DETAILED DESCRIPTION

[0070] The following definitions are used in the description and examples to assist in understanding the scope of the present invention.

[0071] “Chimeric RNA” as used herein means an RNA molecule constructed from at least two polynucleotide sequences that covalently linked together and that derived from at least two different RNA molecules that may or may not be in the same or different species.

[0072] “Guide sequence” as used herein means a polynucleotide sequence that is complementary to a target sequence.

[0073] “Lethal protein” means proteins that may kill the cell in which the protein is produced, if produced in a lethal amount. Lethal proteins include proteins that induce apoptosis, such as Bax, Bcl-Xs, Bad and Bak, Fas, and Caspl, and proteins that inhibit viral replication, inhibit proliferation or inhibit protein synthesis both at the level of transcription or translation. Further specific examples of toxic proteins are full length Tiam, Rac, Rho, and Ras.

[0074] “siRNA” as used herein means a double stranded short interfering RNA molecule of no larger than about 23 nucleotides in length. The scientific literature describes siRNA as mediating the sequence specific degradation of a target mRNA.

[0075] “sRNA” as used herein means a single or double stranded RNA molecule of less than about 25 nucleotides. sRNA comprises both stRNA and siRNA molecules.

[0076] “stRNA” as used herein means a single stranded small temporal RNA molecule that is complementary to a 3′ untranslated region in RNA in a host cell.

[0077] “Stem-loop” as used herein means a single stranded polynucleotide including two sequences of base pairs that complement each other and that permit the formation of a complementing duplex structure in the single-stranded polyribonucleotide, and a non-complementing loop sequence linking said two sequences of base pairs. The complementing base pairs making up the stem portion of the loop consist of at least two, and more preferably at least three base pairs in length. In certain special embodiments, where the stem base pair(s) correspond to the complementing sequence of the first and third sequences, one or more of the second sequence complementing stem base pairs can double as a first and third sequence complementing base pair. Under these special circumstances, the stem portion of the second sequence would be considered to contain only one complementing base pair, or no complementing base pairs.

[0078] “Target sequence” as used herein means a polyribonucleotide sequence present in RNA in a host cell.

[0079] “Transfecting” as used herein means any way of introducing a nucleic acid into a cell as is known by a person skilled in the art. It includes but is not limited to transduction by e.g. calcium phosphate or liposomes based reagents, infection by e.g. viral vectors, phages, electroporation, via a soaking process, or introduction of the nucleic acid using a physical method like micro-injection or DNA coated particle bombardment.

[0080] The self-complementing single stranded polynucleotide according to the present invention comprises a first guide sequence and a second sequence capable of forming a stem-loop structure within said second sequence when said second sequence is RNA. A preferred embodiment of the polynucleotide includes a third sequence, which complements the first guide sequence and is covalently linked to the distal end of the second sequence. In the most preferred self-complementary polynucleotide of the present invention, all nucleotides in said first and third sequences base pair. The preferred self-complementing polynucleotides comprise a second nucleotide sequence that comprises a stem-loop forming region derived from RNA molecules other than mRNA.

[0081] The present invention provides for either the first or third sequences to be a guide sequence that functions to direct the stRNA, siRNA, sRNA or chimeric RNA encoded by the single stranded polynucleotide to an RNA having a complementary sequence in the host cell system. The first and third polynucleotide sequences have a length consisting of about 17 to about 23 nucleotides, and preferably from about 19 to about 22 nucleotides, and most preferably about 19 or about 21 nucleotides, all of which correspond to a sequence found in a specific RNA. The RNA in the host cell may be a RNA molecule such as mRNA, tRNA, snRNA, rRNA, mtRNA, or structural RNA, or an RNA found in the host cell, which RNA is present as a result of a viral, bacterial or parasitic infection. Preferred RNA molecules are mRNA molecules. The RNA in the host cell may be a known RNA coding for a known RNA molecule or a protein of known function, or may not be known to be associated with any particular protein or cellular function.

[0082] Preferred stem-loop sequences are based on stem-loop regions known to those persons skilled in the art to be present in RNA molecules such as, tRNA, snRNA, rRNA, mtRNA, or structural RNA sequences. Persons skilled in the art can readily identify stem-loop RNA structures using predictive computer modeling programs such as Mfold (M. Zuker, D. H. Mathews & D. H. Turner Algorithms and Thermodynamics for RNA Secondary Structure Prediction: A Practical Guide In RNA Biochemistry and Biotechnology, 11-43, J. Barciszewski & B. F. C. Clark, eds, NATO ASI Series, Kluwer Academic Publishers, (1999)), RNAstructure (Mathews, D. H.; Sabina, J.; Zuker, M.; and Turner, D. H., “expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structures”, Journal of Molecular Biology, 1999, 288, 911-940), RNAfold in the Vienna RNA Package (Ivo Hofacker, Institut fur theoretische Chemie, Währingerstr. 17,A-1090 Wien, Austria), Tinoco plot (Tinoco, I. Jr., Uhlenbeck, O. C. & Levine, M. D. (1971) Nature 230, 363-367), ConStruct, which seeks conserved secondary structures (Lück, R., Steger, G. & Riesner, D. (1996), Thermodynamic prediction of conserved secondary structure: Application to RRE-element of HIV, tRNA-like element of CMV, and mRNA of prion protein. J. Mol. Biol. 258, 813-826; and Luck, R., Gräf, S. & Steger, G. (1999), ConStruct : A tool for thermodynamic controlled prediction of conserved secondary structure. Nucleic Acids Res. 21, 4208-4217), FOLDALIGN, (J. Gorodkin, L. J. Heyer and G. D. Stormo. Nucleic Acids Research, Vol. 25, no. 18 pp 3724-3732, 1997a; and J. Gorodkin, L. J. Heyer, and G. D. Stormo. ISMB 5; 120-123, 1997b), and RNAdraw (Ole Matzura and Anders Wennborg Computer Applications in the Biosciences (CABIOS), Vol. 12 no. 3 1996, 247-249).

