUACGCGGAAUACUUCGA(dT)5-3′;


 


SEQ ID NO: 11:


3′-(dA)5GCAUGCGCCUUAUGAAGCU-5′;


 


(dA)8-GL3Luc-(dT)8 dsRNA(SEQ ID NO: 12/SEQ ID 


NO: 13):


SEQ ID NO: 12:


5′-CUUACGCUGAGUACUUCGA(dT)5-3′;


 


SEQ ID NO: 13:


3′-(dA)5GAAUGCGACUCAUGAAGCU-5′;


 


(dA)8-GL2Luc-(dT)8 dsRNA(SEQ ID NO: 15/SEQ ID 


NO: 16):


SEQ ID NO: 15:


5′-CGUACGCGGAAUACUUCGA(dT)8-3′;


 


SEQ ID NO: 16:


3′-(dA)8GCAUGCGCCUUAUGAAGCU-5′.






FIG. 6: Oligomerization of dsRNA promoted by intermolecular interactions using a linker interacting with symmetric 3′ overhangs of dsRNAs duplexes mediates efficient GL3Luciferase silencing when complexed with jetPEITM.
A549-GL3Luc cells were transfected with (dT)5-GL3Luc-(dT)5 dsRNA (SEQ ID NOs: 5 and 11), and (dT)8-GL3Luc-(dT)8 dsRNA (SEQ ID NOs: 12 and 14) duplexes without or with (dA)15 (SEQ ID NOs: 17) and (dA)25 (SEQ ID NO: 18) linkers, respectively, complexed with jetPEITM. Luciferase gene expression was measured after a 48 h incubation period. Experiments were made in triplicate and the luciferase silencing efficiency was calculated from the endogenous luciferase level of nontransfected A549-GL3Luc cells normalized by the content of protein in cell lysates.









(dT)5-GL3Luc-(dT)5 dsRNA(SEQ ID NO: 5/SEQ ID 



NO: 12):



SEQ ID NO: 5:



5′-CUUACGCUGAGUACUUCGA(dT)5-3′;


 



SEQ ID NO: 12:



3′-(dT)5GAAUGCGACUCAUGAAGCU-5′;


 



(dT)8-GL3Luc-(dT)8 dsRNA(SEQ ID NO: 12/SEQ ID 



NO: 14):



SEQ ID NO: 12:



5′-CUUACGCUGAGUACUUCGA(dT)8-3′;


 



SEQ ID NO: 14:



3′-(dT)8GAAUGCGACUCAUGAAGCU-5′;


 



(dA)15(SEQ ID NO: 17):



5′-(dA)15-3′;


 



(dA)25(SEQ ID NO: 18):



5′-(dA)25-3′.






FIG. 7: Oligomerization of dsRNA promoted by intermolecular interactions using a linker interacting with symmetric 3′ overhangs of dsRNAs duplexes mediates efficient GL3Luciferase silencing when complexed with a cationic lipid formulations such as jetSi-ENDOTM or RNAiFect.
A549-GL3Luc cells were transfected with (dT)2-GL3Luc-(dT)2 siRNA (SEQ ID NOs: 1 and 2), (dT)5-GL3Luc-(dT)5 dsRNA (SEQ ID NOs: 5 and 11), and (dT)8-GL3Luc-(dT)8 dsRNA (SEQ ID NOs: 12 and 14) duplexes with (dA)15 (SEQ ID NO: 17) and (dA)24 (SEQ ID NO: 18) linkers, for the sequences SEQ ID NOs: 5+11 and 12+13, respectively, complexed with jetSi-ENDOTM (a) and RNAiFect (b). Luciferase gene expression was measured after 48 h incubation period. Experiments were made in triplicate and the luciferase silencing efficiency was calculated from the endogenous luciferase level of nontransfected A549-GL3Luc cells normalized by the content of protein in cell lysates.









