In recent years, functional nucleic acids controlling the expression of particular genes in vivo have received attention as novel pharmaceutical drugs or diagnostic drugs comparable to compounds and antibodies. Various studies and developments toward medical applications thereof are underway around the world.
The known functional nucleic acids include, for example: small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), and micro RNAs (miRNAs), which post-transcriptionally suppress the expression of target genes by gene silencing mediated by RNA interference (RNAi); nucleic acid aptamers, which suppress the functions of target substances such as transcription factors by specifically binding thereto; antisense nucleic acids, which suppress the translation of target mRNAs by binding thereto; decoy DNAs containing regulatory regions such as transcription factor-binding domains as decoy sequences, wherein the decoy DNAs capture target substances, thereby suppressing gene expression caused by the transcription factors; and UI adaptors, which specifically inhibit polyadenylation in the mRNA precursors of target genes to destabilize the mRNA molecules and then direct the degradation thereof. All of them are expected as the next-generation pharmaceutical drugs or diagnostic drugs. Among them, RNAi by siRNAs or shRNAs is in the limelight as powerful gene expression control tools capable of suppressing the desired gene expression, because of their target specificity, wide applications, and reliable effects.
Allele-specific gene silencing (or allele-specific RNAi: ASP-RNAi), which is the application of RNAi, can specifically suppress the expression of a desired allele and can therefore specifically suppress only the expression of a mutant allele causing a disease without influencing the expression of wild-type alleles. Hence, this method is considered exceedingly useful in disease therapy.
For example, fibrodysplasia ossificans progressiva (FOP) known as an intractable autosomal dominantly inherited disease is caused by a point mutation that substitutes guanine (G) at the 617th position by adenine (A) or a point mutation that substitutes G at the 1067th point by A on its causative activin-like kinase 2 (ALK2) gene. Since an allele having any of these point mutations is dominant, even a heterozygote having wild-type ALK2 gene is known to develop FOP (Non Patent Literatures 1 to 3). Unfortunately, an effective method for suppressing the onset or progression of this disease has not yet been found. The same holds true for many other autosomal dominantly inherited diseases caused by point mutations. In this context, if ASP-RNAi can suppress only the expression of a mutant allele having a point mutation and permit the expression of wild-type alleles, the onset of autosomal dominantly inherited diseases including FOP can be suppressed. In addition, the progression of these diseases can be prevented for patients who have already developed the diseases.
Such a mutant allele having a point mutation and the wild-type allele differ in their nucleotide sequences only by one to several bases. Thus, the development of siRNAs or shRNAs having exceedingly high specificity is essential for achieving such ASP-RNAi. Nevertheless, regions for designing siRNAs or the like are inevitably limited, because a point mutation site and its neighboring nucleotide sequences are to be used as a target region. Hence, effective methods for selecting target sequences of siRNAs known in the art cannot always be applied to the design. As a result, the design may disadvantageously fail to constantly produce highly specific and effective siRNAs or the like.
In an attempt to develop siRNAs or the like having exceedingly high specificity, it is disadvantageously difficult to correctly discriminate between mutant alleles and wild-type alleles for ASP-RNAi and quantitatively evaluate suppressive effects on their respective expressions.
Non Patent Literature 4 discloses a reporter system using luciferase gene. The literature also proposes a guideline for the design of siRNAs that involves quantifying a suppressive effect on the expression of a mutant allele using this system and bringing about ASP-RNAi on the basis of the obtained results. The accurate evaluation of an ASP-RNAi effect usually requires accurately discriminating between wild-type alleles and mutant alleles and precisely suppressing the expression of the mutant alleles of interest. This requires evaluating suppressive effects on the respective expressions of wild-type alleles and mutant alleles within the same cell. However, the reporter system used in the literature independently detects only the suppression of the expression of a mutant allele. The influence of the siRNAs on wild-type alleles was not sufficiently verified. Thus, the guideline for allele-specific siRNAs obtained using the reporter system disclosed in the literature was not sufficient in terms of accuracy and effects.
Meanwhile, the present inventors have developed a reporter allele evaluation system using Photinus luciferase gene and Renilla luciferase gene having high substrate specificity and disclosed it in Non Patent Literature 5. According to the system, siRNAs and expression vectors each carrying the sequence of a target mutant allele or the sequence of a non-target wild-type allele inserted to the 3′ UTR of the luciferase gene are cotransfected into cultured cells, and luciferase activity derived from each expression vector can be detected on the basis of luminescence, thereby specifically quantifying suppression efficiency for the respective expressions of the mutant allele and the wild-type allele by the siRNAs within the same cell. Thus, use of the reporter allele evaluation system allows more accurate evaluation of an ASP-RNAi effect or development of siRNAs or the like having a higher ASP-RNAi effect.
Non Patent Literature 6 describes, as a specific example of ASP-RNAi evaluation using the reporter allele evaluation system, results of designing an siRNA or shRNA against a mutant allele of human prion gene so that its sequence artificially contains a mismatch mutation at one position and variously changing its base length and the position of the mismatch mutation. The method of this literature produces a relatively marked ASP-RNAi effect. This experiment, however, employed only the human prion gene. In general, the effect of RNAi largely depends on the targeting nucleotide sequence against the target gene, i.e., the target gene-homologous nucleotide sequence incorporated in the siRNA or shRNA. Even if the introduced mismatch mutation can improve an ASP-RNAi effect, this effect is likely to be unique to the targeting nucleotide sequence used in the experiment and cannot be generalized. Accordingly, the results of Non Patent Literature 5 have not led to a guideline for the design of siRNAs or shRNAs having a high ASP-RNAi effect.
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