[0083] RNA stem-loop structures are also found in databases such as the Small RNA Database (Karthika Perumal, Jian Gu, Yahua Chen and Ram Reddy Department of Pharmacology, Baylor College of Medicine, USA), Database of non-coding RNAs (Erdman V A, Barciszewska M Z, Szymanski M, Hochberg A. the non-coding RNAs as riboregulators (2001) Nucleic Acids Res. 29: 189-193), large subunit rRNA database (Wuyts J., De Rijk P., Van de Peer Y., Winkelmans T., De Wachter R. (2001) The European Large Subunit Ribosomal RNA database. Nucleic Acids Res. 29(1): 175-177), the small subunit rRNA database (Wuyts, J., Van de Peer, Y., Winkelmans, T., De Wachter R. (2002) The European database on small subunit ribosomal RNA. Nucleic Acids Res. 30, 183-185), snoRNA Database for budding yeast (Lowe and Eddy, Science 283: 1168-1171, 1999) for Archaea (Omer, Lowe, Russel, Ebhardt, Eddy and Dennis Science 288: 517-522, 2000), for Arabidopsis thaliana : (Brown, Clark, Leader, Simpson and Lowe RNA 7:1817-1832, 2001), tRNA sequences and sequences of tRNA genes (Mathias Sprinzi, Konstantin S. Vassilenko, http://www.uni-bayreuth.de/departments/biochemie/trna/), the 5S ribosomal RNA database (Szymanski M, Barcizewska MZ, Erdman VA, Barciszewskij, “5S ribosomal RNA database” (2002) Nucleic Acid Res. 30: 176-178), The Nucleic Acid Database Project (NDB) at Rutgers University (http://ndbserver.rutgers.edu/NDB/), The RNA Structure Database (www.RNABase.org)

[0084] Using these programs, one can identify an RNA stem-loop sequence, which can then be modified to eliminate multiple loop regions to result in a shorter, more easily synthesized stem-loop sequence. More preferred stem loop sequences are derived from the let-7 nucleotide sequence, or some portion thereof, or an artificially generated polynucleotide sequence based thereon

[0085] Most preferred stem-loop regions consist essentially of a let-7 sequence found in the host cell or some portion thereof, some other naturally occurring RNA sequence or some portion thereof, or an artificial polynucleotide sequence capable of forming a loop structure when such polynucleotide is RNA. By eliminating the multiple loop segments of these sequences that are predicted by the aforesaid computer programs, stem loop sequences that are more easily synthesized and handled are prepared. Most preferred stem loop sequences are derived from the let-7 nucleotide sequences.

[0086] The loop structure is preferably about 4 to about 30 nucleotides in length, a more preferred length is about 4 to about 13 nucleotides, and a most preferred length is about 6 to about 12 nucleotides in length. A special embodiment of loop sequences comprise those artificial sequences based on known RNA loop sequences consisting of about 11 to about 16 nucleotides. Examples of preferred loop sequences are listed in FIGS. 10 and 11 .

[0087] A particularly preferred polynucleotide of the present invention further comprises a fourth nucleotide sequence consisting essentially of an RNA sequence, a single stranded DNA equivalent thereof, wherein said fourth sequence is covalently linked to a free end of either the first or third sequence, and wherein said RNA sequence is capable of being cleaved enzymatically in the host cell thereby resulting in the in situ preparation of a RNA polynucleotide that has first or third sequences with a free 3′ and 5′-end.

[0088] A further embodiment of the present polynucleotide invention further comprises a fifth nucleotide sequence consisting essentially of an RNA sequence, or a single stranded DNA equivalent thereof, which is covalently linked to a free end of said first or third sequence. The fifth RNA sequence is capable of being cleaved enzymatically in the host cell thereby resulting in the in situ preparation of a RNA polynucleotide that has first and third sequences with a free 3′ and 5′-end.

[0089] Said fourth and fifth nucleotide sequence can be preferably derived from precursor RNAS, such as ribozymes, precursor tRNA, precursor rRNA, precursor microRNAs, RNAs recognized by ribozymes, or RNAs recognized by RNase P. Ribozymes cleave themselves such that a free 3′-or 5′-end at the said first or third nucleotide sequence is produced. Alternatively, ribozymes cleave the RNA sequences recognized by them, thereby producing free 3′-or 5′-end at the said first or third nucleotide sequence. Enzymes present in the host cell process precursor RNAs. Such fourth and fifth nucleotide sequences are preferably designed such that they are cleaved by enzymes present in the host cell.

[0090] The aforesaid fourth and fifth sequences may also be derived from “overhang” sequences found naturally, such as sequence that extend beyond the complementing portion of RNAs in the “microRNA” family. MicroRNAs (mRNAs) belong to an expanding class of non-coding RNAs of 21-24 nucleotides with let-7 RNA and lin-4 RNA as founding members. MicroRNA molecules are found in the genomes of a variety of species, including worms, flies, humans and plants, and are typically expressed from approximately 70 nts long hairpin-structured RNA precursors. These hairpin-structured precursors can also exist in a cluster of several precursors. Normally, after processing, only one strand of the duplexed region of the precursor accumulates in the cell as 21-24 nucleotides RNA. Examples of identified mRNAs are described in Lagos-Quintana, M et al. Science ( 2001) 294: 853, Lau, et al. Science (2001) 294: 858, Lee and Ambros Science ( 2001) 294: 862.

[0091] A most preferred dsDNA polynucleotide according to present invention comprises a fourth sequence that functions to permit the directional cloning thereof into a DNA vector, as described in more detail below. The dsDNA polynucleotide sequences may contain restriction sites at either end that are susceptible for cleavage by one restriction enzyme -or two different restriction enzymes to enable efficient cloning. The resulting termini of the dsDNA oligonucleotides preferably have overhanging nucleotide sequences (either 5′ or 3′ overhanging) that match the vector insertion sites. The dsDNA polynucleotide containing the cleavable restriction sites at the termini can be generated by standard molecular biological techniques, for example, by annealing two complementary ssDNA oligonucleotides. Alternatively, two annealed DNA oligonucleotides, with only restriction sites at their 5′ termini, can be enzymatically extended at their complementary 3′ termini to make the fully complementary double stranded DNA. Furthermore, the person skilled in the art is able to utilize blunt end cloning techniques and PCR to develop alternative routes of synthesis to achieve the directional cloning described herein.

[0092] The aforesaid fourth and fifth sequences may or may not be transcribed from the DNA sequence present in a DNA vector of the present invention, but may function as part of the upstream promoter or downstream termination signal. Consequently the fourth sequence may incorporate the start signal for RNA polymerase to transcribe, and the fifth sequence comprise a stop signal, such as a multiple “T” sequence, that transcribes into an RNA fifth sequence of multiple “U” nucleotides. Upon transcription the fourth sequence may not be transcribed into RNA except for one to about five, and more preferably one to about three “G” nucleotides.

[0093] In a particular aspect of the present invention, the process of preparing the self-complementing polynucleotide uses an intermediate polynucleotide consisting essentially of a first sequence consisting of about 17 to about 23 nucleotides, said first sequence covalently linked to a second sequence capable of forming a loop structure, wherein said first sequence consists essentially of a RNA sequence, a single stranded DNA equivalent thereof, or a RNA or DNA sequence complementary to said RNA sequence. mRNA sequences, that are sequences that code for protein, are a special embodiment of the methods and compositions according to the present invention.

