METHODS AND SUBSTANCES FOR DIRECTED RNA EDITING

Abstract

The invention relates to methods and substances for the targeted alteration of genetic information on an RNA level. The substances are artificially produced guide RNAs, which are capable of recruiting endogenous editing enzymes, such as hADAR enzymes, in particular hADAR2 and hADAR1, in order to introduce targeted point mutations in selected mRNAs. The guide RNA consists of multiple segments and is constructed in such a way that individual nucleotides from different segments pair to form a double helix, and the nucleotides of a determined segment form a hairpin structure within the guide RNA. The invention also relates to the method for directed RNA editing, wherein the guide RNA is transfected into the cells in which the RNA editing is to be carried out. The substances and the method can be used for repairing individual, e.g. disease-relevant point mutations, such as those leading to premature stop signals. An advantage of the invention is that endogenous editing enzymes are also used in order to introduce targeted point mutations into the RNA. Only the short guide RNA, used for recruiting endogenous editing enzymes, must be artificially produced for each specific problem and ectopically expressed.

Claims

1. A guide RNA for targeted RNA editing having at least the following nucleotide segments coupled to one another, listed from the 5 end: Segment A: Nucleotide sequence having a length of three to five bases, wherein a guanosine is always in position 1 and a uridine is always in position 2; Segment B: Nucleotide sequence having a length of three to five bases, wherein an adenosine is always in position 1; Segment C: Nucleotide sequence having a length of eight to ten bases, wherein a guanosine is always in position 1; Segment D: Nucleotide sequence UAUGCUAAAUG or UAUGCUCAAUG; Segment E: Nucleotide sequence having a length of eight to ten bases, wherein a guanosine is always in the last position; Segment F: Nucleotide sequence having a length of three to five bases, wherein a cytosine is always in the last position; Segment G: Nucleotide sequence having a length of three to five bases, wherein an adenosine is always in the next-to-the-last position and a cytosine is in the last position; Segment H: Nucleotide sequence having a length of five to nine bases, wherein a cytosine or a uridine is always in the last position; Segment I: Nucleotide sequence having a length of eight to twenty bases, wherein individual nucleotides of the segments A and G, B and F, or C and E pair to form a double helix and the nucleotides of segment D form a hairpin structure.

2. The guide RNA according to claim 1, wherein segment A has the length of four nucleotides, and in particular has the GUGG nucleotide sequence.

3. The guide RNA according to claim 1, wherein segment B has the length of four nucleotides, and in particular has the AAUA nucleotide sequence.

4. The guide RNA according to claim 1, wherein segment C has the length of nine nucleotides, and in particular has the GUAUAACAA nucleotide sequence.

5. The guide RNA according to claim 1, wherein segment E has the length of nine nucleotides, and in particular has the UUGUUAUAG nucleotide sequence.

6. The guide RNA according to claim 1, wherein segment F has the length of four nucleotides, and in particular has the UAUC nucleotide sequence.

7. The guide RNA according to claim 1, wherein segment G has the length of four nucleotides, and in particular has the CCAC nucleotide sequence.

8. The guide RNA according to claim 1, wherein segments H and I are constructed such that, they pair with the mRNA to be edited and place the base to be edited in an A:C mismatch pair.

9. The guide RNA according to claim 1, wherein it contains one of the sequences SEQ ID NO: 1 through SEQ ID NO: 7.

10. The guide RNA according to claim 1, wherein a hairpin structure, especially a BoxB motif, is appended on the 3 end.

11. A method for directed RNA editing in which the guide RNA according to claim 1 is transfected into the cells in which the RNA editing is to be carried out.

12. The method according to claim 11, wherein the transfection occurs by means of a plasmid that codes for the guide RNA and at least one U6 promotor for the transcription of the guide RNA.

13. A method for targeted alteration of genetic information on the RNA level comprising use of the guide RNA according to claim 1.

