JP2008306962A - Hiv-1-specific rna interference molecule - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an RNA interference molecule having low off-target effects and exhibiting effective RNA interfering actions on HIV-1 (human immunodeficiency virus-1) which is a highly diverse virus. <P>SOLUTION: All the partial sequences of 21 bases are formed on the basis of 495 kinds of genomic sequences of HIV-1 group M, and RNA interference molecules satisfying design conditions are selected according to an siRNA (small-interfering RNA) design guideline by Ui-Tei et al. to study the siRNA target cleavage assay and proliferation inhibitory effects on the HIV-1. Thereby, an effective RNA interference molecule can be constructed by using a preservation region at the maximum level as the target for the HIV-1. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to RNA interference molecules that specifically exhibit interference effects on the highest degree of conservation regions in anti-HIV-1 genomic sequences. More specifically, the present invention relates to an RNA interference molecule that exhibits an interference effect specifically for HIV-1 and does not exhibit an off-target effect, a search method thereof, and a design method of the RNA interference molecule.

  RNA interference (RNAi) is a phenomenon in which double-strand RNA (dsRNA) introduced into a cell suppresses gene expression by specifically cleaving mRNA having a homologous base sequence. is there. The molecular mechanism of RNAi is considered to be part of the defense mechanism against viruses and transposons in vivo, and is important in the control of gene expression by microRNA (microRNA; miRNA) that is expressed in vivo. Have a role. In addition, since an arbitrary gene can be knocked down easily by artificially introducing dsRNA into a cell, RNAi has attracted attention as an effective means for analyzing the function of the gene.

  In order to conduct RNA interference experiments in mammals, a method is generally used in which a chemically synthesized short-interfering RNA (siRNA) of about 21 base pairs homologous to a target gene or a vector expressing siRNA is introduced into cells. Used. However, any siRNA sequence does not function in cells, and 70 to 80% of randomly designed siRNAs do not exhibit RNA interference. In order to sufficiently suppress the target gene, a method has been reported in which dsRNA for a target gene is synthesized by in vitro transcription and siRNA mixtures having various sequences cleaved by a dsRNA cleaving enzyme are used (Non-patent Document 1). .

  As for highly effective siRNA, 16 types of siRNAs against firefly luciferase gene were randomly designed, and the results of measuring the RNA interference effect in various cultured cells using firefly luciferase activity as an index have been reported. When siRNAs were arranged in descending order of the RNA interference effect in mammals, a certain pattern was observed. That is, highly effective siRNAs are 1) the 5 ′ end of the antisense strand is adenine (A) or uracil (U), and 2) the 5 ′ end of the sense strand is guanine (G) or cytosine (C). It was. In addition, 3) 4 or more out of 7 bases of 5 ′ of the antisense strand were common in that they were A or U. On the other hand, it was confirmed that siRNA which does not show an effect has a symmetrical characteristic that does not satisfy all the conditions of 1) to 3) (Non-patent Document 2).

  Moreover, since the siRNA is as short as about 21 base pairs, there is a high probability that a homologous portion exists in a gene completely unrelated to the target gene. In such a case, there is a possibility that the expression of a gene unrelated to the target is suppressed by siRNA. This phenomenon is called the off-target effect of siRNA, and is a serious problem in gene function analysis using siRNA and application of siRNA to medicine. In order to avoid the off-target effect, it is desirable to select siRNA that minimizes homology to all genes other than the target. In general, it is considered that a designed siRNA sequence is searched by BLAST, and an unrelated gene is not hit. However, searching for individual siRNA sequences by BLAST requires time and labor, and there is a serious drawback that searching for short sequences such as siRNA is often overlooked. An siRNA design web server is also released as a program for solving such drawbacks (http://design.RNAi.jp/).

  Regarding hepatitis C virus (HCV), there is a report on a dsRNA molecule that exhibits RNA interference (Patent Document 1). Here, siRNAs that inhibit viral replication are disclosed, particularly in infected cells, and particularly preferred dsRNA molecules correspond to HCV nucleic acids and have been reported to inhibit HCV replication in hepatocytes. In another aspect, there is also disclosed a modified siRNA that is resistant to nuclease degradation. However, in this document, regarding inhibition of HCV replication, there is no mention of siRNA having an effective RNAi action, and no problem with the off-target effect is pointed out.