GL3Luc siRNA(SEQ ID NO: 1/SEQ ID NO: 2):



SEQ ID NO: 1:



5′-CUUACGCUGAGUACUUCGA(dT)2-3′;



 



SEQ ID NO: 2:



3′-(dT)2GAAUGCGACUCAUGAAGCU-5′;



 



(dT)5-GL3Luc-(dT)5 dsRNA(SEQ ID NO: 5/SEQ ID 



NO: 11):



SEQ ID NO: 5:



5′-CUUACGCUGAGUACUUCGA(dT)5-3′;



 



SEQ ID NO: 11:



3′-(dT)5GAAUGCGACUCAUGAAGCU-5′;



 



(dT)8-GL3Luc-(dT)8 dsRNA(SEQ ID NO: 12/SEQ ID 



NO: 14):



SEQ iD NO: 12:



5′-CUUACGCUGAGUACUUCGA(dT)8-3′;



 



SEQ ID NO: 14:



3′-(dT)8GAAUGCGACUCAUGAAGCU-5′;



 



(dA)15(SEQ ID NO: 17):



5′-(dA)15-3′;



 



(dA)25(SEQ ID NO: 18):



5′-(dA)25-3′.






Materials and Methods
Chemicals and Oligonucleotides
Oligonucleotides were chemically synthesised and PAGE purified by Eurogentec (Belgium). Oligonucleotides were annealed in 1× Annealing buffer (50 mM KAcetate, 50 mM MgAcetate) (Eurogentec) for 2 min. at 95° C., followed by 2-4 hours incubation at room temperature.
jetSi-ENDOTM (cationic lipid reagent for siRNA transfection) and jetPEITM (cationic polymer, linear polyethylenimine derivative, for nucleic acid transfection) were from Polyplus-Transfection (France). RNAifect was from Qiagen (United State).
Cell Culture
A549 (human lung carcinoma, ATCC N° CCL-185) cells stably expressing the GL3 luciferase (Photinus pyralis luciferase under the control of SV40 elements) were obtained after stable transfection of pGL3Luc plasmid (Clontech). A549-GL3Luc cells were grown in RPMI (Eurobio, France) and supplemented with 10% fetal bovine serum (FBS, Perbio, France), 2 mM glutamax (Eurobio), 100 units/ml penicillin (Eurobio), 100 μg/ml streptomycin (Eurobio) and 0.8 μg/ml G418 (Promega). Cells were maintained at 37° C. in a 5% CO2 humidified atmosphere.
Transfection Experiments
One day before transfection, 2.5×104 cells were seeded in 24-well tissue culture plate in 1 ml fresh complete medium containing 10% FBS. Before transfection, complexes of dsRNA/transfection reagent were prepared. The desired amount of oligonucleotides, dsRNAs with or without oligonucleotide linkers, and transfection reagent were diluted in 150 μl of serum-free medium for jetSi-ENDOTM or 150 μl of NaCl 150 mM for jetPEITM (for a triplicate experiment). Three and 2 μl of jetSi-ENDOTM and jetPEITM were used per μg of dsON, respectively. The solutions were mixed with a Vortex for 10 seconds, and left for 10 minutes at room temperature. The transfection reagent was added to the dsRNAs solution, homogenized for 10 seconds with a Vortex and left 30 minutes at room temperature. Before adding the transfection complexes, the complete medium with serum was removed and replaced with 0.5 ml of serum-free medium. Then, 100 μl of complexes solution was added per well and the plates were incubated at 37° C. After 2 h of incubation, the complete medium was removed and replace with 1 ml of complete medium containing 10% serum. For RNAifect, the desired amount of dsRNAs and oligonucleotide linkers was diluted in 300 μl of serum free medium (for triplicate experiment). Then, the transfection reagent was added to the siRNA mixture (3 μl of RNAifect per μg of dsON). The solution was mixed with a vortex, 10 seconds and left for 15 minutes at room temperature. Before adding the transfection complexes, the complete medium with serum was removed and replaced with 0.3 ml of complete medium with serum. 100 μl of complexes solution were added per well and the plates are incubated at 37° C. After 24 h, the culture medium was removed and replaced by 0.5 ml of complete medium containing 10% serum. For all transfection protocol, the plate was further incubated at 37° C. for 24 or 48 h.
Luciferase and Protein Assay
Luciferase gene expression was measured using a commercial kit (Promega, France). After removing the complete medium, three washings with 1 ml of PBS solution were made. Then, 100 μl of 1× lysis buffer were added per well, and the plate was incubated at room temperature for 30 minutes. The lysates were collected and centrifuged at 14,000 g for 5 minutes. The luciferase assay was assessed with 5 μl of lysate after injection of 100 μl of luciferin solution. The luminescence (RLU) was monitored with an integration over 10 seconds with a luminometer (Berthold, France). Results are expressed as light units integrated over 10 seconds (RLU), per mg of cell protein using the BCA assay (Pierce, France).
Results
As a target model of endogenous gene, we used the A549 cells stably expressing the GL3 luciferase (Photinus pyralis luciferase under the control of SV40 elements). A well-defined chemically produced siRNA, directed against GL3 luciferase mRNA was transfected with a typical cationic lipid-based delivery reagent (jetSi-ENDOTM) and a typical cationic polymer-based delivery reagent (jetPEITM) in the nanomolar concentration range of siRNA. The sequence-specific classical GL3Luc siRNA was a short dsRNA of 19 nucleotides matching the GL3Luc mRNA and comprising identical (i.e. noncomplementary) 3′-overhangs of 2 deoxyribonucleotides (dT) according to the definition of preferable siRNA mediating RNAi in mammalian cells (Elbashir et al., 2001). The silencing efficiency of GL3 luciferase presented in the FIG. 1 reached 70% and more than 80%, 24 and 48 h after transfection, respectively, when the transfection was performed with jetSi-ENDOTM and at 75 nM of siRNA. The low silencing level of GL2Luc siRNA, used as unrelated sequence, confirmed a sequence-specific RNAi. The sequence-specific silencing of GL3 luciferase was also observed when the transfection was performed with jetPEITM, yet, with a lower efficiency and duration than transfection with the cationic lipid derivative.
