Nucleic acid molecule, expression cassette, expression vector, eukaryotic host cell, induction method of RNA interference in eukaryotic host and use of the nucleic acid molecule in therapy of diseases induced by expansion of trinucleotide CAG repeats

Abstract

Subjects of the invention are: nucleic acid molecule, expression cassette, expression vector, eukaryotic host cell, induction method of RNA interference in eukaryotic host and use of nucleic acid molecule in therapy of diseases induced by expansion of trinucleotide CAG-type repeats. Solution relates to the new concept of treating hereditary human neurological diseases caused by expansion of CAG-type trinucleotide repeats using RNA interference technology.

Claims

1. A nucleic acid molecule composed of a duplex and loop, in which one of the duplex strands, the guide strand, comprises a sequence chosen from SEQ ID NO: 1 and SEQ ID NO: 2, and the other strand of the duplex, the passenger strand, is at least 80% complementary to the guide strand, wherein the nucleic acid molecule forms a hairpin structure in a cell.

2. The molecule according to claim 1, characterized in that the duplex region is 19-30 base pairs long.

3. The molecule according to claim 2, wherein the first and the second strand of the duplex are connected by a loop 4 to 15 nt long.

4. The molecule according to claim 2, wherein the loop is specified by a sequence SEQ. ID NO. 23.

5. The molecule according to claim 3, wherein the molecule comprises modified CUG-type repeats in the guide strand having 1, 2, 3, or 4 substitutions causing formation of non-canonical base pairs by interaction with targeted CAG sequences in transcripts.

6. The molecule according to claim 1, wherein the molecule comprises 5 and 3 flanking sequences derived from natural miRNA, wherein precursor flanking sequences of natural length are shortened or not.

7. The molecule according to claim 6, wherein the molecule comprises a loop sequence derived from a natural miRNA.

8. An expression cassette comprising a regulated or constitutive promoter, wherein the promoter is functionally connected to a sequence encoding the nucleic acid molecule of claim 1.

9. The expression cassette according to claim 8, wherein the promoter is a polII or polIII RNA promoter.

10. An expression vector, comprising the expression cassette specified by claim 8.

11. A eukaryotic host cell, comprising the nucleic acid molecule of claim 1.

12. A cell comprising the expression cassette of claim 8.

13. A eukaryotic host cell, comprising the expression vector of claim 10.

14. An RNA interference induction method comprising delivery of a nucleic acid molecule of claim 1 to a eukaryotic host.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows examples of the sequence and structure of the shRNA- and sh-miR-type interfering RNAs.

(2) Interfering RNAs are formed in cells as a result of transcription. They are forming hairpin-type structure with double-stranded stem and loop. Interfering RNA of shRNA-type contain few uridyl residues on the 3 end and sequence of the guide strand, which recognises the sequence of interest (red colour), is located preferentially in the 3 arm of the hairpin stem. sh-miR type interfering RNA contains additional loop and flanking 5 and 3 sequences of the natural precursor microRNA.

(3) FIG. 2 shows interfering RNA processing in HEK293 cells by Dicer RNase. Molecules of interfering RNA directed against mutant CAG tracts in transcripts are introduced to the cells in the form of expression cassettes containing, besides sequence coding shRNA or sh-miR, also reporter gene sequence, such as copGFP or GFP. Using method of high-resolution northern-type hybridisation, formation of transcripts in cells (pre-) or their cutting by Dicer RNase to the pool of short RNA duplexes (siRNA) has been proved. Both the transcript being formed as well as short siRNA duplexes are heterogeneous. M1 and M2 stands for RNA length markers; siA2synthetic siRNA transfected into cells.