14. The method according to claim 13 for the repair of individual point mutations.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In the figures, the broken line represents the constructed inventive guide RNA, while the solid line represents the target mRNA to be edited; the asterisk marks the nucleotide to be edited in the target mRNA.

[0040] FIG. 1: depicts the structure of a natural editing motif of the GluR2 transcript with the bound ADAR2, which has one deaminase domain and two dsRNA binding domains (dsRBD1 and dsRBD2).

[0041] FIG. 2: depicts the general structure of the inventive guide RNA. The letters A through I mark the individual segments of the guide RNA;

[0042] FIG. 3: depicts the structure of the preferred embodiment of the inventive guide RNA;

[0043] FIG. 4: depicts the structure of the constructed guide RNA for the targeted editing of W58X stop codon in the GFP gene as an exemplary embodiment according to the SEQ ID NO 1;

[0044] FIG. 5: depicts the structure of the constructed guide RNA for the targeted editing of W58X stop codon in the GFP gene as an exemplary embodiment according to the SEQ ID NO 3.

[0045] FIG. 6: depicts the structure of the constructed guide RNA for the targeted editing of the R407Q mutation in the CAG codon in the PINK1 gene as an exemplary embodiment;

[0046] FIG. 7: depicts the structure of the constructed guide RNA for the targeted editing of the W437X mutation in the UAG codon in the PINK1 gene as an exemplary embodiment;

[0047] FIG. 8: depicts the structure of the constructed guide RNA for the targeted editing of the W417X mutation in the UAG codon in the luciferase gene as an exemplary embodiment;

[0048] FIG. 9: depicts the structure of the constructed guide RNA for the targeted editing of a disease-relevant mutation R521H in the CAC codon in the FUS gene as an exemplary embodiment.

DETAILED DESCRIPTION OF THE INVENTION

[0049] HEK 293T cells were used for transfecting the guide RNA according to the invention. For each experiment, 1.75*10cells were prepared in a 24-well plate format on the day prior to the transfection. Plasmids that are specific for human cell lines were used for the experiments. Lipofectamine 2000 (Invitrogen) was used as the transfection reagent.

Targeted Editing of W58X Stop Codon in the GFP Gene

[0050] W58X is a mutation in the TGG codon and leads to the TAG stop codon. The guide RNAs constructed for this exemplary embodiment are illustrated in FIG. 4 and FIG. 5. [0051] a) Standard HEK293T cells as described in the foregoing. [0052] b) HEK293T cells with inducible ADAR2 protein expression (genomically integrated ADAR2). This integrated cell line, in which ADAR2 is induced, has approx. 20-times lower expression of the protein than the cells in the preceding examples, which after transfection each express ADAR2 with 300 ng plasmid. In this example, therefore, the intracellular concentrations of the ADAR2 proteins correspond more closely to those that may be found in a natural cell. [0053] a) The cells were cotransfected with the following plasmids: [0054] 300 ng plasmid that codes for W58X. The plasmid is a pcDNA3.1 vector that includes a CMV promotor for gene expression of W58X; [0055] 300 ng plasmid that codes for hADAR2. The plasmid is a pcDNA3.1 vector that includes a CMV promotor for gene expression of hADAR2; [0056] 1600 ng plasmid that codes for the guide RNA. The plasmid is a 2.1-U6 hygro vector that includes a U6 promotor for transcription of the guide RNA. [0057] b) The cells were cotransfected with the following plasmid and hADAR2 expression was induced with doxycycline: [0058] 1300 ng plasmid that codes for the guide RNA.

[0059] Both examination by microscope and RNA isolation were used to evaluate whether the editing of the W58X GFP mRNA took place.

[0060] Successful editing was evidenced by microscope using fluorescent GFP signal within the cells. The editing result is considered positive if the W58X GFP is edited and therefore corrected. Therefore fluorescent cells seen by microscope denote successful editing.