In order to improve the silencing efficiency of dsRNA mediating RNAi, we used a dsRNA (SEQ ID NOs: 5 and 6) of 19 nucleotides matching the GL3 Luc mRNA and comprising 3′-overhangs with 5 deoxythymidine nucleotides at the end of the antisens strand and 5 deoxyguanosine nucleotides at the end of sense strand. These 3′ overhangs can promote a 3′ overhang-3′ overhang interaction leading to intermolecular oligomerization of dsRNA into longer dsRNA. After transfection of A549-GL3Luc cells with (dA)5-GL3Luc-(dT)5 dsRNA (SEQ 5 and 6) complexed with jetPEITM (FIG. 2), a high luciferase silencing is observed (>80% at 50-75 nM of dsRNA, 24 and 48 h post-transfection). (dA)5-GL3Luc-(dT)5 dsRNA (SEQ ID NOs: 5 and 6) mediated a better luciferase gene silencing than standard siRNA transfected with both jetSi-ENDOTM and jetPEITM reagents. Gene silencing with (dA)5-GL3Luc-(dT)5 dsRNA (SEQ ID NOs: 5 and 6) was particularly efficient at 10 nM concentration 48 h post-transfection where GL3Luc siRNA was unable to silence luciferase expression when introduced by either delivery reagents used (FIG. 2).
A single nucleotide substitution in the sequence-specific (dA)5-GL3Luc-(dT)5 dsRNA (SEQ 5 and 6) was introduced at the position 9 (A versus G in the antisens strand) to abolish the specific recognition of GL3Luc mRNA target. This single-mutated sequence, (dA)5-GL3Luc-(dT)5-Mut dsRNA (SEQ ID NOs: 7 and 8), was introduced into A549-GL3Luc cells with jetPEITM. It was unable to silence luciferase expression (FIG. 3) 48 h post-transfection in the concentration range of 5 to 50 nM. As other control of selectivity, (dA)5-GL2Luc-(dT)5 dsRNA (SEQ ID NOs: 9 and 10), matching the unrelated GL2 luciferase, was transfected and was also unable to silence luciferase expression (FIG. 3).
The length of 3′ overhangs of oligomerizable dsRNAs was studied using 5 or 8 nucleotides at the 3′-protusions of the duplexes. Both (dA)5-GL3Luc-(dT)5 dsRNA (SEQ ID NOs: 5 and 6) and (dA)8-GL3Luc-(dT)8 dsRNA (SEQ ID NOs: 12 and 13) showed efficient and comparable level of silencing 48 h post-transfection when introduced with jetPEI in A549-GL3Luc cells (FIG. 4). As controls, (dT)5-GL3Luc-(dT)5 dsRNA (SEQ ID NOs: 5 and 11) and (dT)8-GL3Luc-(dT)8 dsRNA (SEQ ID NOs: 12 and 14), which are unable to promote their oligomerization, were much less efficient to silence luciferase expression compared the results obtained with oligomerisable dsRNAs, SEQ ID NOs: 5 and 6 and 7 (FIG. 4). A control of silencing selectivity was performed with oligomerizable dsRNAs having 5 or 8 nucleotides at the 3′-end of each strand of duplexes but matching the GL2 sequence. Both (dA)5-GL2Luc-(dT)5 dsRNA (SEQ ID NOs: 9 and 10) and (dA)8-GL3Luc-(dT)8 dsRNA (SEQ ID NOs: 15 and 16) were inefficient to silence the endogenously-expressed GL3 luciferase (FIG. 5).
Oligomerization of short dsONs mediating RNAi can be promoted by an oligonucleotide linker which recognizes by base pairing the 3′-overhangs of dsON duplexes by base pairing. As a model, (dT)5-GL3Luc-(dT)5 dsRNA (SEQ ID NOs: 5 and 11) and (dT)8-GL3Luc-(dT)8 dsRNA (SEQ ID NOs: 12 and 14) were introduced into A549-GI3Luc cells with jetPEITM in the presence or absence of poly(dA) nucleotides. Poly(dA) comprising 15 (SEQ ID NO: 17) and 25 (SEQ ID NO: 18) nucleotides in length were used to promote the oligomerization of duplexes of SEQ ID NOs: 5+12 and 12+14, respectively. When the poly(dA) linkers were present, luciferase silencing was highly efficient for both dsRNA duplexes as compared to the silencing efficiencies obtained in the absence of poly(dA) linkers (FIG. 6). The dsRNA with 3′ overhangs with a length of 5 nucleotides showed the best silencing ability in the presence of (dA)15 linker in this example (FIG. 6). Oligomerization of dsRNA mediating RNAi with an oligonucleotide linker thus increased its efficacy.
Composition comprising dsONs oligomerized by an oligonucleotide linker which recognizes by base pairing the 3′-overhangs of dsON duplexes and delivered into cells with a cationic lipid based transfection reagent, such as jetSi-ENDOTM or RNAiFect delivery reagents, mediates specific GL3 luciferase gene silencing in A549-GL3Luc cells. Poly(dA) was used as linker comprising 15 (SEQ ID NO: 17) and 25 (SEQ ID NO: 18) nucleotides in length to promote the oligomerization of duplexes of SEQ ID NOs: 5 and 6, respectively. When the poly(dA) linkers were present, luciferase silencing was highly efficient at the nanomolar level for both dsRNA duplexes as compared to the silencing efficiencies obtained with the typical GL3Luc siRNA (SEQ N° 1) (FIG. 7). Oligomerization of dsRNA mediating RNAi with an oligonucleotide linker increased the gene silencing efficiency as compared to the conventional strategy using siRNA when introduced into cells with a cationic lipid-based delivery system.