[0094] The self-complementing single-stranded polynucleotides may be prepared by chemically synthesis. The process of synthesis requires that the target sequence of the RNA be known, an 17 to 23 nt sequence corresponding thereto prepared, and the synthesis continued to add the stem-loop sequence of about 4 to about 30 nucleotides, more preferably, from 6 to about 13 nucleotides. The isolated synthetic polynucleotide may be used to prepare a vector for use as an intermediate in the practice of the present invention, or further lengthened to include the complementing third sequence.

[0095] In practice, from about two to about five sequences are chosen from a single RNA sequence for the preparation of a corresponding number of self-complementing polynucleotides and vectors containing the same. The present method, described in more detail below, uses this “redundant” set of self-complementing single stranded polynucleotides and vectors including the same, to determine the optimum choice of RNA sequence that targets only one unique RNA that may exist among a family of RNA having homologous sequence regions. Alternatively, one sequence targeted against multiple RNA targets can be designed when knock-down of more than one RNA target, e.g. RNAs belonging to a family, is desired.

[0096] Another method of preparing the self-complementing polynucleotide including said third sequence involves treating a single stranded polynucleotide consisting essentially of a first polynucleotide sequence covalently linked to a second polynucleotide sequence that includes two nucleotide sequences capable of complementary base pairing and thereby forming a stem-loop structure and that has a 3′ OH terminus, under conditions such that said first sequence serves as a template starting at the 3′ OH terminus for the synthesis of a complementary sequence thereto.

[0097] The polynucleotide produced as a result of the extension reaction using the template or by chemical synthesis comprises a first nucleotide sequence and a third nucleotide sequence covalently linked by a second nucleotide sequence capable of forming a stem-loop structure such that all nucleotides in said first sequence and said third sequences are capable of base pairing with each other. The third sequence complementary to said first sequence is covalently linked to the distal end of said second sequence.

[0098] In a special embodiment of the intermediate polynucleotide of the present invention, said second nucleotide sequence contains at least one nucleotide sequence capable of being cleaved enzymatically. A more preferred embodiment comprises a second sequence have at least two enzymatic cleavage sites. A particularly preferred intermediate polynucleotide comprises a second polynucleotide sequence encoding the stem portion of said stem-loop structure including at least one of said enzymatic cleavage sites.

[0099] Another special embodiment comprises a polynucleotide intermediate wherein at least one enzymatic cleavage site is at the 5′ and/or 3′ ends of said second sequence. Enzymatic cleavage sites consist of nucleotide sequences containing at least four to about eight base pairs and are known to persons skilled in the art. Such sequences may add from about two to about twenty additional nucleotides to the length of the second sequence and be substituted for the complementing nucleotides defining the 5′ and 3′ ends of the loop sequences. Such elongated sequences may consist of from about twelve to about 50 nucleotides. Preferred elongated artificial loop sequences consist of from about ten to about 36 nucleotides.

[0100] The present invention also relates to vector constructs comprising the self-complementing polynucleotide and a promoter sequence positioned upstream of the first sequence of said polynucleotide. The self-complementing DNA polynucleotide sequence of the present invention may be inserted preferably into a plasmid DNA vector, an adenovirus DNA viral vector, an adeno-associated virus vector, or a herpes vector, and the RNA self-complementing polynucleotide may be inserted into preferably into a retrovirus vector. The DNA plasmid vector may be delivered alone or complexed with various vehicles. The DNA, DNA/vehicle complexes, or the recombinant virus particles are locally administered to the site of treatment, as discussed below. Preferably, recombinant vectors capable of expressing the present polynucleotides are locally delivered as described below, and persist in target cells. Once expressed, the self-complementing RNA molecule is processed and is guided to the endogenous target RNA, where it functions to degrade the target RNA.

[0101] Preferred promoter sequences include the microRNA promoters such as the let-7 promoter sequences, and the promoters including the pol III promoters and the pol II promoters. The pol III promoters include those promoters selected from the group consisting of 5S rRNA, tRNAs, VA RNAs, Alu RNAs, HI, and U6 small nuclear RNA promoters. The pol II promoters include those such as CMV, RSV, MMLV, tet-inducible, and IPTG-inducible promoters.

[0102] Promoters that may also be used in the expression vectors of the present invention include both constitutive promoters and regulated (inducible) promoters. The promoters may be prokaryotic or eukaryotic depending on the host. Among the prokaryotic (including bacteriophage) promoters useful for practice of this invention are lacI, lacZ, T3, T7, lambda P _r , P _l , and trp promoters. Among the eukaryotic (including viral) promoters useful for practice of this invention are ubiquitous promoters (e.g. HPRT, vimentin, actin, tubulin), intermediate filament promoters (e.g. desmin, neurofilaments, keratin, GFAP), therapeutic gene promoters (e.g. MDR type, CFTR, factor VIII), tissue-specific promoters (e.g. actin promoter in smooth muscle cells, or Flt and Flk promoters active in endothelial cells), including animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift, et al (1984) Cell 38:639-46; Ornitz, et al. (1986) Cold Spring Harbor Symp. Quant. Biol 50:399-409; MacDonald, (1987) Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, (1985) Nature 315:115-22), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl, et al. (1984) Cell 38:647-58; Adames, et al. (1985) Nature 318:533-8; Alexander, et al. (1987) Mol. Cell. Biol. 7:1436-44), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder, et al. (1986) Cell 45:485-95), albumin gene control region which is active in liver (Pinkert, et al (1987) Genes and Devel. 1:268-76), alpha-fetoprotein gene control region which is active in liver (Krumlauf, et al. (1985) Mol. Cell. Biol., 5:1639-48; Hammer, et al. (1987) Science 235:53-8), alpha 1-antitrypsin gene control region which is active in the liver (Kelsey, et al. (1987) Genes and Devel., 1:161-71), beta-globin gene control region which is active in myeloid cells (Mogram, et al. (1985) Nature 315:338-40; Kollias, et al. (1986) Cell 46:89-94), myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead, et al (1987) Cell 48:703-12), myosin light chain-2 gene control region which is active in skeletal muscle (Sani, (1985) Nature 314:283-6), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason, et al. (1986) Science 234:1372-8).

[0103] Other promoters which may be used in the practice of the invention include promoters which are preferentially activated in dividing cells, promoters which respond to a stimulus (e.g. steroid hormone receptor, retinoic acid receptor), tetracycline-regulated transcriptional modulators, cytomegalovirus immediate-early, retroviral LTR, metallothionein, SV-40, E1a, and MLP promoters.