Human immunodeficiency virus (HIV), which causes acquired immunodeficiency syndrome, is highly mutated, and this is not only the anti-HIV drug resistance but also the acquired immune response of the host against HIV-1 infection and vaccine It is a cause to avoid. It has been reported that RNA viruses such as HIV and HCV have extremely high genome diversity, and that when one kind of siRNA is used alone, a mutant virus resistant to the siRNA can easily appear (non-patented). References 3, 4). Therefore, provision of effective siRNA is desired as one of the countermeasures against HIV-1, which is a highly diverse virus.
Nature Methods. 2: p.779-784 (2005) Biotechnology Journal, 1-2 p.23-28 (2006) J. Virol. 77, 11531-11535 (2003a) J. Virol. 78, 2601-2605 (2004) JP 2006-320327 A

  An object of the present invention is to provide an RNA interference molecule that exhibits an RNA interference action effectively against HIV-1, which is a highly diverse virus, and has a low off-target effect.

  As a result of intensive research in order to solve the above problems, the present inventors have determined that a partial sequence of 21 bases (hereinafter simply referred to as “21-mer”) based on 495 species of HIV-1 group M genome sequences. By selecting a 21-mer that meets all the design requirements for constructing a highly effective RNA interfering molecule and examining the target cleavage assay and the growth inhibitory effect of HIV-1 As a result, an RNA interference molecule effective against the above can be constructed, and the present invention has been completed.

That is, this invention consists of the following.
1. An RNA interference molecule comprising a base sequence of at least 21 bases, targeting a conserved region of HIV-1 and exhibiting an RNA interference effect specifically for HIV-1.
2. The RNA interference molecule according to claim 1, wherein the conserved region of HIV-1 is a region that conserves at least 70% of the sequence of HIV-1.
3. The RNA interference molecule according to claim 1 or 2, wherein the conserved region of HIV-1 is selected from untranslated regions.
4). HIV-1 untranslated regions include TATA sequences necessary for viral gene expression, poly A signal (AAUAAA) region, primer activation signal (PAS) region necessary for reverse transcription, primer bindig site (PBS) region, Packaging signal (Ψ) region necessary for packaging, central poly purine tract (cPPT) region, central terminal sequence (CTS) region, and 3 'poly purine tract (3'PPT) region necessary for genomic + strand DNA synthesis The RNA interference molecule according to claim 3, which is a region selected from any one of the above.
5. An RNA interference molecule that targets the base sequence shown in any of SEQ ID NOs: 2 to 235 in the sequence listing and exhibits an RNA interference effect specifically for HIV-1 in the sequence of HIV-1.
6). In the sequence of HIV-1, SEQ ID NOs: 7, 11, 12, 14, 17, 23, 61, 65, 71, 99, 101, 106, 107, 110, 127, 136, 137, 139, 159, 171, 174 The RNA interference molecule according to claim 5, which exhibits an RNA interference effect specifically for HIV-1 targeting a base sequence portion shown in any one of 197, 218-235.
7). The method for searching for an RNA interference molecule according to claim 1.
8). The method for designing an RNA interference molecule according to any one of claims 1 to 6.
9. A method for inhibiting HIV-1 proliferation, comprising using one or more RNA interference molecules according to any one of claims 1 to 6.
10. The method for inhibiting HIV-1 proliferation according to claim 1, wherein the sense strand uses one or more RNA interference molecules corresponding to siRNA having the base sequence shown in any one of SEQ ID NOs: 237 to 277.
11. One type of RNA interference molecule corresponding to siRNA having a sense strand consisting of any one of SEQ ID NO: 239, SEQ ID NO: 242, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 251-254, SEQ ID NO: 266-268, or The method for inhibiting HIV-1 proliferation according to claim 10, wherein a plurality of types are used.

  The HIV-1 specific RNA interference molecule of the present invention can effectively suppress the proliferation of HIV-1 despite the diversity of the HIV-1 genome. This can contribute to the screening of anti-HIV-1 therapeutics and the development of anti-HIV-1 therapeutics using RNA interference molecules.