Sequences
















SEQ ID NOs: 1 and 2: 
5′-CUUACGCUGAGUACUUCGA(dT)2-3′


GL3Luc siRNA
3′-(dT)2GAAUGCGACUCAUGAAGCU-5′


 


SEQ ID NOs: 3 and 4: 
5′-CGUACGCGGAAUACUUCGA(dT)2-3′


GL2Luc siRNA
3′-(dT)2GCAUGCGCCUUAUGAAGCU-5′


 


SEQ ID NOs: 5 and 6]: 
5′-CUUACGCUGAGUACUUCGA(dT)5-3′


(dA)5-GL3Luc-(dT)5 dsRNA
3′-(dA)5GAAUGCGACUCAUGAAGCU-5′


 


SEQ ID NOs: 7 and 8: 
5′-CUUACGCUAAGUACUUCGA(dT)5-3′


(dA)5-GL3Luc-(dT)5 Mut dsRNA
3′-(dA)5GAAUGCGAUUCAUGAAGCU-5′


 


SEQ ID NOs: 9 and 10]: 
5′-CGUACGCGGAAUACUUCGA(dT)5-3′


(dA)5-GL2Luc-(dT)5 dsRNA
3′-(dA)5GCAUGCGCCUUAUGAAGCU-5′


 


SEQ ID NOs: 5 + 11: 
5′-CUUACGCUGAGUACUUCGA(dT)5-3′


(dT)5-GL3Luc-(dT)5 dsRNA
3′-(dT)5GAAUGCGACUCAUGAAGCU-5′


 


