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  • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
  • C12N15/1132—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses against retroviridae, e.g. HIV
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  • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
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  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2320/50—Methods for regulating/modulating their activity

Definitions

• the present invention concerns methods and reagents useful in modulating gene expression.
• lhe invention relates to modulating gene expression using one multitargeting interfering RN ⁇ molecule having two strands each of which targets one or more sites on one or more pre-selected RN ⁇ molecules.
• RNA interference agents for example, using double-stranded RNA to repress expression of disease-related genes is currently an area of intense research activity.
• Double-stranded RNA of 19-23 bases in length is recognized by an RNA interference silencing complex (RISC) into which an effector strand (or "guide strand") of the RNA is loaded, This guide strand acts as a template for lhe recognition and destruction of highly complementary sequences present in the transcriptome .
• RISC RNA interference silencing complex
• guide strand acts as a template for lhe recognition and destruction of highly complementary sequences present in the transcriptome .
• interfering RNAs may induce lranslalional repression without mRNA degradation.
• dsRNAs double-stranded RNAs
• dsRNAs double-stranded RNAs
• dsRNAs particularly those designed against one target, may have at least two categories of off-target side effects that need to be avoided or minimized.
• Undesirable side effects can arise through the triggering of innate immune response pathways (eg.
• Inadvertent side-effects can be obtained when the passenger strand of a duplex is loaded and generates suppression of RNA species distinct from those targeted by the putative guide strand.
• Loading bias is well understood and most design processes only select sequences for a RNAi duplex from which only the intended guide strand will be loaded. Tims, some bioinf ⁇ rmatic and/or experimental approaches have been developed to try to minimize off-target effects. Algoritliins for in slllco hybridization arc known, and others have been developed for predicting target accessibility and loading bias in an effort to eliminate or minimize side-effects while maintaining effectiveness.
• RNA molecules for potentially treating human diseases of viral and non-viral origin are in various stages of development.
• the diseases include Age-related Macular Degeneration, Amyotrophic Lateral Sclerosis (ALS), and Respiratory Syncytial Virus (RSV) infection.
• ALS Amyotrophic Lateral Sclerosis
• RSV Respiratory Syncytial Virus
• RNA interference may be useful and potent in obtaining knock-down of specific gene products, many diseases involve complex interactions between ontologically-unrelated gene products. Thus, the use of single-gene targeting approaches may not succeed except where a single or dominant pathophysiologic pathway can be identified and interrupted.
• putative targets can be identified for most diseases. Attempts to confirm that inhibiting single targets in isolation is therapeutically valuable have been disappointing. Indeed, obtaining therapeutic effectiveness is proving lo be extremely challenging, probably because of multiple levels of redundancy in most signaling pathways. For example, many disorders, such as cancer, typo 2 diabetes, and atherosclerosis, feature multiple biochemical abnormalities. In addition, some putative targets may he subject Lo enhanced mutation rates, thereby negating the effects of interfering RNAs on any such target.
• nucleic acid therapeutics such as interfering RNAs are candidates for viral therapy, in part because modern rapid gene sequencing techniques allow viral genome sequences to be determined even before any encoded functions can be assessed, the error-prone replication of viruses, particularly RNA viruses, means that substantial genomic diversity can arise rapidly in an infected population.
• strategies for the development of nucleic acid therapeutics have largely centered on the targeting of highly-conserved regions of the vi ral . genome. It is unclear whether these constructs are efficient at Heating viral infection or preventing emergence of resistant viral clones.
• interfering RNAs which can modulate multiple RNAs or target multiple sites within an RN ⁇ .
• Methods for the design and for making such therapeutic multi -targeting interfering RNAs arc also needed.
• Antiviral interfering RNAs that can be developed rapidly upon the isolation and identification of new viral pathogens and thai can be used to help slow, or even prevent, the emergence of HCW, resistant isotypes are also needed.
• Interfering RNA molecules are now designed and produced with specificity for multiple binding sequences present in distinct genetic contexts in one or more pre-sclccled target RNA molecules and arc used to modulate expression of the target sequences.
• the present invention relates to a inulti targeting interfering RlSA molecule comprising Formula (I):
• X, X', Y, or Y' independently consists of one or more nucleotides and in another aspect ol this embodiment X consists of a third nucleotide sequence that is at least partially complementary to a second portion of the first binding sequence, where the second portion is adjacent to and connected with lhe . 3'-end of said first portion of the first binding sequence, and where X' consists of a fourth nucleotide sequence that is substantially complementary Io the third nucleotide sequence.
• X and X' are completely complementary to each other.
• Tt is also preferred that, X is completely complementary to the second portion of the first binding sequence.
• Y' is designed to consist of a fifth nucleotide sequence that is at. least partially complementary to a second portion of the second binding sequence and the second portion is adjacent to and connected with the 3 '-end of said first portion of the second binding sequence.
• Y consists of a sixth nucleotide sequence that is substantially complementary to the fifth nucleotide sequence.
• Y and Y' are completely complementary to each other. It is also preferred that Y' is completely complementary to the second portion of the second binding sequence.
• S and S 7 are completely complementary l ⁇ each other. It is also preferred that XS is completely complementary to the first portion and the second portion of the first binding sequence. It is also contemplated that Y'S' is completely complementary lo the first portion and the second portion of the second binding sequence. Further, XSY and Y'S'X' can be completely complementary to each other.
• S consists of a first nucleotide sequence of a length of about 8 to about 15 nucleotides and XSY and Y'S'X' preferably include lengths of about 15 to about 29 nucleotides.
• each of XSY and Y'S'X' arc of a length of about 1 1 J to about 23 nucleotides.
• the multitargeting interfering IiMA molecule comprises one or more terminal overhangs and preferably these overhangs consists of I to 5 nucleotides.
• the multitargeting interfering RNA molecule comprises at least one modified ribonucleotide, universal base, acyclic nucleotide, abasic nucleotide or non-ribonucle ⁇ tide and more preferably, the multitargeting interfering RNA molecule comprises at least one 2'-O-methyl ribosyl substitution or a locked nucleic acid ribonucleotide.
• first and the second binding sequences of the multitargeting interfering RN ⁇ molecule are present in distinct genetic contexts in one pre- selected target RNA molecule or alternatively, the first and the second binding sequences fire present in distinct genetic contexts ttt at least two pre-selectcd target RNA molecules.
• ai least one of the pre-selectcd target RN ⁇ molecules is a noti-coding RNA molecule.
• at least one of the pre-selectcd target RNA molecules is a messenger RNA (niRN A).
• at least one of the binding sequences is present in the 3'- J&.15207
• the pre-sdecled larget RNA molecules ai'e involved in a disease or disorder of a biological system and the disease or disorder is preferably that of an animal or a plant.
• Preferred animals include, but are not limited to rat, a mouse, a dog, a cat, a pig, a monkey, and a human.
• the pre-selected target RNA molecules encode a protein of a class selected from the group consisting of receptors, cytokines, transcription factors, regulatory proteins, signaling proteins, cytoskclelal proteins, transporters, enzymes, hormones, and antigens.
• Preferred proteins include those selected from the group consisting of ICAM-I 1 VFXlF-A, MCP-IJL-S, VEGF-B, ICiF- I, Gl ⁇ c ⁇ p, Inppl ⁇ , bPOF, PRTF, VEGF-C, VECTF-D, ⁇ -catenin, ⁇ -ras-B, K-ras-A, HGFK, and TNF alpha and preferably the multitargeling interfering KNA molecule decreases expression of any combination of IC' ⁇ M-1, VEGF-B, VEGF-C, VEGF-D, IL-S, bFGF, PIGF, MCP-I and IGF-I in an expression system.
• the multilargeling interfering RNA molecule decreases expression of any combination of ⁇ -catcni ⁇ , ⁇ -ras, and EGFR in an expression system or decreases expression of both Gluc ⁇ p and lnppl J in an expression system.
• the miiltilargeting interfering RNA targets viral RNA.
• Prcfered viral targets include human immunodeficiency virus (HIV), a hepatitis C virus (HCV), an influenza virus, a rhinovirus, and a severe acute respiratory syndrome (SARS) virus.
• HCV human immunodeficiency virus
• HCV hepatitis C virus
• influenza virus a rhinovirus
• SARS severe acute respiratory syndrome
• the multitargeting interfering RN ⁇ molecule targets hepatitis C virus (HCV) and, an RNA molecule encoding TNFalpha.
• one or more of the pre-selected target RNA molecules preferably comprises one or more RNA molecules selected from a first biological system.
• one or more of the pre-seleetcd target RNA molecules comprises one or more RNA molecules selected from a second biological system thai is infectious to a first biological system.
• the pre-selected target RNA molecules comprise one or more RN ⁇ molecules selected from a first biological system and one or more ⁇ >re-sdecled target RNA molecules selected from a second biological system that is infectious to the first biological system.
• the pre-selected target RNA molecules comprise one or more RNA molecules selected from an animal or a plant and one or more RNA molecules selected from a microbe or a virus that is infectious to the animal or the plant.
• Tine pre-selected target RNA molecules preferably comprises an RN ⁇ molecule encoding a human protein TNFalpha, LEDGF(p75), BAF, CCR5, CXCR4, furio, NFkB, STATl.
• S preferably consists essentially of a nucleotide sequence selected from the group consisting of:
• S' consists essentially of a nucleotide sequence selected from the group
• the multitargeting interfering RNA molecules consist essentially of:
• the invention relates to a biological system comprising a multitargeting interfering RNA molecule comprising Formula (I):
• p consists of a terminal phosphate group that is independently present or absent; wherein S consists of a first nucleotide sequence of ft length ⁇ f about 5 Io about 20 nucleotides that is completely complementary to a first portion of a first binding sequence, and S' consists of a second nucleotide sequence of a length of about 5 to about 20 nucleotides that is completely complementary Io a first portion of a second binding sequence, wherein said first and second binding sequences are present in distinct genetic contexts in at least one pre-selected target RNA molecule, and wherein S and S' are at least substantially complementary to each other but arc not palindromic; and further wherein X, X', Y, or Y', is independently absent or consist 1 ?
• preferred biological systems include virus, microbes, cells, plants, or animals.
• the invention further relates to vectors comprising nucleotide sequences encoding the multitargeting interfering RNA molecules of this invention.
• Preferred vectors include viral vrctors and preferred vectors are those selected from the group consisting of an adeno-associated virus, a retrovirus, an adenovirus, a lentivirus, and an alpha virus. Cells comprising these vectors are also contemplated in this invention.
• the multitargeting interfering RNA molecule is a short hairpin RNA molecule
• those cells containing those vectors or the short hairpin RNA molecules of this invention are also contemplated.
• the invention fuithcr relates to pharmaceutical compositions comprising the multitargcting intci'fci'ing RNA molecules of this invention together with an acceptable carrier.
• Other pharmaceutical compositions include the vectors of this invention together with acceptable carriers.
• the invention in yet another embodiment, relates to a method of inducing RNA interference in a biological system, such as virus, microbes, cells, plants, or animals. These methods include the steps of introducing the multitargcting interfering RNA molecules of the present invention into those biological systems.
• step h j) obtaining a second strand sequence which comprises the second consensus target sequence selected in slcp h) and, adjacent Io and connected with the 5 * -end of the second consensus target sequence, a complement of the consensus 3' flanking sequence of step g), and; k) designing a multilargeting interfering RNA molecule comprising a first strand having the first strand sequence in step i) and a second strand having the second strand sequence obtained in step j).
• the invention further comprises the step of obtaining a collection of candidate sct ⁇ ds of the length n , the steps of: i) generating a first collection of sequences of the length n from each of the nucleotide sequences obtained in step b) above using a method comprising the steps of: !
• step b) beginning at a terminus of each of the nucleotide sequence; 2) sequentially observing the nucleotide sequence using a window size of n; and 3) stepping along the nucleotide sequence with a step size of I; ii) generating a second collection of sequences each of which is completely complementary to a sequence in the first collection; and iii) obtaining the collection of candidate seeds of the length ti from the inspection of the first and the second collections of sequences, wherein a candidate seed and its complete complement arc not palindromic, and each candidate seed and its complete complement occurs at least once in the nucleotide sequences obtained in step b) ⁇ f the method provided above.