(4) FIGS. 3A and 3B show allele-selective silencing of the huntingtin protein and ataxin 3 (Western blotting) and transcript of the HTT gene (RT-PCR) by shRNA-type interfering RNA. A) Human fibroblasts derived from a patient with Huntington disease (17/68 CAG) were transduced using lentiviral vectors encoding shRNA-type interfering RNA. Cells were infected using MOI 10 amount of virus (multiplicity of infection) and analysed 7 days after infection. shLucnegative control, shRNA directed against Luc gene, sh2.4positive control, shRNA directed against specific sequence of the HTT gene; signals intensity from huntingtin protein were normalised to the level of plectin protein and shLuc control, statistical analyses were used for comparison (one-sample t-test). Error bars present standard deviations. Statistically significant values (p-value) are marked with asterisk. B) Human fibroblasts derived from a patient with spinocerebellar ataxia type 3 (18/69 CAG) were transduced using lentiviral vectors encoding shRNA-type interfering RNA (shA2R and shA2R1). Cells were infected using two virus concentrations (MOI 1 and 10) and analysed 7 days after infection. shLucnegative control, shRNA directed against Luc gene, K-cells not transduced with virus. GAPDHreference protein

(5) FIGS. 4A and 4B show shows analysis of interfering RNA silencing selectivity, for RNAs forming mismatches with CAG sequence of interest. a) Western blotting analysis of ATXN3, TBP, FOXP2, EIF2AK3, RPL14 i LRP8 protein levels in HD fibroblasts 72 h after transfection using 50 nM RNA duplexes consisting of modified CUG repeats forming mismatches with CAG sequence of interest (reference reagents: d7, P9b and W13/16). Nucleotide sequences of repeat tracts in genes encoding analysed proteins comprise: tracts for TBP (CAG)3(CAA)3(CAG)9(CAA)(CAG)(CAA)(CAG)20 and FOXP2:(CAG)4(CAA) (CAG)4(CAA)2(CAG)2(CAA)2(CAG)3(CAA)5(CAG)2(CAA)2(CAG)5(CAA)(CAG)5. Creference value defining expression level in cells transfected using control siRNA. Intensiveness of signals were normalised to the level of reference protein GAPDH and compared using statistical test (one-sample t-test). Error bars show standard deviations. Statistically significant values are marked with asterisk (*p<0.05). b) in comparison to unmodified molecules CAG/CUG, modifications of CAG repeats tract in passenger strand of interfering RNA reduced their possibility of binding and non-specific activity in RNAi pathway towards CUG transcripts; mfeminimum free energy.

(6) Below are example embodiments of the present invention described above.

EXAMPLES

(7) shRNA- and Sh-miR-Type Interfering RNA Containing Modified CAG/CUG Sequences in the Structure of Hairpin Stem are Substrates for Dicer RNase.

(8) In order to assess if interfering RNA are further processed in cells by proteins of RNAi pathway, HEK293 cells were transfected using expression vectors encoding shRNA and sh-miR molecules (FIG. 2). After 24 h, total RNA was isolated and separated in polyacrylamide gel and further hybridisation with probe in order to visualise transcripts and their cut products was performed (northern blot analysis). It was determined that interference RNAs produced in cells are cut by Dicer RNase to the pool of short heterogeneous siRNA molecules. Furthermore, RNA-H1 shRNA molecules produced under control of polymerase III promoter are heterogeneous already at the transcript level (marked as a pre-), which is mainly due to presence of different length urydil residues at 3 end.

(9) shRNA-Type Interference RNA Lead to Allele-Selective Silencing of Mutant Proteins Huntingtin and Ataxin-3.

(10) In order to test the efficiency of shRNA-type interference RNA forming selective mismatches with CAG sequence of interest, lentiviral vectors encoding shA2R and shA2R1 molecules were constructed (containing ID. NO. 1 and ID. NO. 2 sequences from the Table). Human fibroblasts derived from patients with Huntington disease and spinocerebellar ataxia type 3 were transduced using lentiviruses in MOI 10 or 1 concentration and analysed 7 days after transduction (FIG. 3). Isolated protein was further analysed using Western Blotting and silencing level of mutant and normal variant was assessed in comparison with reference proteins and negative controls. Both analysed reagents lead to allele-selective silencing of mutant huntingtin, leaving non-mutant protein on normal level (FIG. 3A). HTT gene transcript analysis proved silencing selectivity of mutant form. Silencing efficiency of interfering RNAs was also tested on different model of polyglutamine diseasesSCA3 (FIG. 3B). Analysed molecules lead to allele-selective silencing of mutant ataxin-3 for both analysed virus concentrations (MOI 1 and MOI 10).

