Crispr-cas3 system containing modified crrna

Abstract

The present invention has successfully improved the stability and effectiveness of genome editing in a CRISPR-Cas3 system by: subjecting a loop region and the flanking regions thereof present in crRNA repeat sequence to 2'-O-methyl modification and phosphate thioester modification; and dividing a set of proteins into two groups to express the proteins from two mRNA molecules and incorporating a Cap1 structure into the mRNA molecules encoding the set of proteins. In addition, high-efficiency in vivo genome editing has been successfully achieved by a single injection of lipid nanoparticles containing the CRISPR-Cas3 system into mice.

Description

CRISPR-Cas3 systems containing modified crRNA

[0001] The present invention relates to a CRISPR-Cas3 system comprising a modified crRNA and its medical application.

[0002] Transthyretin amyloidosis (ATTR) is a complex disease characterized by the deposition of amyloid-like deposits of the transthyretin (TTR) protein. ATTR is divided into two main forms: hereditary transthyretin amyloidosis (ATTRv) and wild-type transthyretin amyloidosis (ATTRwt). Transthyretin, primarily synthesized in the liver, forms tetramers to transport thyroid hormones and vitamin A in the blood and cerebrospinal fluid. In ATTRv, genetic mutations in the TTR gene result in structurally unstable TTR tetramers. These unstable tetramers disassemble, and misfolded TTR units aggregate as fibrillar amyloids, which deposit in tissues and organs, causing progressive systemic damage. In contrast, ATTRwt is not caused by a genetic mutation and results in the aggregation of normal wild-type TTR protein. ATTRwt is recognized as a cause of heart failure, primarily in elderly individuals, but diagnosis is challenging due to its non-hereditary nature and overlap with other cardiac diseases. ATTRwt deposits consist of unmutated TTR, primarily affect the heart, and are known as senile systemic amyloidosis. Current treatments for ATTR include symptomatic treatment for each symptom, as well as TTR tetramer stabilizers to improve structural instability and small interfering RNA (siRNA) agents to inhibit TTR production. However, liver transplantation, which is currently performed to maintain normal TTR production, poses the challenge of securing donor organs. siRNA agents have been reported to reduce blood TTR concentrations and improve symptoms, and TTR tetramer stabilizers have also been shown to slow symptom progression, but they require continued administration and are not a fundamental treatment.

[0003] In recent years, development of new therapeutic approaches using genome editing technology to rewrite gene sequences has been progressing. The CRISPR-Cas system is a revolutionary gene therapy tool and has shown promising results in this field. Finn et al. successfully knocked out the Ttr gene in the liver (~70%) and reduced serum TTR levels (>87%) by administering the genome editing tool CRISPR-Cas9 to mice via a lipid nanoparticle (LNP) delivery system targeted to liver cells (NPL 1). A similar method has also been applied to primates, and a phase 1 clinical trial is currently underway in the United States (NCT04601051, NPL 2). As of November 2022, reports have shown that blood TTR levels were sustained by more than 90% for 4 to 6 months after administration. However, the safety of this treatment and its impact on symptoms are not yet fully understood. One concern with gene editing using CRISPR-Cas9 is the possibility of in-frame mutations. Approximately one-third of edits lead to base deletions, potentially introducing new mutations into the TTR gene. Another concern is the potential for off-target mutations in untargeted genes.

[0004] CRISPR-Cas systems are classified into Class 1, which consists of multiple Cas proteins (Cas3, 5, 6, 7, 8, 11), and Class 2, which consists of a single Cas protein such as Cas9. While CRISPR-Cas9 recognizes 20-residue base sequences, the CRISPR-Cas3 system can distinguish longer 27-residue base sequences, demonstrating higher sequence specificity (Non-Patent Document 3). Furthermore, CRISPR-Cas3 can cause large-scale gene deletions without in-frame mutations.

[0005] For this reason, there is a need to develop treatments that utilize these properties of the CRISPR-Cas3 system for various diseases, including transthyretin amyloidosis.

[0006] Finn, J. D. et al. Cell Rep 22, 2227-2235 (2018) Gillmore, J. D. et al. N Engl J Med 385, 493-502 (2021) Morisaka, H. et al. Nat Commun 10, 5302 (2019)

[0007] The present invention aims to provide an improved CRISPR-Cas3 system that can be used to treat various diseases, including transthyretin amyloidosis.