• the step of obtaining a collection of candidate seeds of the length n comprises the steps of: i) obtaining the completely complementary sequence for each nucleotide sequence obtained in step (b) of this designing method; ii) generating a first collection of sequences of the length n from each of the nucleotide sequences obtained in step b) and a second collection of sequences of the length « from each of the completely complementary sequences obtained in the present method, wherein the generating step comprises: 1) beginning at a terminus of the nucleotide sequence of each of the nucleotide sequences obtained in step b) above or each of the completely complementary sequences obtained in this aspect of the invention; 2) sequentially observing the nucleotide sequence using a window size of n; and 3) stepping along the nucleotide sequence with a step siae of 1 ; and wherein following the generating step ⁇ f this aspect the method further comprises iii) obtaining the collection of candidate seeds of the length
• the step of selecting a group of candidate seeds comprises the step of discarding any sequence of the length n that: i) is composed of a consecutive string of 5 or more identical single nucleotides; U) is composed of only adenosine and uracil; iii) is predicted to occur with unacceptable high frequency in the non-target transcript ⁇ me of interest; iv) is predicted to have a propensity to undesirably modulate the expression or activity of one or more cellular component; v) is any combination of i) l ⁇ iv); or vi) is palindromic.
• each of the steps of selecting a first and a second consensus target sequence comprises the step of discarding any sequence that; i) is composed of only a single base; ii) is composed of only adenosine and uracil; iii) has a consecutive string of five or more bases which are cytosine; iv) is predicted to occur with unacceptable high frequency in the non- larget t ⁇ mscriptome of interest; v) is predicted to have a propensity to undesirably modulate the expression or activity of one or more cellular component; or vi ⁇ is any combination of i) to v).
• the designing methods of this invention may further comprise the step of modifying the multitargeting interfering RNA molecule, i) to improve the incorporation of the first and the second strands of the multititrgeling interfering RNA molecule into the RNA induced silencing complex (RISC); ii) to increase or decrease the modulation of the expression of at. least one target RNA molecule; iii) to decrease stress or inflammatory response when the multitargeting interfering RNA molecule is administered into a subject; iv) to alter half life in an expression system; or v) any combination of i) to iv).
• RISC RNA induced silencing complex
• the designing methods of this invention preferably further comprise the steps of making the designed miillilargeting interfering RNA molecule and testing it in a suitable expression system.
• the step of selecting a first consensus target sequence further comprises designing the consensus target sequence where the consensus 3' flanking sequence to the seed comprises a sequence that, is at least partially identical to the 3 1 flanking sequence to the seed in at least one sequence obtained in step b) of the designing steps of this invention.
• the consensus 3 ⁇ -fJank ⁇ ng sequence to the seed can comprise a sequence that is identical to the 3' J&J 5207
• the consensus 3' flanking sequence to the complete complement of the seed comprises a sequence that is at least partially identical to the 3' flanking sequence to the complete complement of the seed in at least one sequence obtained in step b)
• the consensus 3 1 flanking sequence to the complete complement of the seed comprises a sequence that is identical to the 3'-flanki ⁇ ig sequence to the seed in the sequences obtained in step b).
• the complement of the consensus 3' flanking sequence in the step of obtaining a first strand sequence, is a complete complement of the consensus 3' flanking sequence of step h) of the designing method. Or, also preferably, it) the step of obtaining a second strand sequence, the complement of the consensus 3' flanking sequence is a complete complement of the consensus 3' flanking sequence of step g).
• the first strand and the second strand are completely complementary to each other, excepting the overhangs if present or in another aspect in the step of designing a multitargeting interfering
• the first strand and the second strand are incompletely complementary to each other.
• the invention in another embodiment, relates to a method of treating a subject, comprising the step of administering to said subject a therapeutically effective amount of a pharmaceutical composition comprising a multiiargeling interfering RN ⁇ molecule of this invention.
• the method further comprises administering to said subject a therapeutically effective amount of one or more additional therapeutic agents.
• the invention in yet another embodiment, relates to a method of inhibiting the onset of a disease or condition in a subject, comprising administering to said subject a pr ⁇ phylaclically effective amount of a pharmaceutical composition comprising at least one mullitargcting interfering RNA molecule of this invention.
• Other embodiments include processes for making a pharmaceutical composition comprising mixing a multilargeting interfering RNA molecule of this invention and a pharmaceutically acceptable carrier.
• FIG. 1 MultiUtrgeting of VHGF-A and IC]AM- 1 using both strands of a CODEMlR duplex.
• a 12 nt seed region was identified by analyzing the two target transcripts.
• Various permutations of positioning lhc CODEMIR around the seed were investigated and the resulting sequences are listed in Table I - 1.
• CODEMIR-27 and -28 correspond to the duplexes of CODEMTR-16 and -17, respectively, excepting the introduction of wobble base-pairs into the extremities of the duplexes to adjust the loading bias.
• CODEMIR-36 is an example of an incompletely compleinentaiy dupleA formed with guide strands that are fully complementary to the regions of VECiF-A and ICAM-I mRNA targeted by CODEMIRl ⁇ and CODEMIR 17.
• Panel A Further exemplification of multitargetmg using both strands of a CX)DEMIR duplex in which the CODEMlR duplex strands may be completely complementary to each other. Any overhangs present, will be without complementary base pairing.
• Panel. B An example of a CODEMlR showing incomplete complementarity between the two active strands of the CODEMIR. vSuch incomplete complementarity, can derive, for example, by virtue of each strand being completely complementary or almost completely complementary to its respective rargfit.
• Figure 3 Effect of a single blunt-end on VEGF and ICAM suppressive activity of CODEMIR targeting these two proteins.
• ARPE- 19 cells were traiisfccted with 4OnM duplex RNA and VEGF (closed bars) or ICAM (open bars) expression was assayed 4X hours post-trfmsfeelion. Each bar represents the mean of triplicate samples. Error bars indicate standard deviation of the mean. DETAILED DESCRIPTION
• UTR untranslated region
• VlROMIR multitargeting interfering RNA preferentially targeted to viral targets
• an “activity”, a “biological activity”, or a “functional activity” of a polypeptide or nucleic acid refers to an activity exerted by a polypeptide or nucleic acid molecule as determined in vivo or in vilto, according to standard techniques. Such activities can be a direct activity, such us the RNA interfering activity of an iRNA on a target RNA molecule, or Un indirect activity, such as a cellular signaling activity mediated by the RNA interfering activity of an iRNA.
• Bio system means, material, in a purified or unpurified form), from biological sources, including but not. limited to human, animal, plant, insect, microbi al, viral or other sources, wherein the system comprises the components required for biologic activity (e.g., inhibition of gene expression).
• biological system includes, for example, a cell, a virus, a microbe, an organism, an animal, or a plant.
• a “cell” means an autonomous self-replicating unit that may constitute an organism (in the case of unicellular organisms) or is a sub unit ⁇ f multicellular organisms in which individual cells may be specialized and/or differentiated for particular functions.
• a cell can be prokaryolic or cukaryotic, including bacterial cells such as E, coli, fungal cells such as yeast- bird cell, mammalian cells such as cell lines of human, bovine, porcine, monkey, sheep, apes, swine, dog, cat, and rodent origin, and insect cells such as Drosopkila and silkworm derived cell lines, or plant cells.
• the cell can be of somatic or germ line origin, totipotent or hybrid, dividing or non- dividing.
• the cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell.
• Tt is further understood that the term "cell” refers not only to the particular subject cell, but also to the progeny or potential progeny of such a cell. Uecause certain modifications can occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
• the term “complementary” or “complementarity” as used herein w ith respect to polynucleotides or oligonucleotides (which terms are used interchangeably herein) refers to a J&J S207
• RNA-RNA interactions include the standard Watson-Crick pairing (A opposite U or T- and G opposite C) and the non-Watso ⁇ -Crick pairing (including but not limited to the interaction through the
• complementarity between two nucleic acid sequences corresponds to free-energy changes for helix formation. Tims, determination of binding free energies for nucleic acid molecules is useful for predicting the three-dimensional structures of RNAs and for interpreting KNA-RNA associations, e.g., RNAi activity or inhibition of gene expression or formation of double stranded oligonucleotides.
• complementarity can be partial, for example where at least one or more nucleic acid bases between strands can pair according to the canonical base pairing rules.
• C ⁇ AAAC. ⁇ TCAG-3' are partially complementary (also referred to herein as “incompletely complementary 11 ) to each other.
• "Partial complementarity" or “partially complementary” as used herein indicates that only a percentage of the contiguous residues of a nucleic acid sequence can form Watson-Crick base pairing with the s.ame number of contiguous residues in a second nucleic acid sequence. in an anti-parallel fash.io ⁇ .
• nucleotides nut of a total of 10 nucleotides in the first oligonucleotide fom ⁇ ig Watson-Crick base pairing with a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementarity respectively.
• Complementarity cor also be total where each and every nucleic acid base of one strand is capable of forming hydrogen bonds according to the canonical base pairing rules, with a corresponding base in another, a ⁇ tiparallcl strand.
• CTGACAATCG-3' and S'-CGATTGTCAGO' arc totally complementary (also referred to herein as “completely complementary”) to caeh other.
• complete complementarity indicates thai all the contiguous residues of a nucleic acid sequence can form Watson-Crick base pairing with the same number of contiguous residues in a second nucleic acid sequence in an anti-parallel fashion.
• a sequence that is completely complementary to another sequence is also referred to as the complete complement of the other.
• the two strands would be considered to have no complementarity, In certain embodiments herein, at. least portions of two antiparallel strands will have no complementarity. In certain embodiments such portions may comprise even a majority of the length of the two strands.
• strands that are incompletely or partially complementary there may be portions or sections of the strands wherein there are several or even many contiguous bases wldch are completely complementary to each other, and other portions of the incompletely complementary strands which have less than complete complementarity ⁇ i.e. those sections are only partially complementary to each other.
• the percentage of complementarity between a first nucleotide sequence and a second nucleotide sequence can be evaluated by sequence identity or similarity between the first nucleotide sequence and the complement, of the second nucleotide sequence.
• a nucleotide sequence that is X ⁇ o complementary to a second nucleotide sequence is X% identical to the complement of the second nucleotide sequence.
• the "complement of a nucleotide sequence" is completely complementary to the nucleotide sequence, whose sequence is readily deducible from the nucleotide sequence using the rules of Watson-Crick base pairing. J&J 5207
• Consali ⁇ Ti or conserved indicates the extent to which a specific sequence is found to be represented in a group of related target, sequences, regardless of the genetic context of UiC specific sequence.
• Genetic context refers Io lhe flanking sequences that surround a specific identified 5 sequence and that fire sufficiently long to enable one of average skill in the art to determine its position within a genome or RNA molecule relative to sequence annotations or other markers in common use.
• Sequence identity or similarity is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by
• identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case can Tic, as determined by the match between strings of such sequences.
• percent identity or similarity of two amino acid sequences or of two nucleic acids the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid
• PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules.
• the default parameters of the respective programs e.g., X BLAST and NBLAST
• the FASTA method (Atschul el al;, (1990), J. Molec, Biol. 215, 403), can . also be used,
• the percent identity between Iwo sequences is determined using lhe Ncedlema ⁇ and Wunseh (J. MuL Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package.
• the Accelrys GCG GAP program aligns two complete sequences Io maximize the number of matches and minimizes the number of gaps.
• the percent identity between two sequences is determined using the local homology algorithm of Smith and Waterman (.1 MoI Biol. 1981, 147(J): 195-7), which has been incorporated into die BestFit program in the Accelrys GCG software package.
• the BestFit program makes an optimal alignment of the best segment of similarity between two sequences. Optimal alignments are found by inserting gaps to maximize the number of matches. Nucleotide sequences that shim; a substantial degree of complementarity will form stable interactions with each other, for example, by matching base pairs.
• stable interaction with respect to two nucleotide sequences indicates that the two nucleotide sequences have sufficient complementarity and have the natural tendency to interact with each .
• Two nucleotide sequences can form stable interaction with each other within a wide range of sequence complementarity. In general, lhe higher the complementarity the stronger or the more stable the interaction is. Different strengths of interactions may l)e required for different processes. For example, the strength of interaction for the purpose of forming a stable nucleotide sequence duplex in vitro may be different from that for the purpose of forming a stable interaction between an iRNA and a binding sequence in vivo. J&J 5207
• the strength of interaction can be readily determined experimental fy or predicted with appropriate software by a person skilled in the art.
• Hybridization can be used Iu lust whether two polynucleotides arc substantially complementary to each other and to measure how stable the interaction is.
• Polynucleotides that share a sufficient degree of complementarity wilt hybridize to each other under various hybridization conditions, In one embodiment, polynucleotides that share a high degree of complementarity thus form Strang stable interactions and will hybridize to each other under stringent hybridization conditions.
• Stringent hybridization conditions has the meaning known in the art, as described in Sambrook ct al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, c -° ⁇ d Spring Harbor, New. York, (1989).