[0104] In the vector construction, the polynucleotides of the present invention may be linked to one or more regulatory regions in addition to a promoter. Selection of the appropriate regulatory region or regions is a routine matter, within the level of ordinary skill in the art. Regulatory regions other than promoters include enhancers, suppressors, etc.

[0105] In addition to recombinant retrovirus (ssRNA virus) and adenovirus (dsDNA), systems, other viral packaging systems such as ssDNA viruses, for example, adenovirus-associated virus (AAV), are suitable as for use as vector backbone in the present invention. Furthermore, other ssRNA viruses such as, for example, Sindbis virus, HIV, and Semliki Forest viruses, and other dsDNA viruses, such as for example, Epstein Barr virus, herpes simplex virus, baculovirus or vaccinia viruses are useful as vector backbone constructs in the present invention. Each of these systems has a different host range. In the Sindbis virus, of the Alphavirus genus, (Invitrogen, San Diego, Calif.), the polynucleotide is ligated into the multiple cloning site of a Sindbis virus DNA vector, i.e., pSinRepS, operatively linked to the Sindbis subgenomic promoter and polyadenylation site; the polynucleotide replaces the Sindbis virus structural protein genes. For the production of Sindbis virus particles, the recombinant Sindbis vector encoding the oligonucleotide DNA is linearized, transcribed into RNA and co-transfected into vertebrate (BHK-21, Vero) or invertebrate cells (Drosophila) with RNA transcribed from the helper vector, pDH-BB, that encodes the viral structural proteins. Following transfection, the recombinant Sindbis genomic RNA acts as an mRNA, is translated into the Sindbis virus polymerase, and expresses the sRNA from the subgenomic promoter and the structural proteins from the helper RNA. Because of Sindbis virus' host range, the recombinant Sindbis virus can be packaged and used to express the encoded sRNA in mammalian, avian, reptilian, mosquito and Drosophila cells (see for example, Xong, C. et al. (1989) Science 243:1188-1191; Hahn C. S. et al. (1992) Proc. Natl. Acad. Sci. (USA) 89:2679-2683; Huang, H. V. et al. (1993) U.S. Pat. No. 5,217,879; Huang, M. and Sommers, J. (1991) J. Virol. 65:5435-5439). Also Herpes Simplex virus type 1 (HSV-1) can be used. Wild type HSV-1 is a human neurotropic virus, making them especially suitable as a vector for gene transfer to the nervous system. However, non-lytic recombinant HSV-1 has a broad host range. Recombinant HSV-1 viruses can be made replication deficient by deletion of one the immediate-early genes.

[0106] Retroviruses, like murine leukemia virus, are single stranded RNA viruses that are commonly used in the clinical and research area. The major advantage of retroviral gene delivery is their stable integration into target cells. Lentiviruses, like human immunodeficiency virus (HIV), belong to the retrovirus family and have been used as an alternative since lentiviral vectors can infect non-dividing cells as well as dividing cells and integrate with high efficiency (Chang L J, Gay E E, The molecular genetics of lentiviral vectors—current and future perspectives, Curr Gene Ther. 2001 September; 1(3): 237-51). Retroviruses can also be used to transduce sRNA.

[0107] For expression in AAV, the polynucleotide is cloned into an AAV expression vector. To produce recombinant AAV particles, 293 cells are infected with adenovirus type 5; 4 hours later the infected cells are co-transfected with the AAV expression plasmid-oligonucleotide DNA construct and an AAV helper plasmid, pMV/Ad (Samulski et al., (1989) J. Virol. 63:3822-3828). As recombinant AAV is produced, the 293 cells undergo cytopathology, becoming spherical and lose their ability to adhere to a tissue culture surface. Following development of maximal cytopathology the supernatant is harvested and, if necessary, concentrated (Halbert et al. 1997. J. Virol. 71:5932-5941).

[0108] For vaccinia virus expression, a replication competent vaccinia virus can be used. The polynucleotide is operatively linked to a vaccinia virus promoter, for example, P 11. Preferably, vaccinia virus strain MVA is used because it expresses recombinant genes but contains a deletion that renders it replication incompetent in many mammalian cells. Therefore, the polynucleotide can be expressed in target host mammalian cells without the development of vaccinia virus induced cytopathology. The recombinant vaccinia virus is produced by infecting chicken embryo fibroblasts (CEF) with vaccinia and co-transfecting a transfer vector into which has been ligated the polynucleotide of the invention and a marker gene (beta galactosidase) functionally linked to a vaccinia promoter, such as P11, and flanked by genomic sequences. The construct is inserted into the vaccinia genome by homologous recombination. Recombinant viruses can be identified by in situ staining for beta-galactosidase expression with X-gal (Wyatt et al. (1995) Virology 210:202-205).

[0109] Additional vector systems include the non-viral systems that facilitate introduction of DNA encoding the self-complementing single-stranded RNA, or the RNA itself into a patient. For example, a DNA vector encoding a desired sequence can be introduced in vivo by lipofection. Synthetic cationic lipids designed to limit the difficulties encountered with liposome mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Felgner, et. al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7); see Mackey, et al., (1988) Proc. Natl. Acad. Sci. USA 85:8027-31; Ulmer, et al. (1993) Science 259:1745-8). The use of cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Felgner and Ringold, (1989) Nature 337:387-8). Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications WO 95/18863 and WO 96/17823, and in U.S. Pat. No. 5,459,127. The use of lipofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages and directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, for example, pancreas, liver, kidney, and the brain. Lipids may be chemically coupled to other molecules for the purpose of targeting. Targeted peptides, e.g., hormones or neurotransmitters, and proteins for example, antibodies, or non-peptide molecules could be coupled to liposomes chemically. Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, for example, a cationic oligopeptide (e.g., International Patent Publication WO 95/21931), peptides derived from DNA binding proteins (e.g., International Patent Publication WO 96/25508), or a cationic polymer (e.g., International Patent Publication WO 95/21931).

[0110] The more preferred viral vectors useful in the practice of the present invention are the El-deleted adenoviral vectors, with the E1, E2A deleted vectors being most preferred. The more preferred adenoviral vectors include the E1-deleted adenoviral serotype 5 vectors, with the E1, E2A deleted vectors being most preferred. Vectors may also be prepared from other adenoviral serotypes and corresponding packaging cells that include sequences for viral proteins deleted from such vector backbones. The most preferred adenoviral vector/packaging cell combinations are those combinations where the packaging cell and vector do not include any overlapping adenoviral sequences, which overlap would provide the statistical possibility of the production of replication competent adenoviral particles. Preferred packaging cells useful in the production of such vectors include the 293 and 911 cells, with the most preferred cells being the PER.C6 cell line. The modified PER.C6/E2A cell line is a special embodiment complementing the E1, E2A deleted adenoviral vector constructs, with non-overlapping adenoviral E1, E2A sequences, and is most preferred in the practice of the present invention.