[0008] The present inventors conducted extensive research to solve the above-mentioned problems and succeeded in improving the stability and efficacy of genome editing in the CRISPR-Cas3 system by performing 2'-O-methyl and thiophosphate modifications on the loop region of the crRNA repeat sequence and the region outside it, employing a Cap1 structure in the mRNA encoding a group of proteins, and dividing the protein group into two groups and expressing them with two mRNAs. Furthermore, by administering lipid nanoparticles encapsulating this CRISPR-Cas3 system to mice, they succeeded in highly efficient in vivo genome editing.

[0009] Furthermore, in editing the transthyretin gene, an example of a therapeutic target gene, the CRISPR-Cas3 system was found to reduce cytotoxicity, immunogenicity, and off-target mutations compared to the CRISPR-Cas9 system, and genome editing efficiency was significantly improved by using crRNA targeting the 5'UTR region or exon 1 of the gene.

[0010] From the above, the present inventors have found that an improved CRISPR-Cas3 system can provide a treatment that is both effective and safe, and have completed the present invention.

[0011] More specifically, the present invention includes the following aspects.

[0012] (1) A crRNA of the CRISPR-Cas3 system, which has a 5' repeat sequence-spacer sequence-3' repeat sequence structure, in which the ribose of the nucleotide is 2'-O-methyl modified and the phosphodiester bond between the nucleotides is thiophosphate modified in at least a part of each of the following regions (a) and (b):

[0013] (a) a region constituting the loop of the 5' repeat sequence and a region 5' thereof; (b) a region constituting the loop of the 3' repeat sequence and a region 3' thereof; (2) The crRNA according to (1), in which the riboses of at least 50% of the nucleotides in each of the regions (a) and (b) are 2'-O-methyl modified.

[0014] (3) The crRNA described in (1), in which at least four phosphodiester bonds from the 5' end of the region (a) and at least four phosphodiester bonds from the 3' end of the base (b) are thiophosphate modified.

[0015] (4) A CRISPR-Cas3 system comprising the crRNA according to any one of (1) to (3) and mRNA encoding a group of proteins constituting the CRISPR-Cas3 system.

[0016] (5) The CRISPR-Cas3 system according to (4), wherein the mRNA has a Cap1 structure.

[0017] (6) The CRISPR-Cas3 system according to (4), wherein the proteins constituting the CRISPR-Cas3 system are divided into two groups, and the proteins of each group are encoded by a single mRNA.

[0018] (7) A lipid nanoparticle encapsulating the CRISPR-Cas3 system according to any one of (4) to (6).

[0019] (8) The lipid nanoparticle described in (7), wherein the crRNA targets the transthyretin gene.

[0020] (9) The lipid nanoparticle described in (8), wherein the crRNA targets the 5'UTR or exon 1 of the transthyretin gene.

[0021] (10) A therapeutic agent for transthyretin amyloidosis, comprising the lipid nanoparticles according to (8) or (9).

[0022] The CRISPR-Cas3 system of the present invention enables highly efficient genome editing while suppressing off-target mutations not only in vitro but also in vivo. In particular, when applied to the medical field, it can provide gene therapy that is both effective and safe.

[0023] (A) is a diagram showing crRNA targeting the area surrounding exons 1 and 2 of the mouse Ttr gene. (B) is a graph showing the results of comparative quantification of the genome editing efficiency of the CRISPR-Cas3 system using droplet digital PCR (ddPCR) for Hepa1-6 cells transfected with each crRNA. N1-Me-Pseudo UTP-modified Cap1-mRNA for each Cas was introduced into Hepa1-6 cells using three mRNA transfection patterns (all-in-one (AiO), separated (Sep), and divide-in-two (DiT)). Modified crRNA #2 is shown. In the crRNA sequence, an "*" indicates a phosphorothioate modification of the internucleotide phosphodiester bond, and an "- (underline)" indicates a 2'-O-methyl modification of the ribose of the nucleotide. The base sequence of the crRNA is shown in SEQ ID NO: 13. Modified crRNA #2 is shown. In the crRNA sequence, an "*" indicates a phosphorothioate modification of the internucleotide phosphodiester bond, and an "- (underline)" indicates a 2'-O-methyl modification of the ribose of the nucleotide. The base sequence of the crRNA is shown in SEQ ID NO: 13. Modified crRNA #2 is shown. In the crRNA sequence, an "*" indicates a phosphorothioate modification of the internucleotide phosphodiester bond, and an "- (underline)" indicates a 2'-O-methyl modification of the ribose of the nucleotide. The base sequence of the crRNA is shown in SEQ ID NO: 13.