  In the present invention, “RNA interference molecule” refers to a double-stranded polynucleotide exhibiting an RNA interference effect or a molecule capable of expressing the same, and siRNA and a part thereof modified, or substituted with DNA. Is also included. Examples of molecules capable of expressing a polynucleotide include shRNA vectors.

  The target site of the RNA interference molecule of the present invention can be selected by the following method. The HIV-1 specific RNA interference molecule of the present invention targets the highest conserved region of the HIV-1 genome, and the potential target region is the Los Alamos HIV Sequence Database (HIV sequence database, http: Analysis can be performed based on 495 HIV-1 group M genome sequences available from //www.hiv.lanl.gov/) (see Tables 1-1 to 1-10). First, a 21-mer which is a 21 base partial sequence of the HIV-1 genome constituting 495 HIV-1 sequences is generated, and the number of the 495 species common to each 21-mer is examined. Then, a highly conserved region that can be an effective candidate as a target of an RNA interference molecule is identified from the genome sequence.

  For example, when a certain 21-mer is common to 300 of 495 HIV-1 sequences, simply the conservation of the oligonucleotide is 300/495 × 100 = 60.6 (%) And can be calculated. However, since most of the HIV-1 sequences used in this analysis lack the 5 'UTR region sequence, when the conservation degree of the 5' UTR region is calculated, the conservation degree is too low when the denominator is 495. May be evaluated. In this case, a more accurate conservation degree can be calculated by complementing the 3 ′ LTR sequence for the LTR portion in the 5 ′ UTR region and calculating an appropriate value for the denominator.

  First, when 21-mers constituting 495 HIV-1 sequences were generated, a total of 4,417,157 species were obtained, and 1,208,150 species were confirmed excluding sequence duplication. The fact that the total 21-mer generated from 495 HIV-1 sequences is only about a quarter when duplication is removed suggests that the diversity of the HIV-1 genome is remarkably high.

  The conservation of each 21-mer can then be calculated using the siVirus program (Naito et al., Nucleic Acids Res. 34, W448-W450 (2006)).

  Attempts at RNA interference targeting HIV-1 have already been reported by several research groups, but many of the siRNAs used in these studies have been shown to be poorly conserved ( (See Tables 2-1 to 2-3.)

  However, it was confirmed that 21-mer located in a specific region is highly conserved. Many of these regions were within the untranslated region and contained sequences essential for viral gene expression and propagation. That is, TATA sequence region necessary for viral gene expression, poly A signal (AAUAAA) region, primer activation signal (PAS) region necessary for reverse transcription, primer bindig site (PBS) region, packaging necessary for virus packaging The ones located in the signal (Ψ) region, the central polypurine tract (cPPT) region, the central terminal sequence (CTS) region, and the 3 'polyurine tract (3'PPT) region, which are necessary for genome + strand DNA synthesis, are highly sophisticated. It was preserved. The RNA interference molecule of the present invention is a double-stranded polynucleotide that exhibits an RNA interference effect targeting these untranslated regions, a molecule capable of expressing it, siRNA and a part thereof modified, or DNA Can be selected from those replaced with Moreover, the RNA interference molecule of the present invention can be used as long as it targets not only these non-translated regions but also a site having a high degree of conservation. Here, the high degree of conservation is specifically 495 species of HIV-1 group M genome sequences available from the Los Alamos HIV Sequence Database (HIV sequence database, http://www.hiv.lanl.gov/). Among these, the preservation degree is 70% or more, more preferably 80% or more.

  The non-translated regions exemplified above are all regions that exert their functions through the RNA sequence itself or the RNA secondary structure rather than the amino acid sequence. For example, the regions shown in FIGS. 3 and 4 are targeted. be able to. For example, many of the regions that are translation regions and are conserved at the amino acid sequence level allow mutation of the third base of the codon, and thus are not always conserved at the base sequence level.

  As a result of identifying each 21-mer by the above analysis and examining the 21-mer having a conservation degree of 70% or more, the sequences shown in SEQ ID NOs: 2 to 235 in the Sequence Listing are listed (Table 3-1). -3-5, see FIG. Of the RNA interference molecules that target these sequences, 36 species meet the siRNA design guidelines of Ui-Tei et al. And are expected to be effective in mammalian cells. In addition, 30 types can be predicted to be effective in the siRNA design method of Reynolds et al., And 44 types in the design method of Amarzguioui et al.