• An exemplary stringent hybridization condition comprises hybridization in 6x sodium chloride/sodium citrate (SSC) at about 45 0 C, followed by one or more washes in () .2x SSC and 0,1% SDS al 50 - 65 "C.
• SSC sodium chloride/sodium citrate
• ⁇ iitiinatch refers to a nucleotide of either strand of two interacting strands having no corresponding nucleotide on the corresponding strand or a nucleotide of either strand of two interacting strands having a corresponding nucleotide on the corresponding strand that is non-complcmcotaiy.
• a "match” refers to a complementary pairing of nucleotides.
• the te ⁇ n "expression system” refers to any in vivo or in vitro system that can be used to evaluate the expression of a target RNA molecule and or the RNAi activity of a mult ⁇ target ⁇ ng RNA molecule of the invention.
• the "expression system” comprises one or more target RN ⁇ molecules, a multitargeting interfering RNA moleeule targeting the target RNA molecules, and a cell or any type of in vitro expression system known to a person skilled in the art that allows expression of the target RNA molecules and RNAi.
• RN ⁇ includes any molecule comprising at least one ribonucleotide residue, including those possessing one or more natural nucleotides of the following bases: adenine, cytos ⁇ nc, guanine, and uracil; abbreviated A, C, G, and IJ, respectively, modified ribonucleotides, universal base, acyclic nucleotide, abasic nucleotide and non- dbonucleotides.
• “Ribonucleotide” means a nucleotide with a hydroxyl group at the 2' position of a p- D-i ⁇ bo-furanose moiety.
• non-target transcriptome indicates the transcriptome aside from the targeted RNA molecules.
• the non-targeted transcriptome is that of the host.
• the non-targeted transcriptome is the transcriptome of the biological system aside from the given targeted RNA.
• Modified ribonucleotides include, for example 2'deoxy, 2'deoxy-2'-fluoro, 2'O-methyl, 2'O-methoxyethyl, 4'thio or locked nucleic acid (LNA) ribonucleotides. Also contemplated herein is the use of various types of ribonucleotide analogues, and RNA with internucleotide linkage (backbone) modifications. Modified internucleotide linkages include for example, phosphorothioate-modified, and even inverted linkages (i.e. 3 '-3' or 5'-5').
• Preferred ribonucleotide analogues include sugar-modified, and nucleobase-modified ribonucleotides, as well as combinations thereof.
• the 2 '-OH-group is replaced by a subst ⁇ tuent selected from H, OR, R, halo, SII, SR, NH 2 , NHR, NR 2 or ON, wherein R is Cl -C6 alkyl, alke nyl or alJcynyl and halo is F, Cl, Hr, or I.
• the phosphoester gro ⁇ p connecting to adjacent ribonucleotides is replaced by a modified group, e.g. a phosphorothioate group. Any or all of the above modifications may be combined.
• the 5'termini can be OH, phosphate, diphosphate or triphosphate. Nucleobase-modified ribonucleotides, i.e.
• ribonucleotides wherein the naturally-occurring nucleobase is replaced with a non-naturalfy occurring nuclcobase instead, for example, undines or cytidines modified at the S-position (e.g.5-(2-amino)pro ⁇ yJ uridine, and 5-bromo uridine); adenosines and guanosines modified at the 8-position (e.g. 8-bromo guanosine): deaza nucleotides (eg, 7-deaza-adenosine); O - and N-alkylated nucleotides (e.g. N6-methyl adenosine) are also contemplated for use herein.
• undines or cytidines modified at the S-position e.g.5-(2-amino)pro ⁇ yJ uridine, and 5-bromo uridine
• adenosines and guanosines modified at the 8-position e.g
• universal base refers to nucleotide base analogs that form base pairs with each of the natural DNA/RN ⁇ bases with little discrimination between them.
• Non- limiting examples of universal bases include (C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3- nilropyrrole, 4- nitroindole, 5-nitroindole, and 6-nitroindol as known in the art (see for example Loakes, 2001, Nucleic Acids Research, 29, 2437- 2447). J&J 5207
• acyclic nucleotide refers to any nucleotide having an acyclic ribose sugar, for example where any of the ribose carbons (Cl, C2, C3, C4, ⁇ r C5), are independently or in combination absent from the nucleotide,
• RNA sequences As used herein with respect to the listing of RNA sequences, the bases thymidine (''T") and uridine (“U”) are frequently interchangeable depending on the source of the sequence information (DNA or RNA). Therefore, in disclosure of target sequences, seed sequences, candidate seeds, target RNA binding sites, and the like, the base “T” is fully interchangeable with the base “IJ".
• the base "U” cannot be generally substituted with "T” in a functional manner. It is however known jn the art lhal certain occurrences of the base "U” in RNA molecules can be substituted with 1 T" without substantially deleterious effect on functionality.
• T for U in overhangs, such as UlJ overhangs at the 3' end is known to lie silent, or at a minimum, acceptable, and thus is permissible in the interfering RNA sequences provided herein.
• ⁇ "target RNA molecule” or a "pre-selecicd target RNA molecule” as used herein refers to any RN ⁇ molecule whose expression or activity is desired to be modulated, for example decreased, by an interfering RNA molecule of the invention in an expression system.
• a “target RNA molecule” can be a messenger RNA molecule (rnRNA) that encodes a polypeptide of interest.
• a messenger RN ⁇ molecule typically includes a coding region and non-coding regions preceding ("5 1 UTR") and following ("3'UTR") the coding region.
• a “target RNA molecule” can also be a non-coding RNA (ncRNA), such as small temporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA), short interfering RNA (siRNA), small nucleolar RNA (snoRNA), ribosomal RN ⁇ (rRN ⁇ ), transfer RN ⁇ (iRNA) and precursor RNAs thereof.
• ncRNA non-coding RNA
• RNAs can also serve as target RN ⁇ molecules because ncRN ⁇ is involved in functional ⁇ r regulatory cellular processes. Aberrant ncRNA activity leading to disease can therefore be modulated by inultitargetiiig interfering RN ⁇ molecules of the invention.
• the target RNA can further be the genome of a virus, for example a RNA virus, or a replicative intermediate of any virus at any stage, as well as any combination of these, J&J 5207
• the "largel RN ⁇ molecule” can be a RNA molecule thivL is endogenous to a biological system, or a RN ⁇ molecule lhal is exogenous to the biological system, such as a RNA molecule of a pathogen, for example a virus, which is present in a cell after infection thereof.
• a cell containing the target RN ⁇ can be derived from or contained in any organism, for example a 5 plant, animal, protozoan, virus, bacterium, or fungus.
• Non-limiting examples of plants include i ⁇ io ⁇ oeots, dicots, or gyrrm ⁇ spe ⁇ ns.
• Non-limiting examples of animals include vertebrates or invertebrates.
• Non-iimiling examples of fungi include molds or yeasts.
• a "target RNA molecule” as used herein may include any variants or polymorphism of a desired RNA molecule. Most genes are polymorphic in that a low but nevertheless significant
• K rate of sequence variability occurs in a gene among individuals of the same species.
• a KNA molecule may correjate with multiple sequence entries, each of which represents a variant or a polymorphism of the RNA molecule.
• Li designing any gene suppression tool there is tine risk lhal the selected binding seqt ⁇ enoe(s) used in the computer-based design may contain relatively infrequent alleles. As a result, the active sequence designed might be expected to
• J 5 provide the required benefit in only a small proportion of individuals.
• the frequency, nature and position of most, variants are easily accessible to those trained i ⁇ the art,
• sequences with a target molecule that are known to be highly polymorphic can be avoided in the selection of binding sequences during the bioinformatic screen.
• 20 particular target may be used in the design stages of an interfering RNA of the invention to make sure thai the targeted binding sequence is present in lhe majority of allelic variants, with the exception of the situation in which targeting of the allelic variant is desired (that is, when the allelic variant itself is implicated in the disease of interest).
• target RNA molecule comprises at least one targeted binding sequence that is
• the targeted binding sequence can be refined to include any part ⁇ f the transcript sequence (eg 5'UTR, C)RF, 3'UTR) based on the desired effect. For example, trauslali ⁇ nal repression is a frequent mechanism operating in the 3'UTR (i.e. as for microRNA). Thus, lhe targeted binding
• sequence can include sequences in the 3' UTR for effective translational repression.
• target binding sequence shall all mean a portion of * a target RNA molecule sequence comprising & seed sequence and (he sequence flanking either cine or both ends of the seed, said binding sequence predicted to a form stable interaction with one strand of a muititargeting interfering RN ⁇ of lhe invention basod on the complementarity between the said strand and the binding sequence.
• seed or “seed sequence” or “seed region sequence” refers to a sequence of at least about 6 contiguous nucleotides present in & target RNA that is completely complementary to a.portion of one strand of an interfering RNA. Although ⁇ > of more contiguous bases are preferred, the expression “about 6” refers to the fact that, windows of at. least 5 or more contiguous bases or more can provide useful candidates in some cases and can ultimately lead to flic design of useful interfering ItNAs. Thus, all such seed sequences are contemplated within the scope of the instant invention.
• RNA interference or “RN ⁇ i” is used to indicate single or double stranded RNA molecules that modulate the presence, processing, transcription, translation, or half-life of a target RNA molecule, for example by mediating RNA interference ⁇ "RN ⁇ i"), in a sequence-specific manner.
• RNA interference or “RN ⁇ i” is meant to be equivalent to other terms used to describe sequence specific RNA . interference, such as post-transcriptional gene silencing, translational inhibition, or epigenetics.
• RISC-mediated degradation or translational repression as well as transcriptional silencing, altered RN ⁇ editing, competition for binding to regulatory proteins, and alterations of mRN ⁇ splicing. It also encompasses degradation and/or inactivalion of the target RNA by other processes known in the art, including but not limited to nonsense-mediated decay, and translocation to P bodies.
• the interfering RNAs provided herein e.g. CX)DEMUiS and VlROMiRs
• interfering RNAs can be used to manipulate or alter the genotype orphenotype of an organism or cell, by intervening in cellular processes such as genetic imprinting, transcription, translation, or nucleic acid processing (e.g., transamination, meUrylation. ' ctc).
• the te ⁇ n "interfering RNA” is meant to be equivalent to other terms used to describe nucleic acid molecules that iire capable of mediating sequence specific RN ⁇ i, for example short
• interfering RNA siRNA
• dsRNA double- stranded RNA
• miRNA micro-RNA
• sJiRNA short hairpin RNA
• short interfering oligonucleotide short interfering nucleic acid
• short interfering modified oligonucleotide chemically-modified siRNA
• post- transcriptional gene silencing RN ⁇ ptgsRNA
• interfering RNA can also be assembled from a single oligonucleotide, comprising self- complementary regions linked by means of a nucleic acid based or non-nucleic acid-based linker(s).
• the "interfering RN ⁇ ” can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary regions;
• the "interfering RN ⁇ ” can also be a single-stranded polynucleotide having one or more loop structures and a stem comprising self- complementary regions (e.g.
• shRNA short hairpin RNA, shRNA
• the polynucleotide can be processed either in vivo or in vitro to generate one or more double stranded interfering RN ⁇ molecules capable of mediating RNA inactivation.
• the cleavage of the self-paired region or regions of the single strand RNA to generate double- stranded RNA can occur in vitro or in vivo, both of which are contemplated for use herein,
• interfering RNA need not be limited to those molecules containing only RNA, but further encompasses those possessing one or more modified ribonucleotides and non- nucleotides, such as those described supra.
• interfering RNA includes double- stranded RN ⁇ , single-stranded RNA, isolated RNA such as partially purified RN ⁇ , essentially pure RNA, synthetic RNA, recombinantly produced RN ⁇ , as well as altered RNA that differs from naturally occurring RN ⁇ by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the multitargeting interfering RNA or internally, for example at one or more nucleotides of the RNA.
• Nucleotides in the RJSlA molecules of the instant invention can also compri se non-standard nucleotides, such as iion-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally occurring RNA.
• the interfering RNA of the invention also termed “multitargeting interfering RNA” is an interfering double-stranded RNA, each strand of which can form stable interactions with binding sites present in distinct genetic contexts on one or more target RNA molecules.
• multitargeting interfering RNA examples include CODEMlRs, Computationally -DEsigned, Multi- targeting Interfering RNAs, and VlRQMTRs, where the latter multitargeting interfering RN ⁇ molecules are preferentially targeted to viral targets.
• '"Sequence means the linear order in which monomers occur in a polymer, for example, the order of amino acids in a polypeptide or lhe order of nucleotides in a polynucleotide.
• a “subject” as used herein, refers to an organism to which the nucleic acid molecules of the invention can be administered.
• a subject can be an animal or a plant, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment, or any cell thereof.