[0023] Figure 1 shows the results of evaluating the effect on genome editing efficiency by ddPCR analysis after introducing a CRISPR-Cas3 system containing crRNA modified in five patterns into Hepa1-6 cells. Figure 2 shows the administration and sampling schedule for in vivo editing using the CRISPR-Cas3 system in ICR mice. Figure 3 shows the results of measuring serum Ttr concentrations by ELISA one week after administration in ICR mice intravenously administered with various concentrations of the CRISPR-Cas3 system encapsulated in lipid nanoparticles (containing TTR#2 Stem1 ver2 as crRNA and using Sep or DiT as mRNA).Graphs showing the results of measuring serum Ttr levels over time up to 4 weeks after administration to ICR mice. The dose of the CRISPR-Cas3 system encapsulated in lipid nanoparticles was 6 mg / kg. (A) is a graph showing the genome editing efficiency in genomic DNA (gDNA) extracted from liver cells of ICR mice 4 weeks after administration of 6 mg / kg of DiT-Cas3-LNP, and (B) is a photograph showing the results of detecting Ttr in liver tissue by immunostaining. Electrophoresis photographs showing the results of genome editing in ICR mice administered 6 mg / kg of DiT-Cas3-LNP, detected by PCR amplification of the target region. Graphs showing the results of measuring the levels of each cytokine in the serum of ICR mice administered 6 mg / kg of DiT-Cas3-LNP over time using an ECL multiplex assay. Electrophoresis photographs showing the results of detecting Cas3 over time by Western blot analysis in the liver tissue of ICR mice administered 6 mg / kg of DiT-Cas3-LNP. Graphs showing the results of detecting each Cascade RNA and crRNA over time by RT-PCR analysis in the liver tissue of ICR mice administered 6 mg / kg of DiT-Cas3-LNP. A diagram showing crRNA targeting the area around exons 1 and 2 of the human Ttr gene. A diagram showing the pCas3-Cascade-GFP plasmid into which HepG2 cells were also introduced together with each crRNA. A graph showing the results of comparative quantification of genome editing efficiency of the CRISPR-Cas3 system by droplet digital PCR (ddPCR) for HepG2 cells introduced with each crRNA. A graph showing the results of comparative quantification of TTR mRNA expression by RT-PCR for HepG2 cells introduced with each crRNA. 1 shows in vivo editing of the TTR gene by the CRISPR-Cas3 system in mice with a humanized TTR gene. A graph showing the results of measuring serum TTR concentrations by ELISA one week after intravenous administration of the CRISPR-Cas3 system (containing #UT05 or #cr2 as crRNA and DiT as mRNA) encapsulated in lipid nanoparticles.As a control, #NTLA2001 used in the CRISPR-Cas9 system was used.

[0024] The present invention provides modified crRNA in a CRISPR-Cas3 system.

[0025] CRISPR-Cas3 systems are classified into six types: Type IA, Type IB, Type IC, Type ID, Type IE, and Type IF, as well as Type I-G, a subtype of Type IB (see, for example, van der Oost J et al. (2014) Unraveling the structural and mechanistic basis of CRISPR-Cas systems, Nature Reviews Microbiology, Vol. 12 (No. 7), pp. 479-492; Jackson RN et al. (2014) Fitting CRISPR-associated Cas3 into the Helicase Family Tree, Current Opinion in Structural Biology, Vol. 24, pp. 106-114).

[0026] In the CRISPR-Cas3 system, crRNA forms a complex with Cas3 protein and Cascade protein, and the complex acts on target DNA.

[0027] The crRNA of the present invention typically has a structure of "5' repeat sequence-spacer sequence-3' repeat sequence".