[0111] The vectors of the present invention as described herein are also useful in methods of lowering the amounts of RNA or protein translated from RNA in a host cell, or subject, comprising transfecting said cell or subject with a vector that encodes a polynucleotide comprising a promoter operably linked to a first sequence consisting of about 17 to about 23 nucleotides and complementary to about 17 to about 23 nucleotides of said mRNA sequence in said host cell or subject, said first sequence covalently linked to a second sequence capable of forming a loop structure. Preferred vectors according to the present invention comprise the aforesaid first nucleotide sequence and a third nucleotide sequence covalently linked by a second nucleotide sequence capable of forming a stem-loop structure such that all nucleotides in said first sequence and said third sequences are capable of base pairing with each other, and wherein said second nucleotide sequence comprises a stem-loop forming region derived from naturally occurring RNA sequences found in RNA molecules other than mRNA, such as for example, tRNA, snRNA, rRNA, mtRNA, or structural RNA sequences. Preferred stem loop sequences are derived from the let-7 nucleotide sequence, or some portion thereof, or an artificially generated polynucleotide sequence based thereon. The administration of the aforesaid vector to a subject comprises the administration of an amount of vector effective to lower the amounts of said RNA in said transfected cells of said subject.

[0112] The preferred vector of the present invention including a polynucleotide comprising a promoter operably linked to a sequence of a self-complementing polynucleotide, may be prepared by denaturing the self-complementing polynucleotide, converting the resulting denatured polynucleotide into a double stranded polynucleotide, and ligating the double stranded polynucleotide into a vector capable of transfecting a host cell and transcribing said polynucleotide. Alternatively the self-complementing polynucleotide may be chemically synthesized as two single stranded polynucleotides, which are capable of being annealed to each other followed by ligation into the vector. The ligation may be accomplished preferably into an adapter plasmid that may be used to form a transfectable viral vector particle by co-transfection with a helper molecule in a packaging cell line.

[0113] A further embodiment of the vector construct encoding the self-complementing polynucleotide is wherein said second nucleotide sequence contains at least one nucleotide sequence capable of being cleaved enzymatically. A more preferred embodiment comprises a second sequence have at least two enzymatic cleavage sites. In this embodiment, said second sequence, which can be any length, can be removed by enzymatic cleavage and replaced by a stem-loop sequence. In a further embodiment the second nucleotide sequence encodes for a gene useful for the facilitation of cloning stem-loop sequences into the vector. In a further special embodiment the gene useful for the facilitation of cloning stem-loop sequences into the vector is the E. coli ccdB death gene.

[0114] The present vectors may be administered to a patient by a variety of methods. They may be added directly to target tissues, complexed with cationic lipids, packaged within liposomes, or delivered to target cells by other methods known in the art. Localized administration to the desired tissues may be done by catheter, infusion pump or stent, with or without incorporation of the self-complementing polynucleotide in biopolymers. Alternative routes of delivery include, but are not limited to, intravenous injection, intramuscular injection, subcutaneous injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery.

[0115] Preferably, the viral vectors used in the gene therapy methods of the present invention are replication defective. Such replication defective vectors will usually lack at least one region that is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part), or be rendered non-functional by any technique known to a person skilled in the art. These techniques include the total removal, substitution, partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed in vitro (on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with mutagenic agents. Preferably, the replication defective virus retains the sequences of its genome that are necessary for encapsidating the viral particles.

[0116] Certain embodiments of the present invention use retroviral vector systems. Retroviruses are integrating viruses that infect dividing cells, and their construction is known in the art. Retroviral vectors can be constructed from different types of retrovirus, such as, MoMuLV (“murine Moloney leukemia virus” MSV (“murine Moloney sarcoma virus”), HaSV (“Harvey sarcoma virus”); SNV (“spleen necrosis virus”); RSV (“Rous sarcoma virus”) and Friend virus. Lentivirus vector systems such as human immunodeficiency virus (HIV) or equine lentivirus may also be used in the practice of the present invention.

[0117] In other embodiments of the present invention, adeno-associated viruses (“AAV”) are utilized. The AAV viruses are DNA viruses of relatively small size that integrate, in a stable and site-specific manner, into the genome of the infected cells. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies.

[0118] It is also possible to introduce a DNA vector in vivo as a naked DNA plasmid (see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859). Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (see, e.g., Wilson, et al. (1992) J. Biol. Chem. 267:963-7; Wu and Wu, (1988) J. Biol. Chem. 263:14621-4; Hartmut, et al Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990; Williams, et al (1991). Proc. Natl. Acad. Sci. USA 88:2726-30). Receptor-mediated DNA delivery approaches can also be used (Curiel, et al. (1992) Hum. Gene Ther. 3:147-54; Wu and Wu, (1987) J. Biol. Chem. 262:4429-32).

[0119] The present invention, in a particular embodiment, relates to a composition comprising a self-complementing polynucleotide that is used to down-regulate or block the expression of specific polypeptides or specific non-coding RNA molecules. In one preferred embodiment, the nucleic acid encodes a self-complementing sRNA molecule covalently linked by a stem-loop RNA sequence. In this embodiment, the nucleic acid is operably linked to signals enabling expression of the nucleic acid sequence and is introduced into a cell utilizing, preferably, recombinant vector constructs, which will express the polynucleic acid once the vector is introduced into the cell. Examples of suitable vectors include plasmids, adenoviruses, adeno-associated viruses, retroviruses, and herpes viruses.

[0120] The present invention provides biologically compatible compositions comprising the polynucleotides and/or vectors of the present invention. A biologically compatible composition is a composition, that may be solid, liquid, gel, or other form, in which the polypeptide, polynucleotides, vector, or antibody of the invention is maintained in an active form, e.g., in a form able to effect a biological activity. For example, a nucleic acid would be able to replicate, transcribe, translate a message, or hybridize to a complementary nucleic acid; and a vector would be able to transfect a target cell. A preferred biologically compatible composition is an aqueous solution that is buffered using, e.g., Tris, phosphate, or HEPES buffer, containing salt ions. Usually the concentration of salt ions will be similar to physiological levels. Biologically compatible solutions may include stabilizing agents and preservatives. In a more preferred embodiment, the biocompatible composition is a pharmaceutically acceptable composition.