[0061] The cells transfected with the aforesaid plasmids were analyzed in the fluorescence microscope in numerous independent experiments and fluorescent cells were observed, suggesting successful editing. In addition, a positive control, in which the plasmid for the correct wild type GFP was transfected instead of the plasmid for W58X GFP, was analyzed. As expected, fluorescent cells were observed by microscope for the positive control.

[0062] The same experiments were conducted as a negative control, wherein either the plasmid that codes for the guide RNA or the plasmid that codes for the ADAR2 enzyme was omitted. There was no GFP in the fluorescence microscope, i.e., no positive editing was detectable. Nor was any editing detected in the sequencing of these RNA isolation specimens, which suggests the specificity of this method.

[0063] In the analysis of the isolated RNA, first there was reverse transcription to cDNA, which was multiplied then by PCR (polymerase chain reaction) so that sequencing could then be performed. The ratio of adenine to guanine in the sequencing traces at this target position provided information about how strong the editing was. In the experiment, compared to negative controls, the ratio of adenine to guanine in the cells transfected with the aforesaid plasmids was considerably higher, which suggests successful editing. When using the plasmid that codes for the guide RNA with the SEQ ID no. 1, conduct of part a) of the experiment demonstrated an editing yield of 58%, and with the guide RNA with the SEQ ID no. 3 demonstrated an editing yield of 38%. In the conduct of part b) of the experiment, the guide RNA with SEQ ID no. 1 demonstrated an editing yield of 42% and the guide RNA with SEQ ID no. 3 demonstrated an editing yield of 58%.

Targeted Editing of a Disease-Relevant Mutation in the PINK1 Gene

[0064] The PINK1 gene is linked to Parkinson's disease. In this exemplary embodiment, the so-called R407Q mutation was edited in the CAG codon.

[0065] FIG. 6 illustrates the guide RNA constructed for this exemplary embodiment.

[0066] The cells were cotransfected with the following plasmids: [0067] 300 ng plasmid that codes for PINK1 R407Q. The plasmid is a pcDNA3.1 vector having a CMV promotor for gene expression; [0068] 300 ng plasmid that codes for hADAR2; [0069] 1600 ng plasmid that codes for the guide RNA.

[0070] The editing was analyzed by means of RNA isolation. Editing in the cells transfected with all of the aforesaid plasmids reached 35-40% at the target position. In the other exemplary embodiment, the W437X mutation was edited in the TAG codon. In this way a disease-relevant phenotype (loss of mitophagy) could be repaired.

[0071] FIG. 7 illustrates the guide RNA constructed for this exemplary embodiment.

[0072] The cells were cotransfected with the following plasmids: [0073] 300 ng plasmid that codes for PINK1 W437X. The plasmid is a pcDNA3.1 vector having a CMV promotor for gene expression; [0074] 300 ng plasmid that codes for hADAR2; [0075] 1600 ng plasmid that codes for the guide RNA.

[0076] The editing was analyzed by means of RNA isolation. Editing in the cells transfected with all of the aforesaid plasmids reached 30% at the target position. In addition, a PINK1 functionality assay was performed in HeLa cells and the loss-of-function phenotype could only be restored (microscopic analysis) if hADAR2 and the guide RNA were transfected. Restoration of mitophagy in HeLa cells could be demonstrated using the microscope.

Targeted Editing of a Mutation in the Luciferase Gene

[0077] The so-called W417X mutation was edited in the TAG codon in this exemplary embodiment. FIG. 8 illustrates the guide RNA constructed for this exemplary embodiment.

[0078] The cells were transfected with the following plasmids: [0079] 300 ng plasmid that codes for W417X luciferase. The plasmid is a pcDNA3.1 vector having a CMV promotor for gene expression; [0080] 300 ng plasmid that codes for hADAR2; [0081] 1600 ng plasmid that codes for the guide RNA.