[0028] The "repeat sequence" is a sequence that is repeated via a spacer sequence in the CRISPR structure of the bacterial genome from which the CRISPR-Cas3 system is derived. The wild-type repeat sequence differs depending on the subtype of the CRISPR-Cas3 system and the type of bacteria from which it is derived. For example, in the Type IA CRISPR-Cas system derived from Pyrococcus furiosus, it typically consists of the 30-base sequence set forth in SEQ ID NO: 1, and in Synechocystis sp. The type I-B CRISPR-Cas system derived from Neisseria lactamica typically consists of the nucleotide sequence set forth in SEQ ID NO: 2, which has a chain length of 36 bases; the type I-C CRISPR-Cas system derived from Neisseria lactamica typically consists of the nucleotide sequence set forth in SEQ ID NO: 3, which has a chain length of 32 bases; the type I-D CRISPR-Cas system derived from Microcystis aeruginosa typically consists of the nucleotide sequence set forth in SEQ ID NO: 4, which has a chain length of 37 bases; the type I-E CRISPR-Cas system derived from Escherichia coli typically consists of the nucleotide sequence set forth in SEQ ID NO: 5, which has a chain length of 29 bases; the type I-F CRISPR-Cas system derived from Pseudomonas aeruginosa typically consists of the nucleotide sequence set forth in SEQ ID NO: 6, which has a chain length of 28 bases; and the type I-G CRISPR-Cas system derived from Thioalkalivibrio sulfidiphilus typically consists of the nucleotide sequence set forth in SEQ ID NO: In the CRISPR-Cas system, it typically consists of the base sequence set forth in SEQ ID NO: 7, which has a chain length of 36 bases.

[0029] In the crRNA of the present invention, the "repeat sequence" is typically a wild-type repeat sequence, but may contain mutations (addition, deletion, substitution, and / or insertion of bases) as long as it functions by forming a complex with the proteins that make up the CRISPR-Cas3 system.

[0030] In the crRNA of the present invention, the "spacer sequence" is a sequence designed as a sequence complementary to the target DNA. The target DNA may be endogenous DNA or exogenous DNA. Examples of endogenous DNA include genomic DNA in chromosomes, mitochondria, and chloroplasts. Examples of exogenous DNA include reporter genes, marker genes, and genes of viruses, bacteria, protozoa, etc. that infect hosts. When the crRNA of the present invention is used for human gene therapy, the spacer sequence can be designed as a sequence complementary to the human therapeutic target gene.

[0031] In the crRNA of the present invention, the ribose of the nucleotide is 2'-O-methyl modified (a methyl group (-CH ３ ) and the internucleotide phosphodiester bond is modified with a phosphorothioate (substitution of a sulfur atom for the non-bridging oxygen of the phosphodiester bond).

[0032] (a) The region that constitutes the loop of the 5' repeat sequence and the region 5' thereof (hereinafter referred to as the "5' target modification region"); (b) The region that constitutes the loop of the 3' repeat sequence and the region 3' thereof (hereinafter referred to as the "3' target modification region"); In the typical crRNA processing process, a part of the repeat sequence forms a loop structure, and this loop structure is cleaved by the action of a specific Cas (e.g., Cas6, Cas5).

[0033] In the crRNA of the present invention, the "region forming the loop structure" is typically the 5th to 25th nucleotides of the repeat sequence in Type I-A (SEQ ID NO: 1), the 13th to 32nd nucleotides of the repeat sequence in Type I-B (SEQ ID NO: 2), the 2nd to 21st nucleotides of the repeat sequence in Type I-C (SEQ ID NO: 3), the 14th to 34th nucleotides of the repeat sequence in Type I-D (SEQ ID NO: 4), the 5th to 22nd nucleotides of the repeat sequence in Type I-E (SEQ ID NO: 5), the 6th to 20th nucleotides of the repeat sequence in Type I-F (SEQ ID NO: 6), and the 6th to 24th nucleotides of the repeat sequence in Type I-G (SEQ ID NO: 7).