  More specifically, in the HIV-1 sequence, the base sequence part shown in any one of SEQ ID NOs: 2 to 235 in the sequence listing can be targeted. Furthermore, in the sequence of HIV-1, SEQ ID NOs: 7, 11, 12, 14, 17, 23, 61, 65, 71, 99, 101, 106, 107, 110, 127, 136, 137, 139, 159, It is preferable to target the base sequence portion shown in any of 171, 174, 197, and 218 to 235.

  Among the HIV-1 specific RNA interference molecules for the target sites described above, the siRNA design method, in particular, will be described based on the methods of Ui-Tei et al., Reynolds et al., And Amarzguioui et al.

The outline of the siRNA design guidelines of Ui-Tei et al. (Ui-Tei et al., Nucleic Acids Res. 32, 936-948 (2004)) is as follows. The sense strand and antisense strand constituting the siRNA are simply referred to as sense strand and antisense strand, respectively.
(i) The 5 ′ end of the antisense strand is A or U.
(ii) The 5 ′ end of the sense strand is G or C.
(iii) 4 or more bases out of 7 bases at the 5 ′ end of the antisense strand are A or U.
(iv) G / C does not continue for 10 bases or more in the sequence.

The outline of the determination method of Reynolds et al. (Reynolds et al., Nat. Biotechnol. 22, 326-330 (2004)) is as follows, and a total of 6 or more siRNAs are determined to be effective.
(I) +1 point if the number of G / C in the double-stranded part of siRNA is in the range of 7 to 10 bases out of 19 bases.
(II) The number of A or U in 5 bases at the 5 ′ end of the antisense strand is taken as (0-5) score.
(III) Tm of the double-stranded part is less than 20 ° C.
(IV) If the 19th base from the 5 'end of the sense strand is A, +1 point.
(V) If the third base from the 5 'end of the sense strand is A, +1 point.
(VI) +1 point if the 10th base from the 5 'end of the sense strand is U.
(VII) -1 point if the 5 'end of the antisense strand is G or C.
(VIII) -1 point if the 13th base from the 5 'end of the sense strand is G.

The outline of the determination method of Amarzguioui et al. (Amarzguioui et al., Biotchem. Biophys. Res. Commun. 316, 1050-1058 (2004)) is as follows, and a total of 3 or more siRNAs are determined to be effective.
(1) ΔT3: From the number of A or U at the 3 bases at the 5 ′ end of the antisense strand (0-3), the number of A or U at the 3 bases at the 5 ′ end of the sense strand (0-3) The subtracted value is used as the score. (It can take values from -3 to +3.)
(2) +1 point if the 5 ′ end G or C of the sense strand.
(3) -1 point if the 5 'end of the sense strand is U.
(4) If the sixth base from the 5 'end of the sense strand is A, +1 point.
(5) If the 19th base from the 5 'end of the sense strand is G, -1 point.
(6) +1 point if the 5 ′ end of the antisense strand is A or U.

  SiRNA molecules selected by the above design method can be produced by a general method such as oligonucleotide synthesis. In addition, the RNA interference molecule of the present invention includes those in which siRNA and a part thereof are modified, and the siRNA molecule can also contain a modified nucleotide base. For example, the modified base can be a common one already on the market. The use of modified bases can increase the stability of the siRNA molecule and thereby reduce the amount required to produce the desired effect. Moreover, what substituted the siRNA molecule | numerator by DNA can be obtained with the same synthesis method.

  Furthermore, the RNA interference molecule of the present invention includes, for example, a shRNA vector as a molecule capable of expressing the above-mentioned polynucleotide. Specifically, such a vector can be prepared by a method that has already been ordered in the market by, for example, GenScript, iGENE, Ambion, etc.