SEQ ID NOs: 12 + 13: 
5′-CUUACGCUGAGUACUUCGA(dT)8-3′


(dA)8-GL3Luc-(dT)8 dsRNA
3′-(dA)8GAAUGCGACUCAUGAAGCU-5′


 


SEQ ID NOs: 12 + 14: 
5′-CUUACGCUGAGUACUUCGA(dT)8-3′


(dT)8-GL3Luc-(dT)8 dsRNA
3′-(dT)8GAAUGCGACUCAUGAAGCU-5′


 


SEQ ID NOs: 15 + 16: 
5′-CGUACGCGGAAUACUUCGA(dT)8-3′


(dA)8-GL2Luc-(dT)8 dsRNA
3′-(dA)8GCAUGCGCCUUAUGAAGCU-5′


 


SEQ ID NO: 17: (dA)15
5′-(dA)15-3′


 


SEQ ID NO: 18: (dA)25
5′-(dA)25-3′









BIBLIOGRAPHIC REFERENCES


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## Claims

1. A method for in vivo inhibition of gene expression comprising administering to a mammal composition for RNA interference comprising oligomerized double-stranded nucleic acids comprising a plurality of identical or different double-stranded nucleic acids and optionally a linker, wherein each of said identical or different double-stranded nucleic acids consists of a first distinct oligonucleotide and a second distinct oligonucleotide, wherein each of said first and second distinct oligonucleotides consists of 19-28 ribonucleotides at the 5′-end and 3-50 dT or dT and dA nucleotides at the 3′-end, wherein the 19-28 ribonulceotides at the 5′-end of the first distinct oligonucleotide are base-paired to the 19-28 ribonucleotides at the 5′-end of the second distinct oligonucleotide and wherein 3-50 dT or dT and dA nucleotides at the 3′-end of the first distinct oligonucleotide and at the 3′-end of the second distinct oligonucleotide are unpaired overhanging sticky dT or dT and dA nucleotides, such that the unpaired overhanging sticky dT or dT and dA nucelotides at the 3′-end of the first distinct oligonucleotide of one of the plurality of identical or different double-stranded nucleic acids are complementary to the unpaired overhanging sticky dT or dT and dA nucleotides at the 3′-end of the second distinct oligonucleotide of another of the plurality of identical or different double-stranded nucleic acids or to a linker having unpaired overhanging sticky dT or dT and dA nucleotides, and wherein said complementary unpaired overhanging sticky dT or dT and dA nucleotides of the plurality of identical or different double-stranded nucleic acids or of the linker are hybridized to form said oligomerized double-stranded nucleic acids.

2. A method for in vitro inhibition of gene expression comprising delivering to eukaryotic cells a composition for RNA interference comprising oligomerized double-stranded nucleic acids comprising a plurality of identical or different double-stranded nucleic acids and optionally a linker, wherein each of said identical or different double-stranded nucleic acids consists of a first distinct oligonucleotide and a second distinct oligonucleotide, wherein each of said first and second distinct oligonucleotides consists of 19-28 ribonucleotides at the 5′-end and 3-50 dT or dT and dA nucleotides at the 3′-end, wherein the 19-28 ribonulceotides at the 5′-end of the first distinct oligonucleotide are base-paired to the 19-28 ribonucleotides at the 5′-end of the second distinct oligonucleotide and wherein 3-50 dT or dT and dA nucleotides at the 3′-end of the first distinct oligonucleotide and at the 3′-end of the second distinct oligonucleotide are unpaired overhanging sticky dT or dT and dA nucleotides, such that the unpaired overhanging sticky dT or dT and dA nucelotides at the 3′-end of the first distinct oligonucleotide of one of the plurality of identical or different double-stranded nucleic acids are complementary to the unpaired overhanging sticky dT or dT and dA nucleotides at the 3′-end of the second distinct oligonucleotide of another of the plurality of identical or different double-stranded nucleic acids or to a linker having unpaired overhanging sticky dT or dT and dA nucleotides, and wherein said complementary unpaired overhanging sticky dT or dT and dA nucleotides of the plurality of identical or different double-stranded nucleic acids or of the linker are hybridized to form said oligomerized double-stranded nucleic acids.

3. The method according to claim 1, wherein said administering comprises introducing said composition for RNA interference into living cells and liberating said double stranded RNA in cells, which mediates sequence-specific RNA interference and mRNA degradation of one or many targeted genes in animals or humans.

