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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • 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
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/11—Antisense
  • C12N2310/111—Antisense spanning the whole gene, or a large part of it
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]

Definitions

• the present invention relates to the field of gene expression, in particular to inhibiting the expression of a specific isoform of a gene in mammalian cells using double-stranded RNA and to expressing a specific isoform in a clean back ground.
• the double-stranded RNA based technology of the invention has wide applications, such as for determining function of a particular gene isoform or developing therapeutic methods for treating diseases.
• RNA interference RNA interference
• PTGS The mechanism of PTGS involves enhanced mRNA degradation with double-stranded (ds)RNA as the trigger (Cogoni and Macino, 2000, Curr. Opin. Genet. Dev. 10, 638-643; Carthew, 2001, Curr. Opin. Cell Biol. 13, 244-248). A similar phenomenon (quelling) has been observed in Neurospora (Cogoni et al., 1994, Antonie Van Leeuwenhoek 65, 205-209).
• RNAi The mechanism underlying RNAi has been partially elucidated and a 21- to 23-nt-long dsRNA was found to be the intermediate/mediator of mRNA decay (Zamore et al., 2000, Cell 101 , 25-33; Bernstein et al., 2001 , Nature 409, 363-366).
• RNAi RNA interference
• siRNA small interfering double-stranded RNA
• the authors describe that transfection of 21-nucleotide dsRNA (siRNA) can trigger PTGS of both the co-transfected and the endogenous gene in cultured mammalian cells.
• RNA was shown to be cleaved in Drosophila embryonal cell extracts within the region of identity with the dsRNA (Zamore et al., 2000).
• the potency of RNAi in worms and flies suggests that dsRNA acts catalytically and/or is amplified (Zamore, 2001 , Nature Structural Biology 8, 746-750).
• siRNAs are not replicated by endogenous RNA-dependent RNA polymerase in C.
• siRNAs indeed act as primers for cellular polymerases and are extended to form ds RNAs in both C.elegans and Drosophila (Lipardi et al., 2001 , Cell 107, 297-307; Sijen et al., 2001 , Cell 107, 465-476; Nishikura, 2001 , Cell 107, 415-418). Propagation of RNAi to other regions of the mRNA in this way would result in the spread of gene silencing to related sequences, thereby not allowing isoform-specific dsRNA inhibition.
• RNAi double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.
• Cell 101 25-33 Bernstein, E., Caudy, A. A., Hammond, S. M. and Hannon, G. J. (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference.
• RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs.
• the present invention provides a method of expressing a desired isoform of a gene product in a cell absent undesired isoforms of the gene product, comprising the steps of:
• the nucleic acid encoding the desired isoform having a sequence comprising one or more mismatches relative to the double- stranded portion of the nucleic acid, operably linked to a promoter capable of driving expression of the desired isoform in the cell.
• the common nucleic acid sequence is at least 19 consecutive nucleotides in length and is common to all endogenous isoforms of the gene product in the cell of interest.
• the nucleic acid is preferably 19 to 25 nucleotides long and the double-stranded portion of the nucleic acid is preferably 100% identical to the common nucleic acid sequence.
• the at least partially double-stranded ribonucleic acid preferably contains a double-stranded portion of at least 19 nucleotides, more preferably of 19 nucleotides, and at least one single-stranded 3' overhang of two-nucleotides, preferably both upper and lower strands having a single- stranded 3' overhang of two-nucleotides.
• the desired isoform is encoded by a sequence comprising at least one mismatch relative to the double-stranded portion of the nucleic acid to ensure that the nucleic acid of at least partially double stranded RNA character does not affect expression of the desired isoform.
• the desired isoform can therefore be encoded by a sequence having one, two or more mismatches relative to the double- stranded portion of the nucleic acid as long as the altered coding sequence still encodes the desired product.
• at least one codon e.g., two or more
• encoding the desired isoform may differ from the endogenously used codon at the same position. It is well known in the art that many amino acids are designated by more than one triplet codon.
• the desired isoform has an identical protein sequence to the corresponding endogenous isoform (unless an endogenous wild-type sequence is used to correct an endogenous mutant isoform, in which case the wild-type sequence is used), although amino acid substitutions, deletions or additions to the isoform sequence may be tolerated, in particular conservative substitutions.