  The effect of the RNA interference molecule of the present invention of the present invention can be evaluated by the target cleavage activity of the RNA interference molecule of the present invention itself. The off-target effect that the RNA interference molecule of the present invention recognizes a homologous part of a gene different from the original target and suppresses the gene is that the siRNA sequence corresponding to the RNA interference molecule and the target mRNA sequence are The degree of homology between them can be examined and evaluated by the target cleavage activity of siRNA against an arbitrary target sequence.

  Furthermore, the present invention extends to a method for inhibiting HIV-1 proliferation, which uses one or more RNA interference molecules of the present invention. Specifically, there is a method for suppressing HIV-1 proliferation using an RNA interference molecule corresponding to an siRNA in which the sense strand of the siRNA in the RNA interference molecule has a base sequence shown in any one of SEQ ID NOs: 237 to 277 in the Sequence Listing. More specifically, SEQ ID NO: 239 (si510), SEQ ID NO: 242 (si521), SEQ ID NO: 247 (si764), SEQ ID NO: 248 (si770), SEQ ID NOS: 251 to 254 (si2075, si2329, si2330, si2333). And a method for suppressing HIV-1 proliferation using one or more RNA interference molecules corresponding to the siRNA shown in any of SEQ ID NOs: 266 to 268 (si4750, si4751, si4753).

  Furthermore, the RNA interference molecule of the present invention can simultaneously use RNA interference molecules corresponding to a plurality of siRNAs having different targets. When two types of RNA interference molecules of the present invention are used in combination, 97% or more of the HIV-1 strain is effective against various HIV-1 mutant strains (see FIG. 10).

  The present invention covers not only the above-described RNA interference molecules exhibiting RNA interference effects specifically for HIV-1, but also a search method and a design method for the RNA interference molecules, and further HIV-1 using the RNA interference molecules. It extends to methods of inhibiting proliferation.

  The present invention will be described more specifically with reference to the following examples and experimental examples. However, the present invention is not limited to these examples, and various applications can be made without departing from the technical idea of the present invention. Is possible.

(Example 1) Identification of target region of RNA interference molecule for HIV-1 The genome sequence of HIV-1 of the present invention is the Los Alamos HIV Sequence Database (HIV sequence database, http://www.hiv.lanl.gov/ ) Based on 495 types of HIV-1 group M genome sequences available (see Tables 1-1 to 1-10). A highly conserved region that could be an effective candidate for siRNA target was identified from the genome sequence. First, all 21-mers constituting 495 HIV-1 sequences were generated, and it was examined how many of the 495 species were common to one 21-mer.

  Subsequently, the conservation degree of each 21-mer was calculated using the siVirus program (Naito et al., Nucleic Acids Res. 34, W448-W450 (2006)). When the degree of conservation was shown as 0% to 100%, most of them were close to 0%, and it was revealed that most of the HIV-1 genome was not conserved in 21-mer units. (See FIG. 1). Specifically, 21-mer with a preservation degree of 70% or more is only 1.6% of the whole, and even a 21-mer with a preservation degree of 50% or more was only 5.2% of the whole (Fig. 2). In addition, there was no 21-mer having a conservation degree of 100% common to all HIV-1 sequences. These results suggest that it is difficult to efficiently target HIV-1 having high diversity even if siRNA targeting HIV-1 is randomly designed. Trials of RNAi methods targeting HIV-1 have already been reported by several research groups, but many of the siRNAs used in these studies have low conservation (Tables 2-1 to 2- (See 3.)

  When the 21-mer having a conservation degree of 70% or more was examined by the above analysis, the target sequences shown in SEQ ID NOs: 2 to 235 in the sequence listing were confirmed. Of the siRNAs corresponding to these 234 target sequences, 36 species fulfilled the siRNA design guidelines of Ui-Tei et al. And were predicted to be effective in mammalian cells. In addition, 30 types were predicted to be effective in the siRNA design method of Reynolds et al., And 44 types were predicted to be effective in the design method of Amarzguioui et al.

  In order to verify the anti-HIV-1 effect of siRNA, 23 of the 36 siRNAs that meet the siRNA design guidelines of Ui-Tei et al. have the exact sequence matching with the infectious molecular clone pNL4-3 (GenBank: M19921). In addition, 18 kinds were selected that had a medium degree of conservation, met the siRNA design guidelines of Ui-Tei et al., And completely matched the sequence of pNL4-3 (GenBank: M19921), for a total of 41 kinds.