[0121] Such compositions can be formulated for administration by topical, oral, parenteral, intranasal, subcutaneous, and intraocular, routes. Parenteral administration is meant to include intravenous injection, intramuscular injection, and intraarterial injection or infusion techniques. The composition may be administered parenterally in dosage unit formulations containing standard, well-known nontoxic physiologically acceptable carriers, adjuvants, and vehicles as desired.

[0122] Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient. Pharmaceutical compositions for oral use can be prepared by combining active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethyl-cellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinyl-pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e. dosage.

[0123] Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.

[0124] Preferred sterile injectable preparations can be a solution or suspension in a nontoxic parenterally acceptable solvent or diluent. Examples of pharmaceutically acceptable carriers are saline, buffered saline, isotonic saline (e.g. monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, or mixtures of such salts), Ringer's solution, dextrose, water, sterile water, glycerol, ethanol, and combinations thereof. 1,3-butanediol and sterile fixed oils are conveniently employed as solvents or suspending media. Any bland fixed oil can be employed including synthetic mono- or di-glycerides. Fatty acids such as oleic acid also find use in the preparation of injectables.

[0125] The composition medium can also be a hydrogel, which is prepared from any biocompatible or non-cytotoxic homo- or hetero-polymer, such as a hydrophilic polyacrylic acid polymer that can act as a drug absorbing sponge. Certain of them, such as, in particular, those obtained from ethylene and/or propylene oxide are commercially available. A hydrogel can be deposited directly onto the surface of the tissue to be treated, for example during surgical intervention.

[0126] Preferred pharmaceutical compositions of the present invention comprise a replication defective recombinant viral vector and the polynucleotide identified by the present invention. A special embodiment of the composition invention includes also a transfection enhancer, such as poloxamer. An example of a poloxamer is Poloxamer 407, which is commercially available (BASF, Parsippany, N.J.) and is a non-toxic, biocompatible polyol. A poloxamer impregnated with recombinant viruses may be deposited directly on the surface of the tissue to be treated, for example during a surgical intervention. Poloxamer possesses essentially the same advantages as hydrogel while having a lower viscosity.

[0127] The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise a cytotoxic agent, cytokine or growth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0128] The active ingredients of the present invention may also be entrapped in microcapsules prepared, for example, by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.

[0129] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and. gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.

[0130] The present invention provides methods of treatment that comprise the administration to a human or other animal of an effective amount of a composition of the invention. A therapeutically effective dose refers to that amount of the present polynucleotide that ameliorates the symptoms or condition. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

[0131] For any polynucleotide of the present invention, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state, age, weight and gender of the patient; diet, desired duration of treatment, method of administration, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.

[0132] As discussed hereinabove, recombinant viruses may be used to introduce DNA encoding the self-complementing single stranded polynucleotides, as well as the self-complementing single stranded RNA. Recombinant viruses according to the invention are generally formulated and administered in the form of doses of between about 10 ⁴ and about 10 ¹⁴ pfu. In the case of AAVs and adenoviruses, doses of from about 10 ⁶ to about 10 ¹¹ pfu are preferably used. The term pfu (“plaque-forming unit”) corresponds to the infective power of a suspension of virions and is determined by infecting an appropriate cell culture and measuring the number of plaques formed. The techniques for determining the pfu titre of a viral solution are well documented in the prior art.

[0133] Self-complementing polynucleotides according to the present invention may be administered in a pharmaceutically acceptable carrier. Dosage levels may be adjusted based on the measured therapeutic efficacy.

[0134] In another embodiment, the self-complementing polynucleotide is synthesized and may be chemically modified to resist degradation by intracellular nucleases. Synthetic oligonucleotides can be introduced to a cell using liposomes. Cellular uptake occurs when an oligonucleotide is encapsulated within a liposome. With an effective delivery system, low, non-toxic concentrations of the polynucleotide molecule can be used to degrade the target RNA. Moreover, liposomes that are conjugated with cell-specific binding sites may direct a polynucleotide to a particular tissue.

[0135] In another aspect of the present invention, the polynucleotide vector is transferred into the target tissue using one of the vector delivery systems herein. This transfer is carried out either ex Vivo in a procedure in which the nucleic acid is transferred to cells in the laboratory and the modified cells are then administered to the human or other animal, or in vivo in a procedure in which the nucleic acid is transferred directly to cells within the human or other animal. In preferred embodiments, an adenoviral vector system is used to deliver the expression vector. If desired, a tissue specific promoter is utilized in the expression vector as described above.

[0136] Non-viral vectors may be transferred into cells using any of the methods known in the art, including calcium phosphate co-precipitation, lipofection (synthetic anionic and cationic liposomes), receptor-mediated gene delivery, naked DNA injection, electroporation and bio-ballistic or particle acceleration.

[0137] The present invention may be used in in vitro validation of drug targets, screening for novel drug targets by knocking down genes in cellular assays, and in animal studies for in vivo target validation development of therapeutics.

[0138] The subject invention relates also to methods and compositions for the high throughput delivery and expression in a host of guide nucleic acid(s) targeting RNA of known or unknown function. Methods are described for infecting a host with the adenoviral vectors that express the self-complementing RNA molecules including the guide nucleic acid(s) in the host, identifying an altered phenotype induced in the host by the knockdown of the target RNA nucleic acids, and thereby assigning a function to the product(s) encoded by the target nucleic acids. The methods can be fully automated and performed in a multiwell format to allow for convenient high throughput analysis of sample nucleic acid libraries, which samples code for the guide sequences for use in this method.

[0139] The present invention may be used to prepare libraries of vectors, consisting essentially of the polynucleotide constructs as described herein. These libraries may be prepared as single element, compartmentalized, or discrete elements, wherein each element consists essentially of a vector coding for a unique nucleotide sequence. Alternatively, a library comprising pools of vectors may be prepared.

[0140] The libraries may be used to assist in the elucidation of the functions of host cell RNA molecules including the unique polynucleotide residing in each compartment of said library, or in other words, determining the function of a naturally occurring polynucleotide sequence comprising transfecting a host cell with a vector according to the invention, said vector including a polynucleotide sequence complementary to a portion of said naturally occurring polynucleotide and detecting a change in cellular phenotype. Each vector in the library may be introduced into one or more cells and changes in protein expression, or phenotype observed. The vectors may comprise plasmids, naked RNA, or included in a viral vector construct. Alternatively, more than one vector thereby introducing more than one guide sequence can be introduced into a single host cell. Preferred viral vectors include the adenoviral, retroviral and AAV-vectors. More preferred are the adenoviral vectors, and most preferred are the adenoviral vectors that comprise a replication deficient construct that may be multiplied in a packaging cell having complementary sequences to the sequence contained in the vector itself.