• the desired isoform replaces a mutant isoform expressed by the cell, allowing correct gene function.
• the mutant isoform can be oncogenic, apoptotic, tumor suppressive, inflammation inducive or suppressive, or angiogenic, for example.
• the method of the invention is used to determine the function of the desired isoform (i.e, isoform of unknown function).
• the method is useful in all types of mammalian cells, including cancer cells, such as HeLa (cervical cancer), PC3 (prostate cancer), MDA-MB-231 (breast cancer) and MCF-7 (breast cancer), or cells in vivo.
• cancer cells such as HeLa (cervical cancer), PC3 (prostate cancer), MDA-MB-231 (breast cancer) and MCF-7 (breast cancer), or cells in vivo.
• the desired isoform can be transcribed under the control of any promoter, such as an endogenous promoter, a constitutive promoter, an inducible promoter or a tissue-specific promoter.
• the invention also provides methods of assigning function to a desired isoform of unknown (or unrecognized) function. Also provided are kits and materials for carrying out the invention, as well as cells exhibiting isoform-specific expression in a clean genetic background. The invention also encompasses therapeutic uses and compositions based on the methods of the invention.
• the present invention provides a method of expressing a desired isoform of a gene product in a cell absent undesired isoforms of a gene product, by exposing a mammalian cell to at least one nucleic acid, the nucleic acid being at least a partially double-stranded ribonucleic acid and the double- stranded portion having at least 95% sequence identity to a common nucleic acid sequence shared by two or more isoforms of the gene product; and introducing an expression vector encoding a desired isoform of the gene product into the mammalian cell, the desired isoform having a sequence comprising one or more mismatches relative to the double-stranded portion of the nucleic acid, operably linked to a promoter capable of driving expression of the desired isoform in said cell.
• the nucleic acid will typically be a ribonucleic acid (RNA) that in double-stranded form has at least 95%, preferably 98% most preferably 100% sequence identity to a common nucleic acid sequence shared by two or more isoforms of the gene product.
• Preferred RNA molecules for inhibition comprise sequences identical to a common nucleic acid sequence shared by two or more isoforms of the gene product over at least 15 -25 consecutive bases, preferably over at least 19 consecutive bases.
• the common nucleic acid sequence is common to all endogenous isoforms of the gene product expressed by the cell of interest.
• the RNA sequence is preferably chosen to have identity with exon sequences. Gene expression is inhibited in a sequence-specific manner in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for inhibition.
• Sequence identity (and therefore specificity) may be optimized and common nucleic acid sequence determined by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991 , and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith- Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Sequence specificity is important to ensure that the nucleic acid does not cross- react and affect expression of other unrelated gene sequences.
• the nucleic acid will typically be relatively short for use in mammalian cells to allow efficient delivery of the nucleic acid to the cell and also to avoid any protein kinase R (PKR) response of the cell.
• PLR protein kinase R
• the nucleic acid is at least partially double-stranded RNA in character and will typically be 18 to 25 nucleotides long.
• the double-stranded RNA character is provided by sequences at least 18 nucleotides long, preferably 19 nucleotides in length, which are preferably 100% identical to the common nucleic acid sequence shared by two or more isoforms of the desired gene.
• the nucleic acid comprises at least one single-stranded 3' overhang of two-nucleotides, preferably both upper and lower strands having a single-stranded 3' overhang of two-nucleotides.
• the at least partially double-stranded RNA can be formed by a self- complementary RNA strand (such as a transcript having an inverted repeat), or by two or more complementary RNA strands. RNA duplex formation may be initiated either inside or outside the cell.
• the RNA is introduced in an amount that allows delivery of at least one copy per cell, preferably at least 5, 10, 100, 500 or 1000 copies per cell, depending on the application. The amount introduced is dependent on the desired effect and can be easily determined empirically.
• the nucleic acid may comprise nucleotides or linkages other than those that occur naturally in ribonucleic acid, for example, to stabilize the dsRNA from degradation, especially when RNA is delivered to a cell and not produced by the cell.
• oligoribonucleotides or oligonucleotides that comprise one or more modified (i.e., synthetic or non-naturally occurring) nucleotides.