  The target site of the selected siRNA is SEQ ID NO: 7, 11, 12, 14, 17, 23, 61, 65, 71, 99, 101, 106, 107, 110, 127, 136, 137, 139, 159 in the sequence listing. 171, 174, 197, 218-235, the sense strand of siRNA consists of the base sequence shown in SEQ ID NO: 237-277, and the antisense strand is shown in SEQ ID NO: 279- It consists of the base sequence shown in 319. Each of these base sequences is shown in FIG. siRNA itself can also be identified from the number display following si in FIG.

Example 2 Preparation of HIV-1 Specific RNA Interference Molecules The siRNA sense strands shown in FIG. 5 (SEQ ID NOs: 237 to 277 in the sequence listing) were chemically synthesized and annealed into double strands from RNAi (Tokyo). ) Purchased from. siControl (SEQ ID NO: 278) is a negative control that satisfies the guidelines of Ui-Tei et al. and has no homologous sequence within 3 mismatches in all human transcripts.

  HeLa cells were cultured at 37 ° C. with antibiotics added to Dulbecco's modified Eagle medium (DMEM; Gibco) containing 10% non-immobilized fetal bovine serum. Transfection of siRNA into HeLa cells was performed in a 24-well plate using Lipofectamine 2000 (Invitrogen) according to the method described in the manual for the product. Plasmids to be introduced into cells were extracted using Qiagen Plasmid Midi or QIAwell 96 Ultra (Qiagen).

(Experimental Example 1) Measurement of HIV-1 Reverse Transcriptase Activity The 41 types of siRNA obtained in Example 2 were tested for the growth-inhibitory effect against HIV-1 with four types belonging to subtypes B, B ′, C and CRF01_AE. The HIV-1 infectious molecular clone was evaluated. A summary of the four clones used for the evaluation is shown in Table 4. These three subtypes are the main causes of the HIV-1 epidemic in the Asian region, and form clusters that are separated from each other on the base sequence.

  In this experimental example, each infectious molecular clone was introduced into HeLa cells together with each siRNA, and after 48 hours, the supernatant of the medium was collected and the HIV-1-derived reverse transcriptase activity was measured to evaluate the growth of the virus. .

  The result is shown in FIG. 6A. When each siRNA was introduced at 5 nM, 26 out of 41 RNA interference molecules suppressed the growth of all HIV-1 strains to 20% or less. Of the remaining 15 siRNAs, 13 except for si4794 and si4888 were found active in the target cleavage assay. Moreover, since 12 types except si690, si4794, and si4888 suppressed the proliferation of at least 1 sort of HIV-1 strain to 20% or less, those siRNA itself was considered effective.

  However, when a mismatch of 1 to several bases exists between the siRNA sequence and the target sequence, the growth of those HIV-1 strains could not be suppressed. On the other hand, when the base substitution is close to the end of siRNA, the effect of siRNA may be retained. The result that the influence of the mismatch is small at the position close to the end in this way generally coincided with the analysis result of the off-target effect shown in Experimental Example 2 below.

  FIG. 7 shows an alignment of 41 types of siRNA sequences of the present invention and target sequences of each infectious clone. The siRNA sequence was a sense side sequence corresponding to 21 bases of the antisense strand. Although all are RNA molecules, the base sequence is described using 4 characters of ATGC in accordance with each data recorded in GenBank.

  Moreover, about si689 and si690, although the siRNA sequence and the target sequence corresponded completely, the growth of HIV-1 could not be suppressed. This region is tightly closed in two types of secondary structures that can be taken by the HIV-1 5'UTR (Huthoff et al., 2001), both BMH (branched multiple hairpin) and LDI (long distance interaction), It was suggested that siRNA may be preventing access to the target sequence. Furthermore, it was noted that the effect of si575 is different between pNL4-3 and 93IN101. The reason for this is not clear, but one possibility is that each infectious molecular clone has a different secondary structure of the target portion, and siRNA accessibility may be different.