[0141] The present invention provides for the temporary knock-down of proteins, such as lethal proteins, during virus production, thereby allowing the replication and packaging of virus that include sequences encoding for lethal proteins. sRNA can be used to knock-down gene expression during viral production with any virus and in any viral packaging cell line. Accordingly, the present invention relates to a method of producing viral vectors encoding a toxic protein comprising

[0142] (a) introducing into a cell a polynucleotide sequence as described herein having a first sequence that is complementary to the mRNA coding for said toxic protein,

[0143] (b) introducing said viral vector into said cell,

[0144] (c) culturing said cells under conditions allowing expression of said polynucleotide sequence and replication of said viral vector, and

[0145] (d) recovering said viral vectors.

[0146] A preferred method of the present invention uses viral packaging cells that are stably transfected with said polynucleotide.

[0147] Viral production using adenovirus retrovirus or alphavirus benefit from such knock down methods, and examples of packaging cells include adenoviral packaging cells, such as PER.C6 cells, and derivatives thereof, HEK293 cells, 293 and 911 cells, among others. Furthermore, sRNA knock-down methodology is useful for improving recombinant protein production. Such protein production methods benefit from the down-modulation of heterologous protein expression prior to achieving the optimal production cell titre for protein production.

[0148] The present invention may be applied to every viral packaging and protein production system without a need for optimization. Knock-down constructs described herein may be transfected into any selected packaging cell and such transfected cells are used directly. The present invention uses virus constructs that are used directly to infect cells and no further compound is required by the system to induce virus or protein production.

[0149] The sequences between transcription start and the 5′ end of the expression cassette that is used to express exogenous genes can be used to knock down the expression of those exogenous genes during virus production. Polynucleotides of the present invention, which polynucleotides includes guiding sequences targeted against the sequence between transcription start and the 5′ end of the expression cassette, are co-transfected with the viral plasmid(s) carrying the sequence for the toxic protein, into the packaging cells. Alternatively, a vector according to the invention, encoding polynucleotides that down-modulate expression of target sequences, either by inhibiting translation or by break down of the mRNA, are used. The vector can be used transiently or used to generate a stable derivative of the packaging cell line.

[0150] The various aspects of the present invention are further described in the following non-limiting examples.

EXAMPLES

Example 1

A reporter Assay System Based on let-7 Target Sequence to Monitor Repression

[0151] Example 1 describes development of a reporter assay system that provides a method for measuring knockdown of a readily assayed gene. This system is used to determine if siRNAs and chimeric RNAs can decrease expression of the readily assayed luciferase gene. The system consists of two components. The first component is a reporter DNA molecule based on the pGL3 luciferase reporter vector (available from Promega), which has been modified to include a let-7 target sequence derived from the human let-7 sequence found on chromosome 22. These reporter constructs are designated as follows: The names start with a ‘p’ indicating that the construct is in a plasmid, then the name of the reporter gene follows (e.g. GL3 or GL2), after that the target sequence is mentioned starting with a ‘t’ to indicate that it is the target sequence. For example: pGL3-tLet7 describes a plasmid containing the GL3 gene as reporter and Let7 sequences as target for the knockdown RNA. The second component is a siRNA or a plasmid expressing siRNA or chimeric RNAs. siRNAs are double stranded short interfering RNA molecules no larger than about 23 nucleotides in length. Chimeric RNAs as used herein refer to an RNA molecule constructed from at least two polynucleotide sequences that are covalently linked together and derived from at least two different RNA molecules that may be from the same or different species. The scientific literature describes siRNA as mediating the sequence specific degradation of a target mRNA. In the present application these siRNAs are designated as follows: siRNA followed by the name of the target gene, e.g. siRNA GL3.1 is a duplex siRNA targeted against the GL3 gene. In this example a reporter construct, a siRNA, and an internal control used to normalize luciferase activity (Renilla: PRL-TK) are combined together and used to transfect host cells and the luciferase activity measured. If the siRNA knocks down expression of luciferase mRNA, a reduction of luciferase activity is seen relative to controls. The reporter system is described below. The description is meant only as an example and is in no way limiting to the invention.

[0152] The reporter system is based on the pGL3 luciferase reporter vector (Promega). The let-7 target DNA sequence (5′-ACTATACAACCTACTACCTCA-3′ SEQ ID NO: 1) is introduced just outside of the GL3 coding region in a pGL3-reporter vector, such that it expresses a GL3 mRNA that contains the let-7 target sequence.

[0153] A. Construction pGL3-tLet-7 Reporter Constructs.

[0154] The pGL3-control vector (GenBank Accession Number U47296) is linearized at its unique Xba I site, which is immediately 3′ of the GL3 coding sequence. The double stranded let-7 target DNA sequence is generated using complementary DNA oligonucleotides (Oligo 1 and Oligo 2). In order to facilitate cloning into the Xba I site of pGL3, Oligo 1 and Oligo 2 are designed such that upon annealing, the double stranded let-7 target DNA thus generated has 5′ overhangs on each end that are compatible with an Xba I restriction site. Annealing of Oligo 1 and Oligo 2 is accomplished by mixing the oligos in equimolar amounts to a final concentration of 0.5 nmole/μl each in annealing buffer [10 mM Tris-HCl (pH 7.9), 10 mM MgCl ₂ , 50 mM NaCl], followed by incubation of the mixture for 1 minute at 90° C. and 60 minutes at 37° C. The annealed oligos are then ligated into the Xba I site of the linearized pGL3 vector using common techniques as described in Sambrook et al.

[0155] The Let-7 target DNA sequence is SEQ ID NO: 1:

[0156] 5′-ACTATACAACCTACTACCTCA-3′

[0157] Sequences DNA Oligos (5′ to 3′): 1

[0158] The annealed Oligos 1 and 2 give the following double stranded structure: 2

[0159] Underlined nucleotides denote the Xba I compatible ends. The cloning of the oligos into the vector results in an extra Sca I site (bolded nucleotides), which facilitates the selection of clones with the insert and enables the discrimination between clones with an insert in the forward (F) orientation or clones with an insert in the reverse (R) orientation. The original Xba I site from the starting pGL3-control vector is destroyed by the cloning process and thus is absent in the clones with inserts. The clones are tested for presence of the insert by performing PCR on the transformed bacteria directly, using the following primers:

[0160] OLIGO 3 5′-CATCTTCGACGCAGGTGTCGCA-3′ (SEQ ID NO: 4) (position 1668-1689 according to Promega catalog and U47296 sequence)

[0161] OLIGO 4 5′-CCATCGTTCAGATCCTTATCGA-3′ (SEQ ID NO: 5) (position 2210-2189 according to Promega catalog and U47296 sequence)

[0162] The given positions are based on the pGL3-control vector (Accession number U47296).