• nucleotide monomers in a nucleic acid are linked by phosphodiester bonds or analogues thereof.
• Analogues of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, peptide, and the like linkages.
• the reagents employed are commercially available or, in the case of the oligonucleotides, can be prepared using commercially available instrumentation.
• the duplex RNA will comprise ribonucleotide units or other nucleotide units that allow appropriate processing by the cell and efficient inhibition of the isoforms expressed by the cell.
• the nucleic acid may be synthesized either in vivo or in vitro, particularly in vitro when short sequences are employed. Endogenous RNA polymerase of the cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vivo or in vitro, essentially as described below.
• a mammalian cell is exposed to the nucleic acid described above to inhibit expression of specific isoforms expressed in a cell of interest, preferably to inhibit all related isoforms.
• the term "isoform" is well known in the art and is meant to encompass gene products that are produced as a result of differential gene splicing as well as from the use of alternative transcription and translation start sites.
• the term isoforms include any closely related sequences and therefore may include a mutated gene in a cell, such as deleterious point mutations and the like.
• the mutant isoform can be oncogenic, apoptotic, tumor suppressive, inflammation inducive or suppressive, or angiogenic, for example, and its deleterious function corrected by the method of the invention.
• ShcA any gene product produced in multiple forms can be targeted using the methods of the present invention.
• proteins may be therapeutically important proteins, such as enzymes, e.g., kinases (PKB), ras, integrins, E2F, Rb, FGF, other signaling molecules and transcription factors.
• dsRNA The effect of dsRNA on gene expression will typically result in expression of the target isoforms being inhibited by at least 10%, 33%, 50%, 90%, 95% or 99% or more when compared to a cell not treated by this step.
• the mammalian cell can be any cell of interest.
• the cell may be cells from the inner cell mass, extraembryonic ectoderm or embryonic stem cells, totipotent or pluripotent, dividing or non-dividing, parenchyma or epithelium, immortalized or transformed, or the like.
• the cell may be a stem cell or a differentiated cell.
• Cell types that are differentiated include without limitation adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium, dendritic cells, neurons, glia, mast cells, blood cells and leukocytes (e.g., erythrocytes, megakaryotes, lymphocytes, such as B, T and natural killer cells, macrophages, neutrophils, eosinophils, basophils, platelets, granulocytes), epithelial cells, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of the endocrine or exocrine glands, as well as sensory cells.
• leukocytes e.g., erythrocytes, megakaryotes, lymphocytes, such as B, T and natural killer cells, macrophages, neutrophils, eosinophils, basophils, platelets, granulocytes
• epithelial cells ker
• cells that are easily cultured are preferred. These may include cancer cells, for example, including HeLa (cervical cancer), PC3 (prostate cancer), MDA-MB-231 (breast cancer) and MCF- 7 (breast cancer) cells. It will be apparent to one of ordinary skill in the art that the teachings of the specification can easily be applied to other situations, such as cancer cells for the replacement of a mutated "isoform" with one or more desired isoform(s).
• an expression vector encoding a desired isoform of the gene product is introduced into the mammalian cell, the desired isoform having a sequence comprising one or more mismatches relative to the double-stranded portion of the nucleic acid, operably linked to a promoter capable of driving expression of the desired isoform in said cell.
• the nucleic acid encoding the desired isoform preferably comprises a sequence comprising one or more mismatches, preferably two or more, relative to the double-stranded portion of the nucleic acid used to inhibit expression of the endogenous isoforms to ensure that the nucleic acid (RNAi) does not inhibit expression of the desired isoform.
• the expression vector encodes a desired isoform using at least one codon, more preferably at least two codons, that differ(s) from the endogenous sequence coding the corresponding isoform.
• Changes in protein sequence between the endogenous isoform and desired isoform are also encompassed by the present invention, in particular those substitutions, deletion or additions that do not affect gene function, which are preferably conservative substitutions.
• the desired isoform has an identical protein sequence to the corresponding endogenous isoform (although the coding sequence will be different due to codon usage).
• an expression construct comprising at least one regulatory region (e.g., promoter, enhancer, silencer, splice donor and acceptor and polyadenylation signal) operably linked to the DNA coding for the desired RNA transcript(s) may be used to transcribe the RNA strand (or strands).