• ⁇ "vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
• plasmid which refers to a circular double stranded DNA loop into Which additional DNA segments can be inserted.
• Another type of vector is a viral vector, wherein additional DNA segments can be inserted.
• Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.
• bacterial vectors having a bacterial origin of repl ication. and episomal mammalian vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
• certain vectors, expression vectors are capable of directing the expression of genes to which they are operably linked.
• modulate (or modulation of) the expression of an RN ⁇ molecule means any RNA interference mediated regulation of the level and/or biological activity of the RNA molecule.
• RNAi-related post-transcriptional gene silencing sucb as by cleaving, destabilizing the target RN ⁇ molecule or preventing their translation.
• the term “modulate” can mean “inhibit,” but the use of the word “modulate” is not limited to this definition.
• RNA molecule is determined in a suitable expression system, for example in vivo, in one or more suitable cells, or in an acellular or in vitro expression system such as are known in the art. Routine methods for measuring parameters of the transcription, translation, or other aspects of expression relating to RNA molecules are known in the art, and any such measurements are suitable for use herein.
• inhibitt “down-regulate”, “reduce”, or “decrease” or “decreasing” as with respect to a target RNA or its expression it is meant that the expression of the gene or level and/or J&T 5207
• RNA molecules • biological activity of target RNA molecules is reduced below lhat observed in the absence of the nucleic acid molecules (e.g., multitargcting interfering RN ⁇ ) of the invention.
• inhibition, down-regulation or reduction with a rnultitiirgeting interfering RNA molecule is greater than that observed in the presence of an inactive or attenuated molecule.
• inhibition, down- regulation, or reduction with a multitargeting interfering RNA molecule is greater than that observed in the presence of, for example, multiturgeti ⁇ g interfering RNA molecule with scrambled sequence or with mismatches.
• a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence.
• “Inhibit”, “down-regulate”, “reduce”, or “decrease” as with respect to a target RN ⁇ or its expression encompasses, for example, reduction of the amount or rate of transcription or translation of a target RNA, reduction of the amount ⁇ r rate of activity of the target RNA, and/or a combination of the foregoing in a selected expression system.
• RNA refers to any detectable effect the RN ⁇ may have in a cell or expression system, including for example, any effect on transcription, such as enhancing or suppressing transcription of itself or another RNA molecule.
• the measurement of a "decrease" in expression or the determination of the activity of a given RN ⁇ can be performed in vilro or in vivo, in any system known or developed for such purposes, or adaptable thereto.
• the measurement of a "decrease" in expression by a particular interfering RN ⁇ is made relative to a control, for example, in which no interfering RN ⁇ is used. In some comparative embodiments such measurement is made relative to a control in wliich some other interfering RNA or combination of interfering RNAs is used. Most preferably a change, such as the decrease is statistically significant, bayed on a generally nccepte.H test nf statistical significance.
• RNA need only show an aritlimeliu decrease in one such in vitro or in vivo assay to be considered to show a "decrease in expression" as used herein. More particularly, the biological modulating activity of the multitargeting interfering
• RNA is not limited to, or necessarily reliant on, degradation or translation al repression by conventional RISC protein complexes involved in siRNA and microRN ⁇ gene-silencing, respectively.
• short double-stranded and single-stranded RN ⁇ have been shown to have other possible sequence-specific roles via alternative mechanisms.
• short double- stranded RN ⁇ (dsRN ⁇ ) species may act as modulatory effectors of differentiation/cell activity, possibly through binding to regulatory proteins (Kuwabara, 1'., et al., (2004), Celt, 1 16: 779-93).
• dsRNA may lead to the degradation of mRN ⁇ through the involvement of AU- rich element ( ⁇ RE)-binding proteins (Jing, Q- et al., (2005), Cell, 120: 623-34). Further, dsRNA may also induce epigenetic transcriptional silencing (Morris, K.V., et al., (2004) Science, 305: 1289-89), Processing of mRNA can also be altered through A to I editing and modified splicing.
• ⁇ RE AU- rich element
• palindrome or "palindromic sequence” means a nucleic acid sequence thai is completely complementary to a second nucleotide sequence that is identical to the nucleic acid sequence, e.g., UGGCCA.
• the term also includes a nucleic acid molecule comprising of two .nucleotide sequences that arc palindromic sequences.
• Phenotypic change refers to any delectable change to a cell or an organism that occurs in response to contact or treatment with a nucleic acid molecule of the invention. Such detectable changes include, but arc not limited io, changes in shape, size.
• the detectable change can also include expression of reporter genes/molecules such us Green Fluorescent Protein (GFP ) or various tags that are used to identify an expressed protein or any other cellular component that can be assayed.
• GFP Green Fluorescent Protein
• terapéuticaally effective amount means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal, human, or plant lhat is being sought by , researcher, veterinarian. medical doctor or other clinician, which includes ameliorating or alleviation of the symptoms of the disease or disorder being treated. Methods arc known in the art for determining therapeutically effective doses for the instant pharmaceutical composition.
• prophylactically effective amount refers to that amount of active compound or pharmaceutical agent lhat inhibits in a subject the onset of a disorder as being sought by a researcher, veterinarian, medical doctor or other clinician.
• the . interfering RNAs known to- one of ordinary skill in the art are double-stranded polynucleotide molecules comprising two self-complementary strands which are sense and anlisense to the target.
• the iRNA duplex is usually designed such that the ⁇ ntisensc (guide) strand is preferentially loaded into the RISC and guides the RISC-mediated degradation of the target nucleotide sequence following complementary base-pairing.
• the sense (passenger) strand may be degraded in the process of loading into the RISC complex or soon after by endonuclcascs to which single stranded RNA is highly sensitive.
• the relative thermodynamic characteristics of the 5' termini of the two strands of an interfering RNA determine whether a strand serves the function of a passenger or a guide strand during RNAi.
• the present invention provides a multitargeting interfering RNA molecule comprising two strands, each of which is designed against a specific target sequence.
• the JRNA duplex is designed in such a manner that each surand can be loaded into RISCJ complexes and thus both strands function as "guide" strands.
• both strands are loaded to an approximately equal extent into RISC complexes.
• One strand is at least partially complementary to a first portion of a target RNA binding sequence, which is also referred to as the seed.
• the other strand comprises a sequence wliich is at least partially if not completely identical to the seed, this sequence being at least partially complementary to the first portion of a second target RN ⁇ binding sequence.
• the said first and second target binding sequences are present in distinct genetic contexts in at least one pre-sclecfcd target RNA molecule. That is, multiple target RNA binding sites may be present on the same target RNA molecule, on separate RNA molecules, or both.
• the present invention provides a miillilargeting interfering RNA molecule comprising Formula (I):
• p consists of a terminal phosphate group lhitf can be present nr absent from the 5' -end of either strand. Any terminal phosphate group known to a person skilled in the aft can be used.
• Such phosphate group includes, but is not limited to, monophosphate, diphosphate, triphosphate, cyclic phosphate or to a chemical derivative of phosphate such as a phosphate ester linkage,
• S consists of a first nucleotide sequence of a length of about.5 Lo about 20 nucleotides that is completely complementary to a first portion of a first binding sequence, and J&.T 5207
• S 1 consists of a second nucleotide sequence of a length of about 5 to about 20 nucleotides that is completely complementary to a first portion of a second binding sequence, wherein said first and second binding sequences are present in distinct genetic contexts in at least one pre-seleeted target RNA molecule, and wherein S arid S 1 are at least substantially complementary to each other but arc not palindromic.
• S and S' each has a length of, for example, 5, 6, 7, 8, 9, 10, I I , 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides that are at least partially, preferably completely, complementary Lo the first portion of the at least two binding sequences.
• S is completely complementary to a sequence present in one or more pre-selecled target RN ⁇ molecules.
• S' is completely complementary to a sequence present in one or more pre-selected target RN ⁇ molecules.
• S and S' are completely complementary to each other.
• S is partially complementary to a first portion of a binding sequence present in one or more pre-selected target RNA molecules, such as 6 of 7, 7 of 8, 8 of 9, 9 of 10, 10 of 11, 11 of 12, 12 of 13, 13 of 14, 14 of 15, or 15 of 16 consecutive nucleotides of S are completely complementary to the First portion of at ieast one target RNA binding sequence.
• S and the first portion of the distinct binding sequences have lesser overall complementarity such as 10 of 12, 11 of 13, 12 of 14, 13 of 15, or 14 of 16 nucleotides of complete complementarity.
• S' is partially complementary to a first portion of a second binding site.
• Y and Y') in Formula (T) is independently absent or consists of a nucleotide sequence. In particular embodiments, they are developed so as to generate further binding to the target RNA sites.
• the sequences of X and Y' arc at least partially complementary to the second portions of the first and second target RNA binding sequences, respectively.
• the sequences X' and Y are completely complementary to X and Y', respectively, such that XSY and Y'S'X' arc completely complementary.
• Y are incompletely complementary with X and Y', respectively such that XSY and Y'S'X' are incompletely complementary. This may be required, for example, in situations ill which the loading bias of the interfering RN ⁇ duplex, needs Lo be altered through the use of mismatches in the extremity with the higher hybridization energy.
• sequences X and Y' arc designed so as to maximize binding of XS and Y'S' to the first and second portions of a plurality of target RNA binding sites
• the plurality of target sequences e.g. viral isolates
• identity consensus sequences can be generated by hand by examining the alignments of the target RNA sequences.
• all possible base sequences or a subset of putative XS and Y'S' sequences can be generated by computer algorithm.
• Each putative XS and Y'S' sequence is then hybridized in silica using RJNAhyb ⁇ d or a similar program known to one skilled in the art.
• Those putative sequences that are predicted to best bind the c ⁇ irespouding first and second portions of the target RNA binding sites are then prioritized for the next design phase, which includes filtering out putative sequences that have unfavorable characteristics such as more than 4 contiguous C or G bases.
• the sequences of Y and X' are then designed such that they are at least partial Iy complementary to Y' and X, respectively.
• Overhangs if required may simply be the addition to X' and Y of UU, dTdT or any other base or modified base.
• the bases of flue overhangs are selected so as to further increase the predicted binding of XSY and Y'S'X' to their respective RNA targets. Overhangs may be 1, 2, 3, 4 or 5 bases as required.
• a preferred embodiment is one in which the two strands of the duplex, independently have either partial or complete complementarity to their corresponding at least one target sequence and the two strands are completely complementary to one another, excepting the overhangs when present.
• Another embodiment of the invention is one in which each of the two strands of the duplex independently have either partial or complete complementary to their corresponding at least one target sequence and the two strand? are incompletely cnmp
• interfering RNA can further comprise one or more 5' terminal phosphate group, such as a 5'- phosphate or 5',3'- diphosphate. These may be of use to improve cell uptake,- stability, tissue targeting or any combination thereof.
• X consists of a nucleotide sequence that is at least partially complementary to a second portion of the first binding sequence, said second portion is adjacent to and connected with the 3'-e ⁇ d of said first portion of the first binding sequence, and wherein X' is substantially complementary to X.
• X and X' are completely complementary to each other.
• X is completely complementary to the second portion of the first binding sequence.
• Y' consists of a nucleotide sequence that is at least partially complementary to a second portion of the second binding sequence, said second portion is adjacent to and connected with the 3'-end of said first portion of lhc second binding sequence, and wherein Y is substantially complementary to Y'.
• Y and Y' are completely complementary to each other.
• Y 1 is completely complementary to the second portion of the second binding sequence.
• XSY is at least partially complementary to the first binding sequence to allow stable interaction of XSY with the first binding sequence
• Y'S'X' is at least partially complementary to the second binding sequence to allow stable interaction with the second binding sequence
• XSY and Y'S'X' are at least partially complementary to each other to allow formation of a stable iRN ⁇ duplex.
• XSY is completely complementary to the first binding sequence.
• Y ⁇ S'X' is completely complementary to the second binding sequence.
• XSY and Y'S'X' are completely complementary to each other.
• each strand of a multilargeling interfering RNA molecule of the invention is independently about 17 to about 25 nucleotides in length, in specific embodiments about 17, 18, 19, 20, 21, 22, 23, 24, and 25 nucleotides in length.
• Using shorter length interfering RNA molecules without the need for the generation of multiple active sequences through processing of RNA by enzymes such as Dicer and RNaselll, provides advantages, for example, in reduction of cost, manufacturing, and chance of off-target effects.