[0034] The cleavage sites of the repeat sequences by specific Cas (e.g., Cas6, Cas5) are as follows: Type I-A: between the 22nd and 23rd nucleotides of the repeat sequence; Type I-B: between the 28th and 29th nucleotides of the repeat sequence; Type I-C: between the 19th and 20th nucleotides of the repeat sequence; Type I-D: between the 31st and 32nd nucleotides of the repeat sequence; Type I-E: between the 21st and 22nd nucleotides of the repeat sequence; Type I-F: between the 20th and 21st nucleotides of the repeat sequence; and Type I-G: between the 28th and 29th nucleotides of the repeat sequence.

[0035] In the crRNA of the present invention, the riboses of at least 50% or more (e.g., 60% or more, 70% or more, 80% or more, 90% or more) of the nucleotides in the 5'-target modification region are preferably 2'-O-methyl modified. To avoid negative effects on crRNA processing, it is preferable that the riboses of the two nucleotides in the 5'-target modification region that sandwich the processing cleavage site are not 2'-O-methyl modified. Particularly preferably, in the 5'-target modification region, the riboses of all nucleotides other than the two nucleotides that sandwich the processing cleavage site are 2'-O-methyl modified.

[0036] Furthermore, in the crRNA of the present invention, preferably, in the 3'-target modification region, at least 50% (e.g., 60% or more, 70% or more, 80% or more, 90% or more) of the riboses of the nucleotides are 2'-O-methyl modified. The present inventors have discovered that in the 3'-target modification region, even if cleavage by processing does not occur, the crRNA can form a complex with the proteins that make up the CRISPR-Cas3 system and function (Japanese Patent Application: Patent Application No. 2023-121605). Therefore, in the 3'-target modification region, the riboses of the two nucleotides flanking the processing cleavage site may be 2'-O-methyl modified. Unlike the 5'-target modification region, in the 3'-target modification region, the riboses of all nucleotides may be 2'-O-methyl modified.

[0037] Furthermore, the crRNA of the present invention preferably has at least four phosphodiester bonds (e.g., 7 or more, 10 or more, 15 or more, or 20 or more phosphodiester bonds) from the 5' end of the 5'-target modification region modified with phosphorothioate. To avoid negative effects on crRNA processing, it is preferable that the phosphodiester bonds between the two nucleotides flanking the processing cleavage site in the 5'-target modification region are not modified with phosphorothioate.

[0038] In addition, in the crRNA of the present invention, preferably, at least four phosphodiester bonds (e.g., 7 or more, 10 or more, 15 or more, or 20 or more phosphodiester bonds) from the 3' end of the 3' target modification region are thiophosphate modified. Unlike the 5' target modification region, all phosphodiester bonds in the 3' target modification region may be thiophosphate modified.

[0039] In the crRNA of the present invention, regions other than the 5'- and 3'-target modification regions can be modified with 2'-O-methyl riboses of nucleotides and thiophosphate esters of phosphodiester bonds between nucleotides, as long as their functions are not impaired. Examples of regions other than the 5'- and 3'-target modification regions include spacer sequence regions.

[0040] The CRISPR-Cas3 system of the present invention includes a group of proteins (Cas3 protein and Cascade protein) as components other than crRNA. Typically, Type I-A includes Cas3-HD, Cas3-HEL, Cas5, Cas7, Cas8, and Cas11; Type I-B includes Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11; Type I-C includes Cas3, Cas5, Cas7, Cas8, and Cas11; and Type I-D includes Cas3 and Cas5. Type I-E includes Cas3, Cas5, Cas6, Cas7, Cas10, and Cas11; Type I-F includes Cas2-3, Cas5, Cas6, Cas7, and Cas8; and Type I-G includes Csb2 (Cas6-like), Cas7, Cas8, Cas3, and Cas11. However, it should be understood that Cas11 can exhibit DNA editing activity even when it is excluded from the components of the CRISPR-Cas3 system (for example, it is known that Type I-B and Type I-C can exhibit DNA editing activity, albeit at a lower level than when Cas11 is included, even when Cas11 is not included, and the inventors have confirmed that this is also true for Type I-D). Therefore, the CRISPR-Cas3 system of the present invention also includes a system that does not contain Cas11 in certain subtypes.