• the promoter can be of almost any origin. Preferred are promoters that are active in the chosen host cells like the SV40, beta-actin, metallothionein, T7, polyhedrin and cytomegalovirus promoters.
• the promoter can be a constitutive promoter, an inducible promoter or a tissue-specific promoter, for example, allowing inhibition to be targeted to an organ or cell type; or transcription to be induced upon stimulation of an environmental condition (e.g., infection, stress, temperature, chemical inducers); and/or engineering transcription at a developmental stage or age.
• a knock-in construct can be used to transcribe the nucleic acid of interest under the control of an endogenous promoter, as is known in the art.
• Modified or unmodified RNA can also be chemically or enzymatically synthesized by manual or automated reactions as described above.
• the RNA may be synthesized by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g., T3, T7, SP6).
• Expression vectors may also include sequences allowing for their autonomous replication within the host organism, sequences that encode genetic traits that allow cells containing the vectors to be selected, and sequences that increase the efficiency with which the RNA is transcribed. Stable long-term vectors may be maintained as freely replicating entities by using regulatory elements of viruses. Cell lines may also be produced which have integrated the vector into the genomic DNA and in this manner the transcript(s) is/are produced on a continuous basis.
• an expression vector can further include, if desired, additional sequences operably linked to a promoter, such as a reporter gene (e.g., fluorescent proteins, e.g., green fluorescent protein, ⁇ -galactosidase, alkaline phosphatase, luciferase, CAT, selective gene markers that facilitate the selection of transformants due to the phenotypic expression of the marker gene (e.g., those expressing antibiotic resistance or, in the case of auxotrophic host mutants, genes which complement host lesions), or other useful sequences.
• a reporter gene e.g., fluorescent proteins, e.g., green fluorescent protein, ⁇ -galactosidase, alkaline phosphatase, luciferase, CAT, selective gene markers that facilitate the selection of transformants due to the phenotypic expression of the marker gene (e.g., those expressing antibiotic resistance or, in the case of auxotrophic host mutants, genes which complement host lesions), or other useful sequences
• Nucleic acids can be introduced into a cell by various standard methods in genetic engineering, including physical methods, for example, simple diffusion, by injection of a solution containing the nucleic acid, bombardement by particles covered by the nucleic acid, soaking the cell or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the nucleic acid.
• a particularly preferred method for delivering nucleic acids is the use of electroporation.
• a viral construct accomplishes both efficient introduction of an expression construct into a cell and transcription of RNA encoded by the expression construct.
• lipid-mediated delivery systems such as lipid-mediated delivery systems, chemical mediated transport, such as calcium phosphate transfection, DEAE-dextran transfection, and the like.
• the transfected host cells can be cultured by standard methods in cell culture.
• the methods of the present invention are particularly useful for determining the function of a particular isoform.
• the method further comprises identifying a phenotype of the mammalian cell compared to when the desired isoform is absent, and assigning the phenotype or function to the desired isoform.
• the invention also provides a kit comprising reagents useful in the methods of the invention, which may include a nucleic acid being at least a partially double- stranded ribonucleic acid and the double-stranded portion having at least 95% sequence identity to a common nucleic acid sequence shared by two or more isoforms of the gene product; and an expression vector encoding a desired isoform of the gene product, the desired isoform having a sequence comprising one or more mismatches relative to the double-stranded portion of the nucleic acid, operably linked to a promoter capable of driving expression of the desired isoform in said cell.
• reagents useful in the methods of the invention may include a nucleic acid being at least a partially double- stranded ribonucleic acid and the double-stranded portion having at least 95% sequence identity to a common nucleic acid sequence shared by two or more isoforms of the gene product; and an expression vector encoding a desired isoform of the
• a mammalian cell exhibiting isoform-specific expression achieved by any of the methods of the invention is also provided.
• the isoform may be used to correct aberrant isoforms
• a method for treating a disease comprising administering to a subject in need of treatment an effective amount of a nucleic acid being at least a partially double- stranded ribonucleic acid and the double-stranded portion having at least 95% sequence identity to a common nucleic acid sequence shared by two or more isoforms (in particular the aberrant isoform) of the gene product; and an expression vector encoding a desired isoform of the gene product, the desired isoform having a sequence comprising one or more mismatches relative to the double-stranded portion of the nucleic acid, operably linked to a promoter capable of driving expression of the desired isoform in said cell. Therefore.