(Experimental example 2) Target cleavage assay A target cleavage assay system introduces an expression vector incorporating a sequence of 21 to 40 bases that is a target of siRNA and siRNA simultaneously into a cultured cell, and the target mRNA expressed from the vector is siRNA. By the method of quantifying the efficiency of cleavage by real-time RT-PCR. In order to examine the degree of homology between the siRNA sequence and the target mRNA sequence to produce an off-target effect, the target cleavage activity of siRNA against an arbitrary target sequence was evaluated by a target cleavage assay system. . A pTREC vector was constructed by inserting a Kozak sequence and several restriction enzyme sites between the NheI and XbaI sites of pCI-neo (Promega). The inserted sequence is as follows.
[NheI site] -CACCATGGAATTCACGCGTCTCGAG- [XbaI site] (SEQ ID NO: 321)

  The target mRNA expression vector pTREC (abbreviation of Target RNA Expression Construct) constructed for this assay was used (see FIG. 8). In this assay, the target sequence can be set freely by inserting a synthetic DNA oligonucleotide into the MCS of the pTREC vector, so that the target mRNA sequence can be changed one base at a time for a specific siRNA. Analysis is possible. Since the sequence of mRNA expressed from the pTREC vector is common except for the target portion, siRNA target cleavage activity can be quantitatively evaluated under exactly the same experimental conditions.

  The target sequence of each siRNA was inserted between the EcoRI site and the XhoI site of the constructed pTREC vector. Each construct (0.5 μg / well) was introduced into HeLa cells together with siRNA (final concentration 5 nM). After 24 hours, the cells were collected, and total RNA was extracted with RNeasy96 (Qiagen). The obtained RNA was reverse transcribed with SuperScript (Invitrogen) using oligo dT as a primer to obtain cDNA. Real-time PCR was performed with SYBER Green PCR Master Mix (Applied Biosystems) using ABI PRISM7000 (Applied Biosystems).

  The result is shown in FIG. 6B. When each siRNA was used at 5 nM, 39 of 41 types of siRNA suppressed the target mRNA to 40% or less. Although si4794 and si4888 were expected to be effective, they did not show a sufficient inhibitory effect at a concentration of 5 nM. The reason for this is not clear, but si4794 contains consecutive guanine residues (GGGGGG) and consecutive cytosine residues (CCCCCC) in the sequence, making it difficult to chemically synthesize RNA oligos, and si4888 is called AAAUUUU in the sequence. Since there is a sentence sequence and it is easy to take a secondary structure, there is a possibility that the effective concentration of double-stranded siRNA that correctly formed base pairs in all cases was lowered.

  From the above results, according to the present invention, it was possible to design RNA interference molecules capable of dealing with the high diversity of HIV-1 with high probability. Among the RNA interference molecules of the present invention, siRNAs having a conservation degree of 70% or more and capable of suppressing virus growth to 10% or less were as follows.

si521: Target poly A signal. The degree of preservation was 94%.
si764 and si770: Target Ψ. The degree of preservation was 88%.
si510: Targets TAR-poly A signal. The degree of preservation was 84%.
si2075: Targets a ribosomal slip site. Each degree of preservation was 70%.
si2329, si2330 and si2333: target the protease region. Each degree of preservation was 77%.
si4750, si4751 and si4753: targeting the integrase region. The degree of preservation was 71-74%.

  However, it was impossible to target all HIV-1 registered in the database with one siRNA alone (see FIG. 9). To overcome this, siRNAs corresponding to multiple siRNAs with different targets can be used simultaneously. 97% or more of HIV-1 was found to be effective by combining and simultaneously using two RNA interference molecules of the present invention (see FIG. 10).

  Specifically, si509 and si4888, si510 and si4888, si512 and si4888, si515 and si4888, si1521 and si4888, si509 and si1817, si510 and si1817, si512 and si1817, si509 and si1589, si589 And si1689, si521 and si1689, si689 and si764, si689 and si770, si515 and si1817, si521 and si1770, si521 and si4652, si521 and si4652, si6521 and si7652, si6852 and si18417 , Si521 and si2486, si52 And si4175, si521 and si4794, si521 and si554, si689 and si4809, si689 and si7653, si689 and si7658, si764 and si4888, si770 and si1817, si509 and si2075, si510 and si1275 , Si521 and si2075, si521 and si2329, si521 and si2330, si521 and si2333, si521 and si3000, si521 and si3005, si521 and si4745, si521 and si4750, si521 and si4751 RNA corresponding to each siRNA Effect of over 97.9% was obtained by the combination of Wataru molecule.