[0041] When cleavage of target DNA is not the objective, it is also possible to use a system that does not contain a component having nuclease activity (typically a Cas3 protein, but in Type I-D, Cas10d), or a system that contains a protein in which part or all of the nuclease activity has been eliminated. In this case, the Cas3 protein or Cascade protein can be fused to a heterologous protein having the desired activity to form a chimeric protein. In this embodiment, various editing operations can be performed on the target DNA depending on the activity of the heterologous protein to be fused. Examples of the activity of the heterologous protein to be fused include, but are not limited to, deaminase activity (e.g., cytidine deaminase activity, adenosine deaminase activity), methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, photolyase activity, and glycosylase activity. In this case, a mutant in which the nuclease activity of the Cas3 protein (in the case of type ID, the Cas10d protein) is partially or completely deleted can be used.

[0042] For example, by using a fusion protein of a deaminase with a mutant in which the nuclease activity of Cas3 has been partially or completely eliminated as a component of the CRISPR-Cas3 system of the present invention, precise genome editing can be performed to replace bases without causing deletions at the target site. Techniques for applying deaminase to the CRISPR-Cas system are known (Nishida K. et al., Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems, Science, DOI: 10.1126 / science.aaf8729, (2016)), and can be applied to the CRISPR-Cas3 system of the present invention.

[0043] Furthermore, gene transcription at a target site can be regulated by fusing the Cas3 protein or Cascade protein with a desired transcriptional regulatory protein and using the resulting chimeric protein. Examples of transcriptional regulatory proteins include, but are not limited to, light-inducible transcriptional regulators, small molecule / drug-responsive transcriptional regulators, transcription factors, and transcriptional repressors. In this case, too, a mutant lacking part or all of the nuclease activity of the Cas3 protein (or the Cas10d protein in the case of type I-D) can be used.

[0044] When targeting the chromosomal genome of a eukaryotic cell, it is preferable to add a nuclear localization signal to promote the localization of the Cas3 protein or Cascade protein to the nucleus. The nuclear localization signal can be added to the N-terminus and / or C-terminus of each protein. Similarly, when targeting, for example, the mitochondrial genome and chloroplast genome, it is preferable to add a localization signal that promotes localization thereto.

[0045] In the CRISPR-Cas3 system of the present invention, Cas3 and Cascade may be in the form of a polynucleotide (RNA, DNA) encoding a protein, a vector expressing the protein, or a protein. Examples of RNA include mRNA and circular RNA. In the polynucleotide form, the base sequence may be modified (e.g., codon optimization) to make it suitable for expression in a host cell.

[0046] The RNAs encoding the Cas3 protein and the Cascade protein can be designed to be incorporated into one type of (the same) RNA, or to be partially incorporated into different RNAs, or to be each incorporated into one type of RNA. Similarly, in constructing an expression vector, the DNAs encoding the Cas3 protein and the Cascade protein can be designed to be incorporated into one type of (the same) vector, or to be partially incorporated into different vectors, or to be each incorporated into one type of vector. Furthermore, polynucleotides (RNA, DNA) encoding each protein can be expressed as a single protein by linking them via a polynucleotide encoding an amino acid sequence (such as a 2A peptide) that is cleaved by intracellular proteases, and then the individual proteins can be separated by the action of the protease.

[0047] One preferred embodiment of the CRISPR-Cas3 system of the present invention is a CRISPR-Cas3 system comprising the crRNA of the present invention and mRNA encoding the Cas3 protein and the Cascade protein.

[0048] One preferred embodiment of mRNA is an mRNA having a Cap structure (for example, a Cap1 structure).

[0049] Another preferred embodiment of the mRNA is one in which the proteins constituting the CRISPR-Cas3 system are divided into two groups, and the proteins of each group are encoded by a single mRNA (two mRNAs in total). The division into the two groups is preferably performed so that the total molecular weights of the proteins in each group are similar; for example, Type I-E can be divided into Cas3, Cas5, and Cas11, and the rest.

[0050] Cells in which target DNA can be edited using the CRISPR-Cas3 system of the present invention include prokaryotic and eukaryotic cells. Prokaryotic cells include bacteria and archaea, while eukaryotic cells include animal cells, plant cells, algae cells, and fungal cells.

[0051] For administration to a living body, the CRISPR-Cas3 system of the present invention can be encapsulated in a transport carrier such as a lipid nanoparticle, which is suitable for medical applications (e.g., in vivo therapy).