• the invention provides the use of the nucleic acid as hereinabove described for the treatment of aberrant isoform expression and for the manufacture of
• compositions of the invention may be accomplished orally or parenterally.
• Methods of parenteral delivery include topical, intra-arterial (e.g. directly to the tumour), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.
• these pharmaceutical compositions can contain suitable pharmaceutically acceptable carriers comprising excipients and other compounds that facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration can be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co, Easton PA).
• 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, etc, suitable for ingestion by the patient.
• compositions for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores.
• Suitable excipients are carbohydrate or protein fillers include, but are not limited to 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 carboxymethylcellulose; and gums including arabic and tragacanth; as well as proteins such as gelatin and collagen.
• 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 can be provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, 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 characterise the quantity of active compound (i.e. dosage).
• compositions which 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 a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers.
• the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
• Pharmaceutical formulations for parenteral administration include aqueous solutions of active compounds.
• the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline.
• Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
• suspensions of the active compounds can be prepared as appropriate oily injection suspensions.
• Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
• the suspension can also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
• penetrants appropriate to the particular barrier to be permeated are used in the formulation.
• penetrants are generally known in the art.
• the signaling adaptor/scaffold protein ShcA is a member of the She family, which consists of three genes, ShcA, ShcB/Sli/Sck and ShcC/N-Shc/Rai (Luzi et al., 2000, Curr. Opin. Genet. Dev. 10, 668-674).
• ShcA is ubiquitously expressed, whereas ShcB and ShcC are expressed specifically in the brain (Nakamura et al., 1998, J. Biol. Chem. 273, 6960-6967).
• ShcA There are three isoforms of ShcA, p66ShcA, p52ShcA and p46ShcA, derived from a single gene through differential usage of transcription initiation sites (p66 versus p52/p46) and translation start sites (p52 versus p46), which differ only in the amino terminal regions (Luzi et al., 2000).
• ShcA was chosen to exemplify the invention as different cellular functions have been suggested for each isoform and therefore is of scientific interest to obtain isoform-specific expression of ShcA.
• the invention is not limited in any way to this particular gene family.
• the primary transcript of p52/p46 mRNA contains the entire sequence of p66 mRNA; however, the very 5' region of p66 mRNA is present in the first intron of p52/p46 mRNA but is absent in the latter mRNA, having been spliced out.
• the p46 and p52 isoforms are derived from the same mRNA using different translation initiation sites.
• the p66-siRNA site is in the 5' region only present in p66 ShcA mRNA, while h/m-shc siRNA site is in the second exon which is present in both mRNAs. Note that the sequence of p66-shc siRNA is from a 5' region of human p66ShcA mRNA and is absent in p52/46 ShcA mRNA.
• DMEM Dulbecco's Modified Eagle's Medium
• AMIMED fetal calf serum
• streptomycin 50 units/ml penicillin at 37°C in a humidified C0 2 (5%) incubator.
• DMEM Dulbecco's Modified Eagle's Medium
• streptomycin 0.2 mg/ml streptomycin
• penicillin 50 units/ml penicillin at 37°C in a humidified C0 2 (5%) incubator.
• One day before transfection with siRNA cells were plated in 6-well plates in media without antibiotics at 1.4x10 5 cells per well.
• siRNA h/m-shc siRNA from nt 677-697 (in the PTB domain), 5'-CUA CUU GGU UCG GUA CAU GGG-3' (SEQ ID NO:1) and 5'-CAU GUA CCG AAC CAA GUA GGA-3' (SEQ ID NO:2).
• Sequences were derived from the sequence of human p66ShcA mRNA (accession number: HSU7377) and its complement and each pair has a 3' overhang of 2 nt on each side.
• Designed RNA oligonucleotides were blasted against Database (GEMBL) to ensure gene specificity.