• the interaction between the two strands can be adjusted to improve loading of both strands into the cellular RJSC complex (Klivorova et al. (2003) Celt, 1 15: 209-16; Schwarz et al. (2003) CTeII, 1 15: 199-208), or to otherwise improve the functional aspects of the interfering RNA,
• the skilled aitisan will appreciate that there are routine methods for altering the strength and other properties of the base paired strands through the addition, deletion, or substitution of J&.T 5207
• RNA molecules comprises, for example, a hairpin loop or similar secondary structure that allows the molecule to self-pair to form at least a region ⁇ f double-stranded nucleic acid of Formula (I).
• double-stranded RNA molecules provide certain advantages for use in therapeutic applications.
• blunt-ended molecules are disclosed herein for certain embodiinenls, in various other embodiments, overhangs, for example of 1-5 nucleotides, are present at cither or both termini.
• the overhangs are 2 or 3 bases in length.
• Presently preferred overhangs include 3Mcrminus LRJ overhangs (3'-IJU) in certain embodiments.
• overhangs exemplified for use herein include, bul are not limited to, 3'-AA, 3'-CA, 3'-AU, 3'-UC, 3'-CU, 3'-UG, 3'-CC, 3'-UA, 3'-U, and 3'-A. Still other either 5'-, or more preferably 3'-, overhangs of various lengths and compositions are contemplated for use herein on the RNA molecules provided.
• the multitargeting interfering RNA molecule of the invention comprises one or more terminal overhangs, for example, an overhang consisting I to 5 nucleotides.
• the inul Ii targeting interfering RN ⁇ molecule of the invention comprises at least one modified ribonucleotide, such as one 2'-O-mcthyl ribusyl substitution.
• At least one target RNA molecule is an-mRNA. More specifically, in some embodiments, at least one target encodes a receptor, cytokine, transcription factor, regulatory protein, signaling protein, cytoskeletal protein, transporter, enzyme, hormone, or antigen. As such, the potential range of protein targets in the cell is not, limited, however the skilled artisan will appreciate that certain targets arc more likely to be of value in a particular disease state or process. In addition, the skilled artisan will appreciate that target RNA molecules, whether coding or regulatory, originating from a pathogen (e.g. a virus) fire useful with the inullilargeting interfering RNAs and methods provided herein.
• a pathogen e.g. a virus
• At least one of the binding sequences is in the 3' UTR of an mRNA.
• the inclusion of one target or more targets does not preclude the use of, or intention for, a particular interfering RNA to target another selected target. Such targeting of any additional J&J 5207
• RNA target molecules may result in less, equal, or greater effect in an expression system.
• the multilargeting interfering RNAs ⁇ f the instant invention arc preferably screened for off-target effects, especially those that are likely. For example, reviewing the potential binding to (he entire tran scrip Lome, or as much of it as is known at the time provides a useful approach lo such screening, For example, where a genome has been completely sequciiced, the skilled artisan will appreciate that the entire transcriptome can be conveniently screened for likely off-target effects.
• tissue-specific lranscriptomes eg retina for ocular applications
• non-target transcripts that are identified through bioinformatic approaches from the complete transcriptome may actually not be present in the tissue into which the multitargeting interfering RMA is applied.
• the two strands of a multitargeting interfering RNA of the invention form stable interaction with at least two distinct targeted binding sequences present in distinct genclio contexts on a single target.
• RN ⁇ molecule and thus modulates the expression or activity of the RNA molecule.
• Targeting multiple binding siies on a single target KNA molecule with a single iRN ⁇ provides more effective RN ⁇ i of the target RNA molecule. This approach is particularly useful for the modulation of virus gene expression where the mutation rate is high.
• the two strands of a multitargeting interfering RNA of the invention form stable interaction with at least two binding sequences present in distinct genetic contexts on multiple pre-selectcd target RNA molecules, and thus modulates the expression or activity of multiple pre-selected target RNA molecules.
• Targeting multiple target RN ⁇ molecules with a- single iRN ⁇ represents an alternative to the prototypical one drug one target approach. In considering the complexity of biological systems, designing a drug selective for multiple targets will lead to new and more effective medications for a variety of diseases and disorders.
• RNA molecules that are involved in a disease or disorder of a biological system are prc-selected and targeted by a multilargeting interfering RNA molecule of the invention.
• the biological system can be, for example, a plant, or an animal such as a rat, a mouse, a dog, a pig, a monkey, and a human.
• the pre-sclected target RNA molecules can, for example, encode a protein of a class selected from the group consisting of receptors, cytokines, transcription factors, regulatory proteins, signaling proteins, cytoskcletal proteins, transporters, enzymes, hormones, and antigens.
• the pre-selected larget RNA molecules can, for example, encode a protein selected from the group consisting of 1C AM-I, VEGF-A. MCP-1, IL-8, VEGF- B.
• IGF-I IGF-I, Gluc6p, Inppll, bFGF, PIGF, VEGF-C, VEGF-D, ⁇ -catenin, ⁇ -ras-B, ⁇ -ras-A, EG FR, and TNF alpha.
• the multitargeting interfering RNA molecule of the invention can, for example, modulate expression of any combination of ICAM-1, VEG F-B, VECiF-C, VEGF-D, IL-8, bFGF, PIGF, MCP-I and IGF-I, any combination of 1C AM-I, VEC F-A and IG F-I, any combination of ⁇ -catenin, ⁇ -ras, and EGFR, both lCAM-1 and VEC F-A, or both Gluc6p and Inppll, in a biological system, such as an animal.
• the proselected target RN ⁇ molecule can also be a viral RNA, including a viral RNA encoding a protein essential for the virus.
• Such essential proteins can, for example, be involved in the replication, transcription, translation, or packaging activity of the virus.
• Exemplary essential proteins for a HlV virus are GAG , P OL, VlF, VPR, TAT, NEF, REV, VPU and ENV, all of which can be a pre-selected target molecule of the invention.
• the multitarget ⁇ ng interfering RN ⁇ of the invention can be used Io modulate viral RNA from, including but not limited to, a human immunodeficiency virus (HFV), a hepatitis C virus (HCV), an influenza vims, a rhinovims, and a severe acute respiratory syndrome (S ⁇ RS) virus or a combination thereof,
• HBV human immunodeficiency virus
• HCV hepatitis C virus
• influenza vims a rhinovims
• S ⁇ RS severe acute respiratory syndrome
• the multitargeting interfering RNA of the invention are designed to target one or more target RNA molecules in a first biological system and one or more larget molecules in a second biological system that is infectious to the first biological system.
• die multitargeting interfering RNA of the invention are designed to target one or more host RNA molecules and one or more RNA molecules of a virus or a pathogen for the host.
• the viral RNA is HCV or HIV and the host target RNA includes, but is not limited to, TNFalpha, LEDGF(p75), BAF, CCR5, CXCR4, furin, NFkB or ST ⁇ T1.
• RNA molecules arc provided in the Examples that are functional against specific targets.
• CODEMIRs and/or VIROMIRs are useful for decreasing expression of RNAs, for example, their intended target RNA molecules and data supporting the activity arc also provided herein in the working examples.
• Such molecules can target multiple sites J&J 5207
• RNAs a ⁇ d are useful to decrease the expression of such one or preferably two or more such targeted RN As in an expression system.
• a given multitargeting interfering RNA will be more effective at modulating expression of one of several target RNAs than another.
• the iiiullilargeting interfering RNA will similarly affect all targets in one or more expression systems.
• RNAi efficiency (i) asymmetry of assembly of the RISC causing one strand to enter more efficiently into the RISC than the other strand; (ii) inaccessibility of lhe targeted segment on the target RN ⁇ molecule; (iii) a high degree of off-target activity by the interfering RNA; (iv) sequence- dependent variations for natural processing of RNA, and (v) the balance of the structural and kinetic effects described in (t) to (iv). See Hossbach et al. (2006), RNA Biology 3: 82-89.
• a multitargeting interfering RNA molecule of the invention In designing a multitargeting interfering RNA molecule of the invention, special attention can be given to each of the listed factors to increase or decrease the RNAi efficiency on a given target RNA molecule.
• Another general aspect of the invention is a method for designing a multitargeting interfering RNA, The method of the invention includes various means leading to a multitargeting interfering RN ⁇ that effectively target distinct binding sequences present ill distinct genetic contexts in one or more pre-selected target RNA molecules.
• a multitargeting interfering RNA can be designed by visual or computational inspection of the sequences of the target molecules, for example, by comparing target sequences and their complements and identifying sequences of length n which occur in both the target sequence and the complement oF the target, sequence sets.
• a multitargeting interfering RNA can be designed by visual or computational inspection of the sequences of the target molecules to find occurrences of the sequence of length n and of its complete complement within the set of target sequences.
• all possible sequences nf ⁇ pre-scTccTCd length, n can be generated by virtue of each permutation possible for each nucleotide position to a given length (4 n ) and then examining for their occurrence in the prc-sclceled nucleotide sequences and their complements.
• a mu I ti targeting interfering RNA when there is a pl ⁇ arilty of target sequences, can be designed by visual or co ⁇ ipulational inspection of lhe sequences of the target molecules, for example, by aligning sequences and visually or computationally finding consensus target sequences for the design.
• each strand be capable of modulating expression of its intended target (i.e. each strand is "active" against its target RNA, e.g. by having at least partial complementarity thereto) while simultaneously requiring that each of the strands is at least sufficiently complementary to tbe other that a duplex can form.
• each strand is capable of modulating expression of its intended target (i.e. each strand is "active" against its target RNA, e.g. by having at least partial complementarity thereto) while simultaneously requiring that each of the strands is at least sufficiently complementary to tbe other that a duplex can form.
• n ⁇ strand which is solely a guide strand or solely a passenger strand because each strand serves as both guide strand and passenger strand.
• Such molecules can be designed as single strands with hairpin structures that can, for example, be processed in vivo to become a duplex consisting of two separate strands.
• the invention provides a method for designing a multitargeting 5 interfering KNA molecule, comprising the steps of:
• a) selecting one or more target RNA molecules, wherein the modulation in expression of the target. RN ⁇ molecules is desired; b) obtaining at least one nucleotide sequence for each of the target RNA molecules; 0 c) selecting a length, jn, in nucleotides, for a seed sequence, wherein n about 6 or more; d) obtaining a collection of candidate seeds of iJie length n from each nucleotide sequence obtained in step b), wherein a candidate seed and its complete complement are not palindromic, and the candidate seed occurs at least once in one or more of the nucleotide sequences obtained in step b), and its complete complement occurs at least once in one or 5 more of the nucleotide sequences obtained ⁇ i st.p.p b); e) determining the genetic context of each of the candidate seed and its complete complement, by collecting, for each occurrence of the candidate seed and its complete
• step b selecting a second consensus target sequence, which comprises the complete complement of the seed and a consensus 3' flanking sequence to the complete complement of the seed determined from the sequences obtained in step b); i) obtaining a first strand sequence, which comprises the first consensus target sequence selected in step g) and, adjacent to and connected with the 5'-end of the first consensus- target sequence, a complement of the consensus 3' flanking sequence of step h); j) obtaining a second strand sequence which comprises the second consensus target sequence selected in step h) and, adjacent to and connected with the 5'-end of the second consensus target sequence, a complement of the consensus 3' flanking sequence of step g), and; k) designing a multitargeting interfering RN ⁇ molecule comprising a first strand having the first strand sequence in step i) and a second strand having the second strand sequence obtained in step j).
• the method further comprise repeating steps g) to k) for each seed of length n selected from the group of candidate seeds in step f).
• the method further comprises the step of repeating steps c) to k) for another desired seed length.
• the number of candidate seeds will increase as the length of the seed is decreased.
• finding a candidate seed present in at least one of the selected RN ⁇ sequences and in at least one complement of the selected RNA sequences is an alternative to .finding the candidate seed and i ⁇ .s complete: complement in the selected RNA sequences.
• X. LS, Y, X', S', Y' can be determined and assembled in a number of ways. Often the preference for designing a mullitargeting interfering RN ⁇ molecule of the invention involves; firstly, identifying the seed and its complement, which occur in different genetic contexts; secondly, determining XS and Y'S' so as to bind to their respective target RN ⁇ sequences, and then determining XSY and Y'S'X' wherein Y is the complement of Y' and X' is the complement of X. As an example, XS may be determined as the complement of the seed, (equates to S) together with the complement of a portion of the 3' flanking sequence of the seed (equates to X).
• Y'S' may be determined as the complement of the complement of the seed (equates to S') together with the complement of a portion of the 3' flanking sequence of the complement of the seed (equates to Y').
• the plurality of 3' flanking sequences may be examined to yield consensus 3' flanking sequences.
• X and/of Y' can then be determined as the complements of these consensus 3' flanking sequences. Further modifications can be made to the molecule as described in this specification.
• Target RNA molecules are strategically selected molecules, for example viral or host RNAs involved in disease processes, viral genomes, particularly those of clinical significance, and the like.