(11) Methods

(12) Cell Cultures and Transfection

(13) Fibroblasts derived from the patients with HD (GM04281-17/68 CAG) and SCA3 (GM06153-18/69 CAG) were obtained from Coriell Cell Repositories (Camden, N.J., USA). Cells were cultured in MEM medium (Lonza; Basel, Switzerland) enriched with 8% FBS (Sigma-Aldrich; St. Louis, USA), antibiotics: penicillin, streptomycin, amphotericin B (Sigma-Aldrich) and amino acids (Cat. no. M7145, Sigma-Aldrich). Transfection was performed using Lipofectamine 2000 (Life Technologies; Grand Island, N.Y., USA) according to the manufacturer recommendations.

(14) Western Blotting

(15) Western blot analysis for HTT protein (tract 17/68Q). Briefly, 25 g of total protein was resolved in poliacrylamide gel with SDS (1.5 cm, 4% stacking gel/4.5 cm, 5% separation gel, acrylamide/bisacrylamide ratio 35:1) in XT Tricine buffer (Bio-Rad; Hercules, Calif., USA) under 140 V in water bath. Subsequently, proteins were transferred onto nitrocellulose membrane (Sigma-Aldrich). All immunodetection steps were performed using SNAPid system (Millipore; Billerica, Mass., USA) in PBS/0.9% NaCl/0.1% Tween-20 buffer and 0.25% skimmed milk. Immunofluorescence reaction was detected using ECL Western Blotting Substrate (Thermo Scientific, Rockford, Ill., USA). Protein bands were scanned directly from membrane using camera and analysed using Gel-Pro Analyzer software. Western blot analysis for ATXN3 protein25 g of total protein was separated in polyacrylamide gel with SDS (5% stacking gel, 12% separation gel) in Laemmli buffer under 120V. Other steps of analysis as before.

(16) Northern Blotting

(17) Effector molecules released from vectors have been detected using northern-type hybridisation. Total RNA was isolated from HEK293T cells using TRI Reagent (BioShop; Burlington, Canada) according to the manufacturer recommendations. RNA (35 g) was separated in denaturing polyacrylamide gel (12% PAA, 19:1 acrylamide/bis, 7.5 M urea) in 0.5TBE buffer. RNA was transferred onto GeneScreen Plus (PerkinElmer) hybridisation membrane using semi-dry electrotransfer technique (Sigma-Aldrich). Membrane was hybridised with radioactively labelled DNA probe complementary to interfering RNA molecule. Radioactive signals were detected quantitatively using laser scanner FLA5100 (Multi Gauge v3.0, Fujifilm).

(18) RT-PCR and RNA Isolation

(19) Total RNA was isolated from cells using TRI Reagent (BioShop; Burlington, Canada) according to the manufacturer recommendations. RNA concentration was measured using NanoDrop spectrophotometer. 500 ng RNA was used for reverse transcription reaction, reaction was performed in 55 C. using Superscript III (Life Technologies) and random hexamers (Promega; Madison; WI; USA). PCR products were separated in 1.5% agarose gels in 0.5TBE buffer and were dyed using ethidium bromide.

(20) Plasmids and Virus Vectors

(21) Expression cassettes encoding interfering RNA were containing H1 promoter, sequence of sense strand, loop sequence, antisense strand sequence and terminator sequence consisting of 5 uridines. Vector was also encoding reporter gene sequence copGFP from CMV promoter or GFP from EFla promoter. Expression cassettes encoding interfering RNA were generated using DNA oligonucleotides (Sigma-Aldrich). Oligonucleotides DNA pairs were ligated into pGreenPuro expression plasmid (System Biosciences) and construct sequence was confirmed using sequencing.