  As described above in detail, the siRNA against anti-HIV-1 against the highest degree of conservation region of the present invention can effectively suppress the proliferation of HIV-1 despite the diversity of HIV-1 genome. it can. This can contribute to the screening of anti-HIV-1 therapeutics and the development of anti-HIV-1 therapeutics using the present siRNA.

It is a figure which shows distribution and conservation of 21-mer in 495 kinds of HIV-1 arrangement | sequences. It is a figure which shows the conservation degree of 21-mer in 495 types of HIV-1 arrangement | sequences. It is a figure which shows the BMI (branched multiple hairpin) type secondary structure and the target site of the siRNA of the present invention. It is a figure which shows the LDI (long distance interaction) type secondary structure and the target site | part of siRNA of this invention. It is a figure which shows the base sequence of the target site | part of the RNA interference molecule of this invention, and the sense strand and antisense strand of siRNA molecule. It is a figure which shows the evaluation result of the RNA interference molecule of this invention. A. It is a figure which shows the measurement result of HIV-1 reverse transcriptase activity. B. It is a figure which shows a target cleavage assay result. It is a figure which shows the alignment with respect to each HIV-1 target site regarding the RNA interference molecule of this invention. It is a figure which shows the structure of the pTREC vector used for the target cleavage assay of the RNA interference molecule of this invention. It is a figure which shows the ratio of the mutant strain which recognized the inhibitory effect with respect to the whole each HIV-1 mutant strain when 1 type of RNA interference molecule of this invention was used. It is a figure which shows the ratio of the mutant strain which recognized the inhibitory effect with respect to the whole each HIV-1 mutant strain when two types of RNA interference molecules of this invention were used.

Claims (11)

An RNA interference molecule comprising a base sequence of at least 21 bases, targeting a conserved region of HIV-1 and exhibiting an RNA interference effect specifically for HIV-1. The RNA interference molecule according to claim 1, wherein the conserved region of HIV-1 is a region that conserves at least 70% of the sequence of HIV-1. The RNA interference molecule according to claim 1 or 2, wherein the conserved region of HIV-1 is selected from untranslated regions. HIV-1 untranslated regions include TATA sequences necessary for viral gene expression, poly A signal (AAUAAA) region, primer activation signal (PAS) region necessary for reverse transcription, primer bindig site (PBS) region, Packaging signal (Ψ) region required for packaging, central poly purine tract (cPPT) region, central terminal sequence (CTS) region, and 3 'poly purine tract (3'PPT) region required for genomic + strand DNA synthesis The RNA interference molecule according to claim 3, which is a region selected from any one of the above. An RNA interference molecule that targets the base sequence shown in any of SEQ ID NOs: 2 to 235 in the sequence listing and exhibits an RNA interference effect specifically for HIV-1 in the sequence of HIV-1. In the sequence of HIV-1, SEQ ID NOs: 7, 11, 12, 14, 17, 23, 61, 65, 71, 99, 101, 106, 107, 110, 127, 136, 137, 139, 159, 171, 174 The RNA interference molecule according to claim 5, which exhibits an RNA interference effect specifically for HIV-1 targeting the base sequence portion shown in any one of 197, 218 to 235. The method for searching for an RNA interference molecule according to claim 1. The method for designing an RNA interference molecule according to any one of claims 1 to 6. A method for inhibiting HIV-1 proliferation, comprising using one or more RNA interference molecules according to any one of claims 1 to 6. The method for inhibiting HIV-1 proliferation according to claim 1, wherein the sense strand uses one or more RNA interference molecules corresponding to siRNA having the base sequence shown in any one of SEQ ID NOs: 237 to 277. One type of RNA interference molecule corresponding to siRNA having a sense strand consisting of any one of SEQ ID NO: 239, SEQ ID NO: 242, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 251-254, SEQ ID NO: 266-268, or The method for suppressing HIV-1 proliferation according to claim 10, wherein a plurality of types are used.

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