• RNA is provided above and applies equally to this and other aspects of the invention, as if set out in its entirety here.
• the basis for the selection of a target RNA molecule will be appreciated by those of skill in the art.
• Preferred target RNAs arc those involved in diseases or disorders one wishes to control by the administration of the multitargeting interfering RNA.
• the step of obtaining the sequences for the selected target is conducted by obtaining sequences from publicly available sources, such as the databases provided by the National Center For Biotechnology Information (NCBI) (through the National Institutes of Health (NIH) in the United States), the European Molecular Biology Laboratories (through the European Bioinformatics Institute throughout Europe.) available on the World -Wide Web, or proprietary sources such as fee-based databases and the Jikc. Sequences can also be obtained by direct determination. This may be desirable where a clinical isolate or an unknown gene is involved or of interest, for example, in a disease process. Either complete or incomplete sequences of a target RNA molecule can be used for the design of mult.iTargct.ing interfering RNA of the invention. J&.T 5207
• a plurality of independent target nucleotide sequences arc obtained in step b) for each ⁇ f one or more target RN ⁇ molecules selected in step a).
• Tlie databases described above frequently have multiple sequences available for particular targets. This is especially true where genetic variation is naturally higher, for example with viral sequences.
• lhe plurality of target nucleotide sequences represents strain variation, allelic variation, initiation, or multiple species. The number of such a plurality of sequences may range from several or a low multiple, to numerous - for example dozens or even hundreds or thousands of sequences for a given target. It is especially possible to have such numbers of sequences when working with viral sequences.
• sequences chosen can be further limited based on additional desirable or undesirable features such as areas of low sequence complexity, poor sequence quality, or those that contain artifacts relating to cloning or sequencing such as inclusion of vector-related sequences. Furthermore, regions with extensive inaccessible secondary structure could be filtered out at this stage. Indeed, Luo and Chang have demonstrated that siRN ⁇ targeting accessible regions of mRNA structure such as loops were more likely to be effective than those aligned with stems (Luo & Chang, (2004), Biochetn. Biophyst. Res, Commun., 318: 303-10). The sequences chosen, however, need not be limited, to 3'UTR sequences or regions of low secondary structure.
• the step of selecting a length of n nucleotide bases for a seed sequence is preferably an iterative process that does not require any particular basis or logic at first glance -i.e. the starting seed length may be. any number of bases above about 6.
• the starting seed length may be. any number of bases above about 6.
• the longer the length that is chosen for a seed the less likely that it and its complete complement, will appear in the at least one target RNA, e.g. in a target RNA sequence.
• the shorter the seed sequence length the more frequently it will occur as would be expected.
• an iterative process is used to find the preferred sequences for candidate seeds as described below.
• another value e.g. n+J, n-1
• the process can be repeated to identify candidate seed sequences of length ?J+J, n-1 and so on.
• seed candidates include sequences of a particularly desired or selected length each of which and its complete complement are not palindromic, and wherein the candidate seed occurs at least once in one or more of the nucleotide sequences obtained in step b), and its complete complement occurs at least once in one or more of the nucleotide sequences obtained J&J 5207
• the candidate seeds arc preferably generated by computer, for example by moving stepwise along a. target sequence with a "window" (expressed in te ⁇ ns of a fixed number of contiguous nucleotides ' ) of the desired or selected seed length.
• a “window” (expressed in te ⁇ ns of a fixed number of contiguous nucleotides ' ) of the desired or selected seed length.
• each step is a single base progression, thus generating a "moving window" of selected length through which each target sequence is sequentially viewed.
• Other step distances are contemplated, however, the skilled artisan will appreciate that only a step of one nucleotide will allow the generation of all possible seeds sequences.
• a collection of candidate seeds of the length n can be obtained by the steps of: i) generating a first collection of sequences of the length « from each of the nucleotide sequences chosen for the target molecules, using a method comprising lhe ⁇ steps of:
• a collection of candidate seeds of the length n can be obtained by the steps of: i) oblaini ⁇ u; the completely compiemp.ntary sequence for fiach nucleotide sequence chosen for the target molecules; ii) generating a fi rst col lection of sequences of the length n from each of the nucleotide sequences chosen for lhe target molecules and a second collection of sequences of the length n from each of (he completely complementary sequences obtained in step (i), using S method comprising the steps of: 1 ) beginning al a terminus of the nucleotide sequence of each of the nucleotide sequences chosen for the target molecules or each of lhe completely complementary sequences obtained in step (i);
• the method further comprises the step of discarding candidate seed sequences for which either the seed or its complete complement do not occur with at least a predetermined minimum frequency in the target nucleotide sequences.
• the method ultimately chosen will include one or more of these steps, or all of them as needed.
• the method further comprises the step of discarding any candidate seed sequence that; is composed of only a single base, is composed only of A and U , has a consecutive string of 5 or more C or 5 or more G , is predicted to occur with unacceptable frequency in the non-target traoscriptome of interest; is predicted to have a propensity to undesirably modulate the expression or activity of one or more cellular component (eg. to undesirably activate a cellular sensor of foreign nucleic acid), or any combination thereof.
• Seeds then are selected from the pool of candidate sequences as the ones where the seed is present in one genetic context, and its complete complement is present in a different genetic context in the at least one pre-selecled target sequence. Genetic contexts are determined by collecting, for each occurrence of the candidate seed sequence, a desired amount of the 5' and 3' flanking sequence. The. genetic context of the complement of the seed is determined in a similar fashion.
• a "consensus target sequence" is selected for one or bolh strands of the interfering RN ⁇ .
• Consensus target sequence docs not suggest that there is only one best sequence approximating multiple binding sequences on target molecule(s), rulher a population of one or more alternative sequences may all be consensus target sequences.
• a first consensus target sequence for the first strand of the iRNA comprises a seed sequence and a consensus 3'-flanking sequence to the seed in at. least one of the chosen sequences for the target molecules.
• a second consensus target sequence for the second strand of the iRNA comprises the complete complement of the seed and a consensus 3' flanking sequence to the complete complement of the seed in at least one of the chosen sequences for the target molecules.
• the "consensus 3' flanking sequence" of the seed is readily derived by visual inspection, or through the use of bioinfo ⁇ natic tools or calculations, from (he examination of the genetic context of each occurrence of the seed sequence in the sequences of the target molecules.
• the consensus .T flanking sequence need not be completely identical, but can be identical, to the sequcnce/s flanking the 3' end of the seed of one or more of the target sequences.
• the "consensus 3' flanking sequence" of the complement of the seed is readily derived by visual inspection, or through the use of bioinfoflmatic tools or calculations, from the examination of the genetic context of each occurrence of the complement of the seed.
• the consensus 3' flanking sequence need not be completely identical, but can be identical, to the sequence/s flanking the 3' end of the complement of the seed.
• the consensus target sequence does not include any sequence that is predicted to have a propensity to undesirably modulate the expression or activity of one or more cellular component.
• Consensus target sequences may be determined by eye or by algorithm.
• a computer algorithm can be used to score all possible permutations of paired nucleotides in the positions in which the sequences arc different. This is particularly useful when the target sequences .have some identity beyond lhc seed, but for which an alignment by eye proves difficult.
• This method can be used to determine the consensus target sequence/s, or alternatively, directly design the strands of the candidate nmltitargeting interfering RNA. J&J 5207
• One alternative approach that is particularly useful when a large number of target sequences need to be considered is Io generate all possible permutations of the extension from the seed to a required length, and/or the complete complement of the seed to a required length, thereby . ' generating the putative Y'S' and/or XS of Formula (I) and hybridizing each putative XS and Y'S 1 against the target sequences of interest in silica to determine those which demonstrate the most favorable properties in terms of hybridization to the target.
• Sequences demonstrating strong binding are of particular interest for the mullilargcting interfering RNA.
• the candidate XS and Y' S' are then prioritized for testing not only on this basis but. also taking into account, other features that may be important for the functionality of the in ulti targeting interfering RNA (by, for example, use of appropriate penalty terms).
• the addition of one or two nucleotides to the 5' end of the putative XS or Y'S' that are not complementary to their respective target sequences is considered. This is particularly relevant when an otherwise useful XS or Y'S' is QIV. rich at the 5' end and this is predicted to disfavor loading relative to (he other strand.
• the addition of one or two A/I J nucleotides to the 5 * extremity of the G/C rich XS or Y'S' will most likely promote balanced loading, which is required for optimal activity of the mullitargciing RNA. Because multitargeiing interfering RNAs in most cases tolerate mexcellent ⁇ hes al positions I and 2.
• DNA sequences with stretches of contiguous guanosines are known to produce additional effects not related to targeting of mRN ⁇ .
• most manufacturers recommend not selecting dsRNA duplexes containing long runs of G for their cxperi ments. It was found in this invention that greater than 4 consecutive G greatly reduced the activity of the corresponding CODEMIR (data not shown). Therefore, many seeds could be eliminated if a requirement for 5 or more C is applied.
• One skilled in the ait will recognize that the presence of 5 or more Cs in a seed will correspond to 5 or more Gs in the completely complementary RNA molecule of the invention.
• the method further comprises the step of discarding any consensus target sequence that: is composed of only a single base, is composed only of A.and U, has a consecutive string of 5 or more bases which are C, is predicted to occur with unacceptable frequency in the non-target ( ⁇ iscriptonie of interest, is predicted to have a propensity l ⁇ undesirably modulate the expression or activity of one or more cellular component, or any combination thereof.
• consensus target sequences are intermediates in the design of a mulliUtrgeling interfering RNA ⁇ f the invention,
• the consensus target sequences are used to generate the sequences for the first and the second strand of a muHitargeting interfering RNA of the invention.
• the first strand sequence is designed to comprise the first consensus target sequence and a complement of the consensus 3 1 flanking sequence of the second consensus target sequence, which is adjacent Io and connected with the 5 ! -cnd of the first consensus target sequence.
• the first strand is designed by extending the first consensus target sequence in the S' direction with a complete complement of consensus 3' flanking sequence of the second consensus target sequence.
• the second strand sequence is designed to comprise the second consensus target sequence and a complement of the consensus 3' flanking sequence of the first consensus target sequence, which is adjacent to and connected with the 5'-end of the second, consensus target sequence. !n a particular embodiment, the second strand is designed by extending the second consensus target sequence in the 5' direction with a complete complement of consensus 3' flanking sequence of the first consensus target sequence,
• Hybridization is typically examined from a thermodynamic perspective using RN ⁇ hybrid software (Rehmsmeicr et al., 2004, RN ⁇ , 10: 1507-17) or similar algorithm.
• X and Y' in .Formula (I) arc completely complementary to their respective target sites. Tn the case in which the X and Y', by virtue of being simply complementary to their respective target sites result in very different G/C richness at the two ends, then the loading bias needs to be reduced by either producing mismatches in either X' or Y, depending on the thermodynamic balance.
• several chemical modifications eg LNA, 2'O-methyl and 2'F can be introduced into the "weak" end of the duplex to improve loading balance-.
• m shown in the. Rx ⁇ inplcs. varying the length of the overhang may be used to control the loading balance of (he two strands of the duplex.
• the multitargeting interfering RNA is designed such that there is no loading bias, so that both strands can load equally.
• steps can optionally be added, individually or in combination, Io further the rational process of designing the RNAs - such as Io reduce the number of sequences unlikely to work for the intended purpose, Io increase the effectiveness of the RNAs, to reduce off target effects and the like. Many of these steps can be automated, or require only a limited amount of input from an operator, though the use of bioinformatic computer systems, which as the skilled artisan will appreciate, will facilitate the methods,
• duplex comprises elements predicted or known Io have a propensity to activate a cellular sensor of foreign nucleic acid.
• the designed multitargeting interfering RNA molecule can be modified, for example, i) to improve actual or predicted loading of the strands of the multitargeting interfering RNA molecule into the RNA induced silencing complex (RLSC); Ji) to increase or decrease the modulation of the expression of at least one targeL RNA molecule; iii) to decrease stress or inflammatory response when the multitargeting interfering RNA molecule is J&J 5207
• RLSC RNA induced silencing complex
• the modifying step comprises one or more of altering, deleting, or introducing one or more nucleotide bases to create at least one mismatched base pair, wobble base pair, or terminal overhang, or to increase RISC mediated processing. Techniques for doing so are known in the art. Preferably the modifications are at least initially performed in silica, and the effects of such modifications can be readily tested against, experimental parameters to determine which offer improved properties of the interfering RNA products.
• the methods, through to the step of actually making an RNA are conducted entirely in silica, or by visual inspection and determination.