(22) Virus Assembly and Fibroblasts Transduction

(23) In order to produce lentiviral vectors, plasmids containing expression cassettes with interfering RNA were cotransfected into HEK293TN cells with packaging plasmids pPACKH1-GAG, pPACKH1-REV and pVSV-G (System Biosciences). Medium with lentivirus was harvested on day 2 and 3, filtered, lentivirus particles were concentrated using PEGit Virus Precipitation Solution (System Biosciences). Lentiviral vectors were suspended in Opti-MEM medium (Gibco) and amount of virus particles was tested (TU/ml) using flow cytometry (Accuri C6, BD Biosciences), basing on expression of reporter gene copGFP or GFP. Fibroblasts were transduced using MOI (multiplicity of infection) 1 and 10 in presence of polybrene (4 g/ml).

SUMMARY

(24) In previously described publications and patents there is no solution showing usage of RNAi technology vector reagents for selective silencing of mutant genes containing expanded CAG tracts targeting directly at mutation region. In contrast to previously proposed solutions, interfering RNA being subject of the application acts in highly allele-selective way preferentially silencing expression of mutant alleles aiming at CAG repeats. This effect was obtained thanks to the introduction of specific substitutions into the sequence of interfering RNA, which leads to formation of non-canonical base pairs in interaction with CAG sequence of interest. Interference RNAs pool produced during cellular biogenesis selectively lowers level of mutant proteins leaving wild-type proteins on normal level. Proposed solution reduces also off-target effect, which relies on non-specific activity of interference RNA towards other transcripts containing short CAG and CUG repeats. The use of viral vectors gives an opportunity to deliver interfering RNA to hardly-accessible tissues such as brain. Moreover, in contrast to approaches employing synthetic RNAi molecules, it gives a possibility for a long-term expression of interference RNA in affected tissue without need of multiple administration repeats.

(25) Therapeutic approaches which employ aiming at the repeat sequences in poliQ diseases are more universal in comparison with aiming at the gene-specific sequences, and even more at polymorphic SNP sequences. Versatility of the invention is based on the targeting of interference RNA at mutant CAG-repeat sequences present in at least 9 polyglutamine diseases.

(26) TABLE-US-00001 Sequencelisting Numberof Typeof mismatches mismatches SEQ. withCAG with ID 5-3 guidestrand sequenceof sequenceof No. sequence interest interest 1 CUGCUGCAGCUGCUGCUGCUGC 1 A:A 2 GCUGCUGCAGCUGCUGCUGCU 1 A:A 3 CUGCUGCAGCUGCUGCAGCUGC 2 A:A 4 GCUGCUGCAGCUGCAGCUGCU 2 A:A 5 GCUGCUGCUGCAGCAGCUGCU 2 A:A 6 UGCUGCUGCUGCAGCAGCUG 2 A:A 7 GCUGCUGCUAAAGCAGCUGCU 4 A:A 8 CUGCUGCGGCUGCUGCUGCUGC 1 A:G 9 GCUGCUGCGGCUGCUGCUGCU 1 A:G 10 CUGCUGCAGCUGCUGCGGCUGC 2 A:G 11 GCUGCUGCGGCUGCGGCUGCU 2 A:G 12 GCUGCUGCUGCGGCGGCUGCU 2 A:G 13 UGCUGCUGCUGCGGCGGCUG 2 A:G 14 CUGCUGCGGCGGCUGCGGCUGC 3 A:G 15 GCUGCUGCGGCUGCGGCGGCU 3 A:G 16 UGCUGCUGCGGCGGCGGCUG 3 A:G 17 GCUGCUGCUGCGGCGGCGGCU 3 A:G 18 UGCUGCUGCUGCGGCGGCGG 3 A:G 19 CUGCUGCUGCUGCGGCGGCGGC 3 A:G 20 UGCUGCUGCGGCGGCGGCGG 4 A:G 21 CUGCUGCUGCGGCGGCGGCGGC 4 A:G 22 GCUGCUGCUGGGGCGGCUGCU 4 A:G SEQ. ID No. 23 5 CUUCCUGUCA3

REFERENCES

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