[0163] Using Oligo 3 (SEQ ID NO: 4) and Oligo 4 (SEQ ID NO: 5) as primers and colony DNA as template, PCR products generated from clones without an insert are 543 base pairs; with an insert in either forward (F) or reverse (R) orientation PCR products are 569 base pairs.

[0164] The orientation of the insert is analyzed further by a second round of PCR using two primer combinations:

[0165] 1) Oligo 1 (SEQ ID NO: 2) and Oligo 3 (SEQ ID NO: 4): PCR with let-7 target sequence in R orientation produces a DNA fragment of 297 bp; F orientation will produce no DNA product.

[0166] 2) Oligo 1 (SEQ ID NO: 2) and Oligo 4 (SEQ ID NO: 5): PCR with let-7 target sequence in F orientation gives a DNA fragment of 302 bp; R orientation will produce no DNA product.

[0167] The plasmid generated by successful cloning of let-7 target DNA sequences in the forward orientation into the Xba I site of pGL3 will be known as pGL3-tlet-7F. Similarly, the plasmid generated by successful cloning of let-7 target DNA sequences in the reverse orientation into the Xba I site of pGL3 will be known as pGL3-tlet-7R. Both clones pGL3-tlet-7F and pGL3-tlet-7R are used in further experiments.

[0168] B. siRNAs

[0169] The siRNAs that target pGL3 and pGL2 (used as a negative control; Accession Number X65324) are as described in Elbashir et al. (2001) Nature 411:494-498. siRNAs are double stranded RNAs that include the target sequence and its complement. Two uridine residues are added to the 3′ end of the RNAs. 3

[0170] The siRNA that targets the let-7 target sequence is: 4

[0171] Each RNA oligo pair is annealed as described in Elbashir et al. (2001) Nature 411:494-498 in order to obtain the duplexed siRNA.

[0172] C. Co-Transfection of pGL3-Reporter Constructs and sIRNAs

[0173] The let-7 targeting system is tested by transfecting a DNA/RNA mixture consisting of three components into host cells (for example HeLa or PER.C6/E2A cells):

[0174] 1. Luciferase-based reporter construct

[0175] a. pGL3-control (Promega), or

[0176] b. pGL3-tLet-7F, or

[0177] c. pGL3-tLet-7R

[0178] 2. Internal control for normalization

[0179] a. pRL-TK (Promega; Acc. Number AF025846)

[0180] 3. Duplexed siRNA

[0181] a. siRNA-GL3.1, or

[0182] b. siRNA-let-7.1, or

[0183] c. siRNA-GL2.1

[0184] Day 1:

[0185] HeLa or PER.C6/E2A cells are seeded 20 hrs prior to transfection in 96-well format at 4.5×10 ⁴ cells/100 μl medium (DMEM+10% heat inactivated Fetal Bovine Serum for HeLa cells; DMEM+10% non-heat inactivated Fetal Bovine Serum for PER.C6/E2A cells)/well.

[0186] Day 2:

[0187] Per well DNA/RNA mixtures are prepared in 25 μl (total volume) OptiMEM containing:

[0188] 1. 0.25 μg pGL3-control, or

[0189] 0.25 μg pGL3-tLet-7F, or

[0190] 0.25 μg pGL3-tLet-7R

[0191] 2. 25 ng pRL-TK

[0192] 3. 66.5 ng siRNA-GL3.1, or

[0193] 66.5 ng siRNA-let-7.1, or

[0194] 66.5 ng siRNA-GL2.1, or

[0195] no siRNA

[0196] LipofectAMINE2000 (0.8 μl) and OptiMEM (24.2 μl) are incubated for 7-10 minutes at room temperature and added to each DNA/RNA mixture. This mixture (final volume 50 μl) is incubated for 15-25 minutes at room temperature and subsequently added to the cells from which the medium has been removed. The cells are incubated for 48 hrs in a 37° C. incubator under 10% CO ₂ .

[0197] Day 4:

[0198] The cells are harvested, lysed, and firefly luciferase and renilla luciferase activities measured using the Dual Luciferase kit (Promega) according to the manufacturer's instructions. The absolute firefly luciferase value (luc) of each sample is divided by its internal absolute renilla luciferase value (ren) to obtain the relative luc/ren-value. These relative luc/ren-values are compared to the control sample where no siRNA is included.

[0199] The results of transient transfection on PER.C6/E2A are shown in FIG. 1 . It shows the repression of luciferase activity of pGL3-fusion constructs containing let-7 target sequences by let-7 siRNAs in PER.C6/E2A cells. PER.C6/E2A cells are transiently transfected with the pGL3-fusion constructs containing let-7 target sequences in either orientation (pGL3-tLet-7F, pGL3-tLet-7R) or pGL3-control lacking let-7 sequences, in combination with each of the siRNA duplexes siRNA GL3.1, siRNA let-7.1, siRNA GL2.1, or no siRNA. Co-transfection of siRNA let-7.1 specifically represses luciferase activity of the reporters pGL3-tLet7F and pGL3-tLet7R, but not the pGL3-control. Co-transfection of the positive control siRNA GL3.1 shows repression of all the reporter constructs (pGL3-tLet-7F, pGL3-tLet-7R, pGL3-control).

Example 2

Testing Chimeric let-7 RNAs

[0200] This example describes preparation of let-7-based chimeric RNAs, which are tested for the ability to knock down gene expression in the system described in Example 1. The two complementary RNA strands of the siRNA-duplexes of Example 1 are covalently linked using an RNA loop structure, making a single RNA molecule containing both siRNA strands. This results in a molecule folding into an RNA-duplex with a loop structure on one side of the duplex and a 3′ overhang of 2 uridine residues on the other side of the duplex. Molecules containing this loop structure and the sequences are referred to as chimeric RNAs. The constructs are referred to as follows: loop RNA followed by the gene they are targeted against, e.g. loop RNA GL2.2 is a chimeric RNA molecule containing a loop directed against GL2. The extension ‘.2’ is to indicate that the RNA contains a loop in contrast to the extension ‘.1’ used in Example 1 indicating a duplex RNA without a loop structure. The loop structure used here is the let-7 loop and meant as an example and in no way intended to limit to the invention.

[0201] Such a chimeric RNA molecule is shown below (see also sR-hLet7.2-as below). The complementary RNA regions are shown in uppercase, while the loop region and 3′ uridines are shown in lowercase. The loop region is also underlined.

[0202] Linear