• RNA oligonucleotides were obtained from Microsynth (Balgach, Switzerland). Annealing was performed according to Elbashir et al. (2001 , Nature 411 , 494-498). The complementary two strands (each at 20 ⁇ M) in 200 ⁇ l of annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate) were heated for 1 min at 90°C and then incubated for 1 h at 37°C. An siRNA corresponding to nucleotides 753-773 of the firefly luciferase mRNA (luc siRNA) was used as a negative control.
• annealing buffer 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate
• siRNAs were introduced into HeLa cells using the OligofectAMINE Reagent (Life Technologies) according to the manufacturer's instructions, with 10 ⁇ l of 20 ⁇ M siRNA and 3 ⁇ l of transfection reagent per well. At different times after transfection, whole-cell extracts were prepared and analyzed by Western blotting. Cells were lysed in a buffer containing 120 mM NaCI, 50 mM Tris pH 8.0, and 1% NP40 plus Complete (Roche) protein inhibitor tablets.
• the whole-cell extracts (20 ⁇ g) were analyzed by Western blotting for She A, ⁇ -tubulin and Grb2 using a polyclonal rabbit anti-She antibody (1:250; Transduction Laboratories), mouse monoclonal anti-Grb2 (1 : 1000; Transduction Laboratories) or a mouse monoclonal anti- ⁇ tubulin antibody (1:1000; Sigma).
• An enhanced chemiluminescence detection method (ECL; Amersham) was employed and the membrane was exposed to Kodak Xomat LS film. Quantification of ShcA proteins was done using ImageQuant 5.0.
• the selective inhibition of the ShcA p66 isoform is demonstrated in this example using siRNA specific for p66.
• Cells were left untreated (control) or transfected without siRNA (control) or with h/m-shc siRNA, p66-shc siRNA (from nt 236-256 in the CH2 domain, 5'-GAA UGA GUC UCU GUC AUC GUC-3', SEQ ID NO:3; and 5'-CGA UGA CAG AGA CUC AUU CCG-3', SEQ ID NO:4) and control luc siRNA essentially as described in Example 1.
• Whole-cell extracts were prepared 48 h later and analyzed for ShcA and ⁇ -tubulin levels as in Example 1.
• This example illustrates the specificity of the siRNA effect with the effect of p66- sch si RNA being restricted to the p66ShcA isoform.
• Expression of p52/p46 ShcA was not affected although p66 and p52/p46 mRNAs share sequence identity in most of the region 3' of the siRNA site, indicating that the silencing signal does not propagate to regions of mRNA 3' to the siRNA in mammalian cells.
• the sequence of p66-shc siRNA is present in the primary transcripts of p52/p46 ShcA mRNA but is spliced out of the mature mRNA.
• This Example illustrates a rapid, alternative approach for isoform-specific gene expression.
• the previous Examples show how the adaptor protein ShcA can be suppressed in an isoform-specific manner in a human cell line.
• the siRNA with a sequence shared by the two ShcA transcripts suppresses p66, p52 and p46 (see Example 1).
• an siRNA whose sequence is present only in p66 mRNA suppresses only the p66 isoform (see Example 2), suggesting that the siRNA signal does not propagate to the 3' region of the target mRNA.
• the expression of individual isoforms is achieved by first downregulating all isoforms by the common (h/m she) siRNA (as in Example 1) and then transfecting with an expression vector for the desired isoform harboring silent mutations at the site corresponding to the h/m-shc siRNA.
• This allows functional analylsis of individual ShcA isoforms or indeed any other gene encoding multiple proteins.
• the full-length mouse p46, p52 and p66ShcA cDNAs were isolated from NIH3T3 cells by reverse transcriptase-polymerase chain reaction using the sense primers 5'-CGG AAT TCA TGG GAC CTG GGG TTT CCT ACT-3" (SEQ ID NO:5), 5'- CGG AAT TCA TGA ACA AGC TGA GTG GAG GCG-3' (SEQ ID NO:6) and 5'- CGG AAT TCA TGG ATC TTC TAC CCC CCA AGC CGA AGT A-3' (SEQ ID NO:7), respectively, and the common antisense primer 5'-CGG AAT TCA CAC TTT CCG ATC CAC GGG TTG C-3' (SEQ ID NO:8).
• Full-length ShcA cDNAs were initially cloned into pBluescriptll KS+ and nucleotide sequences verified by the dideoxynucleotide chain termination procedure.