• the method further comprises die step of choosing a new value for the seed length, n, and repeating each of the remaining steps. Tt is clear that the method can be iterative and the benefits of computers for such purposes arc well known.
• Screens that can be considered for the high throughput assessment of candidates include reporter assays, multiplexed ELlSAs, viral replicon systems, dot-blot assays, RT-PC R etc. '
• Candidate mullitargeting interfering RNA are routinely synthesized as double-stranded RNA molecules with 19 bp of complementarity and 3' two nucleotide overhangs.
• the overhang can be any nucleotides or analogs thereof, such as, for example, dTdT or UU.
• other types and lengths of overhangs could also be considered, as could "blunt-ended" duplexes.
• the overhangs are incorporated a priori into the design by having Y and X' being longer than the corresponding X and Y' by the length of the required overhangs.
• multiple multitargeting interfering RNAs can be co-expressed by several strategies, including bul not limited to, expression of individual multitargeting interfering RNAs from multiple expression vectors (plasmid or viral), expression from multiple expression cassettes contained within a single vector, with each expression cassette containing a promoter, a single multitargeting interfering RNA and terminator.
• Multiple multitargeting interfering RNAs can also be generated through a single polyeistronic transcript, which contains a series of multitargeting interfering RNAs.
• the multitargeting interfering RNAs can be expressed sequentially (sense / intervening loop / antisense) or expressed with the sense sequence of each multitargeting interfering RNA sequentially linked 5' to 3', joined directly or with intervening loop / spacer sequence, followed by the antisense sequence of each multitargeting interfering RNAs sequentially linked 5' to 3'.
• multitargeting interfering RNA are typically tested in cell culture; using an appropriate cell line representative of the targeted tissue.
• the specific conditions used are outlined in the specific examples, Multitargeting interfering RNA that lead to reduction in target RNA expression can then be studied further.
• semiquantitative RT-PCR for the target RNA may be performed to establish whether modulation of expression of a target RNA is likely to be mediated by degradation.
• cells are transtccted with the multitargeting interfering RNA at a concentration of 5-40 nM in the culture medium and after 48 hours, are washed, trypsinized and harvested for total RNA using a RNeasy kit (Qiagcn).
• RT- PCR is then performed using primer sets specific for the target RNAs.
• Proteomic and microarray experiments may be used to assess off-targel effects.
• analysis of markers of IFN-response eg STAT 1 , IFNb, IL-8, phosphoEIF etc. cun be performed on treated cells.
• the candidate multitargeting interfering RNA arc tested for non-specific toxic effects by, for example, direct assays of cell toxicity.
• cytotoxicity is the desired outcome and may reflect the successful repression of key oncogenic signaling pathways.
• Multitargeting interfering RNA are additionally assessed for their ability to repress the production of specific target proteins.
• Multitargcting interfering RN ⁇ demonstrating efficacy in this respect are then assessed for additional evidence of off-target, effects, including arrest of non-target protein production and activation of Protein Kinase R (PKR) mediated responses,
• PKA Protein Kinase R
• the RN ⁇ molecule may be expressed from transcription units inserted into vectors.
• the vector may be a recombinant DNA or RNA vector, and includes DNA plasmids or viral vectors.
• the multitargeting interfering RNA molecule expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, lentivirus or alphavirus.
• the vector is an expression vector suitable for expression in a mammalian cell.
• Methods which are well known to those skilled in the art can be used to construct expression vectors containing a sequence which encodes the multitargeting interfering RNA molecule. These methods include in vitro recombinant DNA techniques, synthetic techniques anil in vivo recombination or genetic recombination. Such techniques arc described in Sambrook et al ( 1989) Molecular Cloning, A laboratory manual, Cold Spring Harbor Press, I'lainview N. Y. and Asubel F M el al (19R9) Currant Protocols in Molecular Biology, John Wiley & Sons, New York N. Y. Suitable routes of administration of the pharmaceutical composition of the present invention may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, intravenous and subcutaneous injections.
• the pharmaceutical composition may be administered in n local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a target organ or tissue, such as intramedullary, intrathecal, direct intraventricular, intraperitoneal, J&J 5207
• a target organ or tissue such as intramedullary, intrathecal, direct intraventricular, intraperitoneal, J&J 5207
• the pharmaceutical composition of the present invention may be delivered in a targeted delivery system, for example, in a liposome, coated with target cell-specific antibody.
• the liposomes will be targeted to and taken up selectively by the target cell.
• Other delivery strategies include, but are not limited to, dcndrimers, polymers, nanoparticles and ligand conjugates of the RNA.
• the mullilargcting interfering RN ⁇ molecule of the invention are added directly, or can be complexed with cationie lipids, packaged within liposomes, or otherwise delivered to target cells or tissues.
• the nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump or stent, with or without their incorporation in biopolymers.
• the invention provides biological systems containing one or more mullitargeting interfering RN ⁇ molecule of this invention.
• the biological system can be, for example, a virus, a microbe, a plant, an animal, or a cell.
• the invention also provides a vector comprising a nucleotide sequence that encodes the multitargeting interfering RN ⁇ molecule of the invention.
• the vector is viral, for example, derived from a virus selected from the group consisting of an adeno-ass ⁇ eiatcd virus, a retrovirus, an adenovirus, a lentivirus, and an alphavirus.
• the inoltitargeting interfering RNA can be a short hairpin RNA molecule, which can be expressed from a vector of the invention.
• the invention further provides a pharmaceutical composition comprising a multitargeti/ig interfering RNA molecule of the invention and an acceptable carrier.
• the pharmaceutical composition comprises a vector for a multitargeting interfering RNA molecule of the invention.
• the present invention provides a method of inducing RNA interference in a biological system, which comprises the step of introducing a multitargeting interfering RNA molecule of the invention into the biological system.
• the invention further comprises a method of treating a subject, comprising the step of administering to' said subject a therapeutically effective amount of a pharmaceutical composition comprising a multilargcting interfering RNA molecule of the invention.
• the invention also provides a method of " inhibiting the onset of a disease or condition in a subject, comprising J&.T 5207
• compositions and methods exemplified herein are of use in the treatment of complex miiltigeiu'e diseases in which single gcne-specii ⁇ c therapeutics inay be at a disadvantage because of the multiple redundancies in pathophysiologic signaling pathways.
• a conscious and calculated approach is provided in which key signaling proteins/pathways can be simultaneously targeted with a single agent to generate greatly increased therapeutic potential.
• the targets of interest may be at least partially controlled by a common
• master regulator This is usually a transcription factor.
• down-regulation of IL-8 and MCP-I might be achievable through targeting the nuclear factor NFkappaB.
• this pathway is also involved in the survival of Retinal Pigmented Epithelial cells (RFE) in times of stress and the down regulation of such a cell-survival factor would likely lead to increased death of RPE in diseased eyes. Therefore, the novel approach disclosed herein has the advantage of being amenable to the modulation of specific targets of interest without having to identity suitable target "upstream" pleiolropjc controllers.
• RNA molecules active against several key pathways may derive synergistic activity against, cells reliant: on several of these targeted pathways.
• activity against a greater proportion of the tumour cells will also be lijkcly because of the "multi-targeted" nature of the RNA molecule of the invention .
• targeting of several key pathways will "cover" more of the patient population.
• the targeting of multiple disease-related transcripLs with a single muJtitargeting interfering RNA molecule of the invention preferably allows full use of available RISC as opposed to the J&J 5207
• siRNA molecules which could, in some cases, saturate the available . intracellular machinery
• Targeting multiple sites within the same RN ⁇ target sequence can also be accomplished with the compositions and methods provided herein.
• Many human diseases/including cancer and viral infections are characterized by RNA targets exhibiting high mutation rates. This increases the likelihood of resistance to nucleic acid therapeutics arising in these diseases, due to mutation of the target RNA. Targeting multiple sites within the target RNA decreases the likelihood of such resistance arising, since at least two simultaneous mutations would be required for resistance.
• the muili-targcti ⁇ g approach of mullilargcting interfering RNAs eg CX)DEMIRs or VIROMIRs
• Targcti ng of multiple sites within the same transcript may also produce synergistic effects on the inhibition of viral growth. Further, employing a mechanism or mechanisms requiring only partial complementarity with the target RNA can have an advantage in decreasing the possibility of developing resistance through point mutation.
• the desired targets for any disease entity iaay be identified based on an approach or a mixture of approaches including, but not limited to, validated drug targets from literature and proprietary target discovery processes.
• the target genes are then prioritized based on evidence supporting a key role for their products in the disease process of interest.
• specific attention may need to-be paid to the accuracy an ⁇ Vor relevance of the sequence to the disease of interest.
• targeting cancer it is advisable to avoid mutational "hot-spots”.
• selective targeting of a specific splice variant or isoform may be desired and thus in such embodiments, the target sequence used in multitargeting interfering RNA design is preferably restricted to that isoform or variant present only in diseased tissue.
• the sequences of the target RNA or RNAs arc preferably downloaded from public or proprietary databases or generated from sequencing experiments. J&J 5207
• This sequence was used to design a CODEMIR active against multiple targets, using each strand of (he CODEMlR to target at least one of the target RNAs.
• the sequence identified above and its complement were used as a centrally-located part of a CODEMlR duplex.
• Bach strand of this central duplex was extended in the 5' direction to provide full complementarity to the corresponding target, whereas each strand was extended in the 3' direction so as to be complementary to its opposing strand in the CODEMIR duplex strand.
• CX)DEMIRs - 16 and -17 were tested for activity against both VFXiF-A and ICAM- 1 targets in RPE cells.
• RPE ceils in culture were used to screen the anli-angiogenic CODEMlRs designed, us described above.
• the human cell line, AR1 ⁇ - ⁇ 9, was used, AKPE- 19 cells were grown in Dulbccco's Modified Eagle's Medium supplemented with 1 U% felal bovine serum and 10 mM glutamine.
• ARPE cells were seeded at 4 x 10 3 cells per well in a 96 well tissue culture plate.
• ARPE-19 cells were seeded at 2.5 x 10 4 cells per well in a 24 well tissue culture plate. Cells were transfecled 24 hours after seeding using lipofectamine (InVitrogen) at a ratio of 1 micro!- lipotectamiiie per 20 pmol of CODEMlR RNA duplex or control. siRNA. [n most studies, medium was replaced 24 hours after transfection at which time deferoxamine (130 ⁇ M) or IL-I ⁇ (1 ng/mL) was added tor the VEGF-A and ICAM-I experiments, respectively. Experiments were performed in triplicate and repeated at least twice. The ARPE- 19 cells were assayed to confirm production of both VEGJF-A and ICAM-I . VEGF-A was assayed in the supernatant using a commercially available ELISA assay (R&O)
• the loading bias can be adjusted, tor example, by introducing wobble G:U basepa ⁇ rs into the extremities of the duplex, or by expanding the CODEMlR to a 22-base duplex with symmetrical extremities. Variations of each of these types were explored.
• CODEMIR-26 is a 22-base duplex that has 4 identical binding nucleotide pairs at each of the two termini of the duplex. As shown in Figure 1, CODEMIR-26 exhibited greatly increased ICAM-I targeting compared to that of CODEMIR- 16. Thus, the adjustments to the sequence were able to correct the loading bias observed with CODEMIR-16.
• CODEMIRs -27 and -28 were designed to test whether disrupting strong G:C pairs at an end of the duplex region would also successfully overcome the loading bias observed with CODEMIR16.
• the substitution of a C with a CJ in the 3'tcrminal region of the guide strands targeting VECiF-A was successful in changing the bias (e.g. CODEMIR-27).
• CODEMIR-28 had similar activity to CODEMlR-27 where changes were made at the other end of the CODEMlR. J&J 5207
• bot.li strand loading bias and target activity can be controlled by introducing mismatches to disrupt the end of the duplex that is inefficiently loaded and simultaneously increase hybridization to the target.
• CODF.MIR-36 a variant of CODEMIR- 16
• both .strands were designed to be entirely complementary to the respective target sequences; the resulting incompletely complementary duplex features several mismatches at lhe 5' extremity of the IC ⁇ M-1 guide sequence.
• the results tor CODEMlR-36 (see Table 1-1) are shown in Figure 1.
• the multitargeting interfering RNA (CODEMlRs) herein disclosed would be expected to be effective in multiple angiogenic diseases of the eye. This is because secreted VEGF-A plays a major role in all of these diseases (Witmev et al (2003) Prog Relin Eye Res, 22, 1-29), although ICAM-I overexpression may be an early initiating event, particularly for diabetic retinopathy and macular edema (Funatsu et ah, (2005) Ophthalmology, 1 12, 806-16.; Jousscn ct al. (2002) Am J Pathol, 160, 501-9 ; Lu et at (1999) Invest Ophthalmol Vis Sd, 40, 1808-12.