4. The method according to claim 2, wherein said method mediates sequence-specific RNA interference and mRNA degradation of one or many targeted genes in eukaryotic cells in culture.

5. The method according to claim 1, wherein said mammal is in need of treatment of a cancer or a viral infection and said cancer or viral infection is treated by said administering.

6. The method according to claim 5, wherein said cancer is bladder cancer, prostate cancer or leukemia.

7. The method according to claim 5, wherein said viral infection is human immunodeficiency virus infection or hepatitis virus infection.

8. A method for in vivo inhibition of gene expression comprising administering to a mammal a composition for RNA interference comprising oligomerized double-stranded nucleic acids comprising a plurality of identical or different double-stranded nucleic acids and optionally a linker, wherein each of said identical or different double-stranded nucleic acids consists of a first distinct oligonucleotide and a second distinct oligonucleotide, wherein each of said first and second distinct oligonucleotides consists of 19-28 ribonucleotides at the 5′-end and 3-50 dT or dT and dA nucleotides at the 3′-end, wherein the 19-28 ribonulceotides at the 5′-end of the first distinct oligonucleotide are base-paired to the 19-28 ribonucleotides at the 5′-end of the second distinct oligonucleotide and wherein 3-50 dT or dT and dA nucleotides at the 3′-end of the first distinct oligonucleotide and at the 3′-end of the second distinct oligonucleotide are unpaired overhanging sticky dT or dT and dA nucleotides, such that the unpaired overhanging sticky dT or dT and dA nucleotides at the 3′-end of the first distinct oligonucleotide of one of the plurality of identical or different double-stranded nucleic acids are complementary to the unpaired overhanging sticky dT or dT and dA nucleotides at the 3′-end of the second distinct oligonucleotide of another of the plurality of identical or different double-stranded nucleic acids or to a linker having unpaired overhanging sticky dT or dT and dA nucleotides, and wherein said complementary unpaired overhanging sticky dT or dT and dA nucleotides of the plurality of identical or different double-stranded nucleic acids or of the linker are hybridized to form said oligomerized double-stranded nucleic acids and a transfection agent or formulation.

9. A method for in vitro inhibition of gene expression comprising delivering to eukaryotic cells a composition for RNA interference comprising oligomerized double-stranded nucleic acids comprising a plurality of identical or different double-stranded nucleic acids and optionally a linker, wherein each of said identical or different double-stranded nucleic acids consists of a first distinct oligonucleotide and a second distinct oligonucleotide, wherein each of said first and second distinct oligonucleotides consists of 19-28 ribonucleotides at the 5′-end and 3-50 dT or dT and dA nucleotides at the 3′-end, wherein the 19-28 ribonulceotides at the 5′-end of the first distinct oligonucleotide are base-paired to the 19-28 ribonucleotides at the 5′-end of the second distinct oligonucleotide and wherein 3-50 dT or dT and dA nucleotides at the 3′-end of the first distinct oligonucleotide and at the 3′-end of the second distinct oligonucleotide are unpaired overhanging sticky dT or dT and dA nucleotides, such that the unpaired overhanging sticky dT or dT and dA nucleotides at the 3′-end of the first distinct oligonucleotide of one of the plurality of identical or different double-stranded nucleic acids are complementary to the unpaired overhanging sticky dT or dT and dA nucleotides at the 3′-end of the second distinct oligonucleotide of another of the plurality of identical or different double-stranded nucleic acids or to a linker having unpaired overhanging sticky dT or dT and dA nucleotides, and wherein said complementary unpaired overhanging sticky dT or dT and dA nucleotides of the plurality of identical or different double-stranded nucleic acids or of the linker are hybridized to form said oligomerized double-stranded nucleic acids and a transfection agent or formulation.

10. The method according to claim 8, wherein said transfection agent is a linear polyethyleimine.

11. The method according to claim 9, wherein said transfection agent is a linear polyethyleimine.

12. The method according to claim 8, wherein said transfection formulation comprises at least a delivery reagent and other components stabilizing the formulation targeting tissues or organs of said mammal or increasing transfection efficiency.

13. The method according to claim 9, wherein said transfection formulation comprises at least a delivery reagent and other components stabilizing the formulation, targeting the cells or increasing transfection efficiency.

14. The method according to claim 8, wherein said transfection agent or formulation is not immunogenic.

15. The method according to claim 1, wherein said composition is delivered to cytoplasm.