• An HA-tagged expression vector pcDNA3HA was constructed by inserting the overlapping oligonucleotide pair 5'-CCC ACC ATG GCT TAC CCA TAC GAT GTT CCA GAT TAC GCT G-3' (SEQ ID NO:9) and 5'-AAT TCA GCG AAT TCT GGA ACA TCG TAT GGG TAA GCC ATG GTG GGG TAC-3' (SEQ ID NO:10) into the Kpnl-EcoRI site of pcDNA3 (Invitrogen).
• the overlapping oligonucleotide pair 5'-CTC CTC CAG GAC CTG AAC AAG CTG AGT G-3' (SEQ ID NO:11) and 5'-CAC TCA GCT TGT TCA GGT CCT GGA GGA G-3' (SEQ ID NO:12) was used to mutate methionine 65 (start site for ⁇ 52) to leucine in p66HA, resulting in p66HA-m1.
• Another overlapping oligonucleotide pair 5'-CCA ACG ACA AAG TCC TGG GAC CCG GGG-3' (SEQ ID NO:13)and 5'-CCC CGG GTC CCA GGA CTT TGT CGT TGG-3' (SEQ ID NO:14), was used to mutate the initiation sites for p46 in both p66HA-m1 and p52HA, resulting in p66HA-ML and p52HA-ML.
• Silent mutations were introduced into these vectors at the sites corresponding to h/m-shc siRNA as above using the overlapping oligonucleotide pair 5'-GGG GTT TCC TAC TTG GTC CGC TAC ATG GGT TGT C-3' (SEQ ID NO:15) and 5'-CAC AAC CCA TGT AGC GGA CCA AGT AGG AAA CCC C-3' (SEQ ID NO:16) to give p46HA-sm, p52HA-ML-sm and p66HA- ML-sm. Note that proteins expressed from these vectors are identical to the parent proteins.
• HeLa cells were first transfected with no siRNA (mock) or with h/m-shc siRNA to downregulate all three endogenous ShcA proteins (isoforms) essentially as described above. Two days later, the cells were transfected with an empty expression vector, pCDNA3, or an expression vector for the desired isoform of wild-type mouse ShcA or silent mutant ShcA using Lipofectamine 2000 (Life Technologies) according to the manufacturer's instructions. One day later, whole-cell extracts were prepared and analyzed by Western blotting for the ShcA expression level. Membranes were blotted with polyclonal anti-ShcA and anti- ⁇ tubulin antibodies.
• siRNA can efficiently, specifically and rapidly downregulate the level of an endogenous protein in mammalian cells.
• the effect of siRNA was restricted to mRNAs containing a sequence essentially identical to the siRNA used. That the primary action of siRNA on mRNA, which is most likely an endonucleolytic attack, does not propagate to other regions of the target mRNA was inferred from the following observations: (1) the effect of p66- shc siRNA was restricted to p66ShcA (Example 2)and (2) h/m-ShcA siRNA targeted wild-type ShcA mRNAs but not mutant mRNAs (Example 3). The second observation was with ectopically expressed ShcA mRNAs.
• sequences of wild-type ShcA mRNA and mutant ShcA mRNA for each isoform were identical except for two nucleotides at the siRNA recognition site located in the middle of the mRNA. These two nucleotide cahgnes were sufficient to avoid the siRNA effect on the expression of the desired isoform. If the silencing signal would spread either 5' or 3' of the siRNA, ShcA expression from both wild-type and mutant mRNAs would have been suppressed, as at least some of these sequences would be identical. That expression from wild-type mRNAs but not from mutant mRNAs was suppressed strongly argues for stringent specificity of siRNA-mediated mRNA decay and no propagation of RNAi in mammalian cells.
• siRNA-mediated mRNA degradation is confined to the cytoplasm and that the target mRNA is restricted to those mRNAs containing a sequence essentially identical to the siRNA used.
• These specific features of siRNA-mediated gene knock-down can be employed over a short time period in conjunction with specific expression vectors to establish conditions for expression of the ShcA gene in an isoform- specific manner. This knockdown-in method should be applicable and useful for the study of any gene expressed as multiple isoforms.

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