[0052] Components of lipid nanoparticles typically include ionizable lipids, phospholipids, cholesterol, and PEGylated lipids. Examples of ionizable lipids include SM-102, ALC-0315, cKK-E12, C12-200, MC3, DLinDMA, DLin-MC3-DMA, DLinC2DMA, ICE, HGT5000, HGT5001, OF-02, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA, DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, HGT4003, and JK-102-CA. Examples of phospholipids include 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol sodium salt (DPPG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn 1-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium (DOPG), 1-palmitoyl-2-oleyl-sn-glycerol-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-stearyl-2-oleyl-phosphoethanolamine (SOPE), and sphingomyelin. PEGylated lipids include, for example, DMG-PEG, ALC-0159, M-DTDAM-2000, C8-PEG, DOGPEG, ceramide PEG, and DSPE-PEG.

[0053] The molar ratio of each component in the lipid nanoparticles is, for example, 35-50% ionizable lipid, 0.5-10% phospholipid, 40-50% cholesterol, and 0.5-3% PEGylated lipid.

[0054] Transporters incorporating the CRISPR-Cas3 system can be used as therapeutic agents for a variety of diseases. Examples of target diseases include, but are not limited to, transthyretin amyloidosis described in this example, as well as (1) cancers such as prostate cancer, lung cancer, brain tumors, and head and neck cancer, (2) monogenic diseases such as congenital immunodeficiency (ADA deficiency), Leber's disease, LPL deficiency, hemophilia, beta thalassemia, adrenal leukodystrophy, and dystrophy, (3) viral infections such as HIV / AIDS, hepatitis B virus, and hepatitis C virus, (4) cardiovascular diseases such as arteriosclerosis obliterans, angina pectoris, and myocardial infarction, (5) neurological diseases such as Parkinson's disease, Alzheimer's disease, and ALS, and (6) eye diseases such as retinitis pigmentosa and age-related macular degeneration.

[0055] The therapeutic agent can be administered parenterally (for example, intravenously, intramuscularly, intraperitoneally, intraarticularly, intradermally, subcutaneously, etc.) or orally, depending on the purpose of treatment.

[0056] As shown in the examples, in the in vivo treatment of transthyretin amyloidosis, the CRISPR-Cas3 system can be equipped with crRNA targeting the transthyretin gene. The crRNA can target both the coding and non-coding regions of the transthyretin gene, but preferably targets the non-coding region, 5'UTR, or the coding region, exon 1. The CRISPR-Cas3 system is preferably encapsulated in lipid nanoparticles as a delivery vehicle. Components of the lipid nanoparticles can include ionizable lipids, phospholipids, cholesterol, and PEG-DMG (e.g., in a molar ratio of 50:3:47:1.5).

[0057] The present invention will be described in more detail below with reference to examples.

[0058] A. Materials and Methods (1) crRNA Design and Plasmid Construction Five crRNAs targeting the first or second exon of the mouse Ttr gene (ENSMUST00000075312.5) were designed (Table 1).

[0059]

[0060] In addition, based on the genomic sequence of the human TTR gene (ENST00000237014.8), 10 crRNAs targeting the first or second exon were designed (Table 2).

[0061]

[0062] For each crRNA, two complementary 5'-phosphorylated oligonucleotides containing the crRNA sequence and a BbsI restriction endonuclease site overhang were synthesized, duplexed, and subcloned into the pCas3-Cascade-GFP plasmid digested with BbsI-HF (New England Biolabs, R3539L). The resulting construct was purified using the NucleoSpin® Gel and PCR Clean-up Kit (MACHEREY-NAGEL, 740609). The G211 gRNA for CRISPR-Cas9 was similarly subcloned into the pSpCas9(BB)-2A-GFP plasmid (pX458, Addgene, #48138). Each construct was verified by Sanger sequencing to confirm that the crRNA or gRNA sequence had been properly subcloned.

[0063] In addition, crRNA containing 2'-O-methyl and thiophosphate modifications was synthesized by Integrated DNA Technologies (Coralville, IA, USA).