• CODEMlRs multitargeting interfering RNA
• CODEMlRs are able to suppress both VEGF-A and ICAM- 1 production by human retinal epithelium ceils ( ⁇ RPE-19 cell line). These cells are a major contributor to the production of these proteins in these ocular angiogenic diseases (Matsuoka et al., (2004) BrJ Ophthalmol, 88, 809-15, Yeh et al. (2004), Invest Ophthalmol Vis Sci, 45, 2368-73). RPE cells arc also the primary site of uptake of foreign nucleic acids in the eye and, for these two reasons, are flic appropriate cell model for evaluation of anti-angiogcnic CODEMlRs in ophthalmology.
• CODEMLRs may also be suitable for the treatment of complex metabolic diseases such as type 2 diabeLes.
• Two potential gene targets for the treatment of this disease are glucose-6-phosphalase and lnppi I . Full transcript sequences were examined.
• Candidate CODEMIRs from the best contiguous region of identity are shown for each case in Table 2- Jt.
• Some modification of the CODBMlRs can be performed to tune the hybridization of the CODEMIR duplex, thereby affecting the loading bias.
• Introduction of mismatches is one way of achieving this (for example see: Ohnishi et al. (2005) Biochem Diophys Res Cotnmun. 329:516-21) and these mismatches can be chosen also for their ability to increase binding of the 3' Tegion of the effector strands to their respective target transcripts ( Figure 2B).
• the CODHM ⁇ R duplex is then no longer composed of two strands with complete complementarity, similarly to CODKMlR-36.
• multitargeting interfering RNA ' molecules will comprise the sequence corresponding to the complement of the seed.
• these complementary sequences arc CUGGGCGAGGCAG (SEQ ID NO: 21) and (5U(KiAlJGUGGAG. (SEQ ID NO: 22) J&J 5207
• the invention can be used to target proteins of interest that are likely to be mutated in chronic forms of disease. Mutations may be particularly prevalent in cancer and viral disease in which drug-resistant forms often cvoJve.
• VIROMlRs were designed to target multiple sites in the Human Immunodeficiency Vims (HIV). The requirement for simultaneous mutation at several sites, in order to overcome the effects of such a VlROMIR, is likely to provide a high genetic; hurdle to the emergence of resistant viral clones or quasispecies.
• the genome of the HXB2 strain of HIV I serotype B (GenBank Accession K03455) was used as the principal sequence of interest and was examined with bioinformatics methods detailed elsewhere in this application to find seeds occurring at more than one location.
• HlV 1 clade B isolates in the LAJNL database as of 1 August 2005 which contain full sequences for any of the GAG, BNV, POL, TAT, VIF, VPR, VPU and N.EF genes were used in these analyses.
• a 17-base seed and its complete complement were found in the HIV reference strain genome as shown below:
• K03455r is a partial sequence of the complement of the reference strain genome.
• an effective RNA therapeutic of the invention should provide broad coverage of the affected population and it is obviously desirable to target sequences that are highly represented in this patient population. Therefore, the seed presented above might not cover a sufficient proportion of the population. Nevertheless, due to its unique size it was considered further in the exemplification of the invention.
• Thr pNL4.3 assay is widely used in the field of IJIV research as a valuable, validated screen for drugs active in HlV and was used by us to test candidate VIROMIRs.
• the sequences of the HIV component of the pNL4.3 plasmid and that of the reference IUV strain (K03455) used in the design of the VlROMIR. Therefore, comparison of the sequence of the reference strain and the srquence of Lhe pNL4.3 plasmid was carried out to confirm that the above-mentioned VIROMIR was targeting a sequence also present in the testing system.
• Other testing systems such as viral challenge assays, fusion reporters, viral pseudoparticles among others, each representing any multitude of therapeutically relevant or irrelevant sequences could equally be considered.
• VIROMlR duplex (VM011) targeting these two seed sites is:
• the encoded RNA folds into a hairpin structure, which is modified by the cellular Drosha and Dicer proteins to generate active VIROMIR RNA duplex(es).
• the skilled artisan will also recognize that a number of variations of the design of the shRN ⁇ construct, could be considered. These include but. are not limited to: length, sequence and orientation of the shRNA duplex components (guide strand, passenger strand, precursors), length and sequence of the loop, choice of promoter, initiator and terminator sequences as well as the cloning strategies used to assemble the final construct.
• HEK-293 cells were .seeded at density of 2 X 10 ⁇ 5 cells in 1 ml Oplimcrn medium / well in a 12-well plate. Cells were transfecled 24 hr later with 200 ⁇ L J&J 5207
• DN ⁇ Lipofcctamine mi* (2 ⁇ ()ng pNL4.3 plasmid, 67ng VIROMIR pSII.- construct in lOO ⁇ L complexed with 2.7 ⁇ L Lipofectamine 2000 in Optimcra).
• the production of p24 was assayed by collection of the supernatant after a further 24 hours of incubation, The production of p24 was expressed as a percentage of the production from cells trail sfected, with the empty control plasmid.
• VMOI l did not. have any appreciable activity in this assay- (data, not shown), perhaps reflecting the lack of equivalent loading, as predicted from the analysis of the ends of the duplex.
• mullitargcting interfering RNAs can be utilized to target both the genome of the infectious agent and one or more key host "drivers" of the disease.
• TNF-alpha is considered a major disease-associated factor in Hepatitis C Virus infection and its sequelae. Investigation of the genome of IICV and the TNF-alpha mRNA was undertaken.
• This seed wan selected because it is present in the HCV genome with a conservation of 94% in 155 isolates of genotypes Ia and Ib. This seed is actually present in two sites in MC V. We then considered the nucleotides in the 3' direction from this seed to establish which site should be primarily considered in the design of the duplex.
• the extended sequences for the sites (+6 bases to the 3' end) were as follows:
• the .seed is in the 5'NTR of HCV and 3'UTR of TNFalpha. Shown below is the location of the seed in the HCV sequence and in the antiparallcl sequence f ⁇ r TNFalpha:
• the required extension for the duplex of the required length is, for example, 5 bases in the 3' direction of the target and 5 bases in the 5' direction as the .seed is usually in the middle of the double stranded duplex (excluding the overhangs). By putting it in the middle, the resulting two strands will have an equivalent portion of complete complementarity with their respective targets when the process outlined below is followed. This should ensure that. binding of the resulting two strands should be comparable. With a seed of, for example, ten nucleotides, the extension by 5 on each side would create a duplex of 20, whereas extension of 4 plus 5 or 5 plus 4 would yield a duplex of 19 nucleotides. Other permutations are equally permissible depending on the lengths of the seed and the desired duplex.
• a means of generating one of the strands of the duplex is as follows:
• the two guide strands have predicted binding to the two targets of;
• the duplex could be extended, with further complementarity U) the HCV sequence, possibly:
• a duplex for which the TNFa- targeting strand is mutated but still capable of binding to the target and the corresponding strand is changed, to match could be:
• the first 5 base pairs of the duplex arc equally balanced at the two ends, without appreciably compromising binding to the target as shown below (note wobble- base pair with TNFa-targeting strand).
• overhangs can be added. It may be beneficial to make those complementary to the intended target so as to enhanced improved binding of the tail region.
• the added bases may be selected so as to provide predicted binding to a specified further region in the target RNA. For example, in the above in silico binding result a large bulge is predicted to be formed from the binding of the first guide strand to the HCV target RNA. The choice of overhangs could be guided by the desire to reduce the length of that bulge. Other alternatives are Io add bases complementary to the target to provide an extension of the binding indicated in silico. So, as an example, the duplex generated above could be further extended using the information from the in silica hybridization to further define the bases required.
• .synthetic multitargeting interfering RNA duplexes wi.II comprise the seed and its corresponding complement (5' AGGGCUCCAGGCG 3' (SEQ ID NO: 63) or 5' GCUCGCCGACKJAG 3"). (SEQ ⁇ D NO: 64) ,)&J 5207
• RISC loading is impaired in the absence of 3 1 overhangs.
• Wc have investigated the use ⁇ f a single biunt-end to prevent loading of one strand of a CODRMIR; a technique that is potentially useful for promoting loading of the guide strand.
• CODEMIR-17 which has a strong preference for loading of the TCAM targeting guide strand.
• the variant CODEMJR -103 (Table 5-1) was designed to include a blunt-end at the 5' end of the fCAM- 1 guide strand.
• This CODHMIR demonstrated increased VEGF suppressive activity, and decreased ICAM-I suppressive activity (Figure 3), which is consistent with altered strand loading.
• ARPE-19 cells were lransfected with 4OnM duplex RHA and VHGF (HLLSA) or ICAM. (FACS) expression was assayed 48 hours post-transfcction. Each bar in Figure 3 represents the mean of triplicate samples. Error bars indicate standard deviation of the meaji.
• Example 6 Activity of CODEMfRs ami VIUOMi I Rs in vivo '
• the activity of CC)DHMIRs and other mulritargcting interfering RNA of the invention could be tested in various preclinical models known to those skilled in the art.
• CODEMIRs-26-28 could be tested in a retinopathy of prematurity model. This model is well known to those working in. the field of ocular angiogenesis and is used extensively as one of several models for the development of drugs active against the diseases of interest. (AMD, diabetic retinopathy etc).
• the .study could comprise of a suitable number of mouse or rat neonate pups equally divided into treatment groups.
• the treatment groups could include negative controls such as vehicle, irrelevant or scrambled sequence controls plus a number of multitargeting interfering RNA.
• negative controls such as vehicle, irrelevant or scrambled sequence controls plus a number of multitargeting interfering RNA.
• siRNA to VEGF as known comparators.
• a model beginning on Day 1 of life, litters are exposed to cycles of hypcroxia followed by several days of room air. The injections could be performed on the last day of cycling, prior to the 4 day normoxia period. vSeveral days later, animals could be injected with F.lTC-dextran and sacrificed. Fluorescence images of the retinal flat mounts could used to estimate the extent of neoangiogeuesis in each animal.
• measurement of the production of the target RNA molecules or their encoded proteins in this casc.VEGF and ICAM
• CODEMIRs could alternatively be evaluated in vivo for inhibition of disease-related angiogenesis using the laser-induced Choroidal Neovascularization (CNV) model in rats or primates.
• CNV laser-induced Choroidal Neovascularization
• animals under general anaesthesia have their pupils dilated and retina photographed.
• CNV is induced by krypton laser photocoagulation. This is performed using laser irradiation to cither the left or alternatively, the right eye of each animal from all treatment groups through a slit lamp. A total of 6-11 laser burns are generally applied to each eye surrounding the optic nerve at the posterior pole.
• the tnultitargeting interfering IiNA are injected into the affected eyes.
• the suitable time can bo the day following laser induction, or for an assessment against established CNV, the injections can be performed several days or weeks following injury.
• Intravitreal injections of the oligonucleotides are performed by J&.J 5207
• the test compounds are placed in the .superior and peripheral vitreous cavity.
• the tlcoangiogcncsis is evaluated by either imaging - and/or direct sampling (eg histology, im ⁇ nunohistochejnis ⁇ ry). In all cases, the assessment of CNV is best, performed by a skilled operator blinded to the actual treatment to ensure a lack of bias in the recording of the information.
• ⁇ n example of a direct imaging method is Colour Fundus Photography (CFP). Again, under anaesthesia as described above, the pupils are dilated. The fundus is then photographed with a camera using the appropriate film.
• CFP Colour Fundus Photography
• fluorescein angiography is used to image the vessels and areas of vascular leakage in the retina. This is performed on all of flic animals following the intraperitoneal or intravenous injection of sodium fluorescein. The retinal vasculature is then photographed using the same camera as used for CFP but with a barrier filter for fluorescein angiography added. Single photographs can be taken at 0.5-1 minute intervals immediately after the administration of sodium fluorescein. The extent of fluorescein leakage is scored by a irained operator. The mean severity scores from each of the lime points are compared by a suitable statistical analysis and differences considered significant at p ⁇ 0.05. In addition, the frequency of each lesion score is counted, tabulated and represented graphically.
• rats can be euthaiiascd at sclccicd time points following treatment (for example 7, 14 and 28 days post injection) and eyes examined by conventional histopathology. A reduction in the number and severity of lesions is expected to be seen with samples treated by active oligonucleotides of the invention.
• Other non-limiting examples including testing the m ⁇ ltitargcting interfering RNA of the invention in other preclinical models such as those that are well known to those skilled in the art.
• a non-exhaustive list includes pulmonary fibrosis (bleomycin induced), paw inflammation (carrageen), joint arthritis, diabetes, viral infection . , tumour xenografts etc.

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