[0082] The editing of the W417X luciferase mRNA was analyzed by means of RNA isolation. Editing in the cells transfected with all of the aforesaid plasmids reached 48% at the target position. In the negative control, the plasmid having the specific guide RNA was omitted during the transfection. There was no editing in the negative control, which suggests the exclusivity of the guide RNA in the editing.

Targeted Editing of a Disease-Relevant Mutation in the FUS Gene

[0083] This gene is linked to ALS (amyotrophic lateral sclerosis), a fatal neuro-degenerative disorder. The mutation of this gene is called R521H and includes a CAC codon, while the functional FUS has a CGC codon.

[0084] FIG. 9 illustrates the guide RNA constructed for this exemplary embodiment.

[0085] This gene was edited in the PCR reaction vessel. To this end, 350 nM purified ADAR2 protein, 125 nM specific 16 nt long guide RNA, and 25 nM of the mutated R521H FUS mRNA was used. The sequencer result demonstrated that 51% of the adenosines were successfully edited to inosine.

Targeted Editing of Endogenous Transcripts by Transfecting the Guide RNA

[0086] Six different genes (-actin, GAPDH, GPI, GUSB, VCP, RAB7A) that are expressed endogenously at different strengths were edited. These were not disease-relevant mutations that have been addressed, but instead depict the attainability of naturally expressed mRNAs. The following cells were used for transfection:

a) standard HEK293T cells (as above) and
b) HEK cells having inducible ADAR2 protein expression (genomically integrated ADAR2). This integrated cell line, in which ADAR2 is induced, has approx. 20-times lower expression of the protein than the cells in the foregoing examples that express ADAR2 after transfection at 300 ng plasmid each. In this example, therefore, the intracellular concentrations of the ADAR2 proteins thus correspond more to those found in a natural cell.

[0087] The guide RNA structures of the flexible part H+I for the specific target genes are provided in Table 2.

TABLE-US-00001 TABLE2 ListoftheguideRNAsthatwereusedfor editingendogenoustargetgenes. NameoftheguideRNA SequenceofH+ I5.fwdarw.3 TAG#1-Actin ACGCAACCAAGUCAUA TAG#3-Actin GCAAUGCCAUCACCUC TAG#1GAPDH AGGGGUCCACAUGGCA TAG#2GAPDH GGCUCCCCAGGCCCCU TAG#1GPI UGCCGUCCACCAGGAU TAG#1GusB CAGAUUCCAGGUGGGA TAG#2GusB UCCCUGCCAGAAUAGA TAG#1VCP CUCCGCCCACCAAAUG TAG#2VCP CCCAAACCACAACAGA TAG#3VCP ACCCACCCACCCAGGU TAG#1RAB7A CUGCCGCCAGCUGGAU TAG#2RAB7A AGGGAACCAGACAGUU
a) The cells were cotransfected with the following plasmids: [0088] 300 ng plasmid that codes for hADAR2; [0089] 1300 ng plasmid that codes for the guide RNA.

[0090] The editing was analyzed by means of RNA isolation. The ratio of adenine to guanine in the cells transfected with these plasmids was considerably higher than in the negative controls, suggesting successful editing.

b) The cells were cotransfected with the following plasmid and hADAR2 expression was induced with doxycycline: [0091] 1300 ng plasmid that codes for the guide RNA.

[0092] The editing was analyzed by means of RNA isolation. The ratio of adenine to guanine in the cells transfected with these plasmids was considerably higher than in the negative controls, suggesting successful editing.

TABLE-US-00002 Gene Part a) of experiment Part b) of experiment -Actin TAG#1 26% 16% TAG#3 24% 14% GAPDH TAG#1 19% 10% TAG#2 20% 11% GPI TAG#1 16% 12% GUSB TAG#1 10% 10% TAG#2 23% 18% VCP TAG#1 23% 23% TAG#2 16% 15% TAG#3 12% 13% RAB7A TAG#1 31% 38% TAG#2 28% 35%

REFERENCES

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