[0064] (2) Cell Culture. The mouse liver tumor cell line Hepa1-6 (BRCB1638) and the human hepatocellular carcinoma-derived cell line HepG2 (RCB1886) were provided by the RIKEN Cell Bank. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific) and 0.1 mg / mL penicillin-streptomycin (Nacalai Tesque) on collagen I-coated dishes (IWAKI) at 37°C in a humidified atmosphere of 5% CO2.

[0065] (3) Plasmid transfection and FACS isolation of GFP-expressing cells. Hepa1-6 cells were electroporated using 4D-Nucleofector and SF Cell Line 4D-Nucleofector solution (Lonza) with program CM-138. After 48 hours, GFP-positive cells were isolated using a BD FACS Sorparia (BD Biosciences). Genomic DNA from GFP-positive cells was extracted using NucleoSpin® Tissue XS (MACHEREY-NAGEL, 740901), and RNA was extracted using the RNeasy Mini Kit (QIAGEN, 74104).

[0066] HepG2 cells were transfected with plasmids using lipofectamine 3000 (Thermo Fisher Scientific). Forty-eight hours after lipofection, cells were detached using trypsin-EDTA solution (Nacalai Tesque, 32777-44). Single-cell suspensions were prepared using a 35 μm cell strainer (Falcon, 352235), and GFP-positive cells were isolated using a BD FACS SORPAria (BD Biosciences). Genomic DNA from GFP-positive cells was extracted using NucleoSpin® Tissue XS (MACHEREY-NAGEL, 740901), and RNA was extracted using RNeasy Mini Kit (QIAGEN, 74104).

[0067] (4) Evaluation of genome editing efficiency PCR primers were designed around the target site and amplified using Quick Taq® HS DyeMix followed by agarose gel electrophoresis. For quantitative analysis of genome editing efficiency, droplet digital PCR (ddPCR) was performed. RT-qPCR was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and a CFX96 Deep Well Real-Time PCR Detection System (Bio-Rad).

[0068] (5) Capture Sequencing of Cas3 and Cas9 POT Regions Capture sequencing of Cas3- or Cas9-POT regions was performed according to a previously described method (Morisaka, H. et al. Nat Commun 10, 5302 (2019)). The target regions were selected as follows: The probes around the on-target region of Ttr covered from 90 kb upstream to 10 kb downstream of the PAM. The probes for the POT region of CRISPR / Cas3 covered from 4.5 kb upstream to 0.5 kb downstream around the potential PAM. The probes for CRISPR / Cas9 covered 1 kb upstream and downstream around the potential PAM. For NGS, genomic DNA was extracted and cleaved with the SureSelect Enzymatic Fragmentation Kit (Agilent, CA, US), and DNA libraries were prepared using SureSelectXT-HS2 reagents (Agilent) and a custom probe kit designed by SureDesign.

[0069] (6) RNA transfection and flow cytometry. GFP and Cas3, 5, 6, 7, 8, and 11 mRNAs were modified by Elixergen Scientific and transfected into Hepa1-6 cells using Lipofectamine Messenger MAX. GFP signals were detected using an Attune NxT flow cytometer. The viability of transfected cells was assessed by PI staining.

[0070] (7) LNP Formation. mRNA was loaded onto LNPs using a microfluidic mixing method. An ethanol solution containing ionizable lipids, phospholipids (DSPC), cholesterol, and PEG-DMG at a constant molar ratio (50:3:47:1.5) was prepared at a total lipid concentration of 8 mM. RNA cargo (Cas9 mRNA and sgRNA at a 1:1 weight ratio, or Cas3-5-11, Cas6-7-8, and crRNA at a 3:3:1 weight ratio) was dissolved in 50 mM acetate buffer (pH 4.0) to a concentration of 81.5 μg / ml of RNA cargo. The lipid ethanol solution and RNA solution were rapidly mixed using a bath on a glass-based iLiNP at a total flow rate of 5 mL / min and an RNA-to-lipid flow ratio of 3. The nitrogen-to-phosphate ratio was adjusted to 6. The resulting LNP solution was dialyzed against 20 mM Tris-HCl buffer (9% sucrose, pH 7.40) using Slide-A-Lyzer™ G3 Dialysis Cassettes (MWCO 20 kDa, Thermo Fisher Scientific) for at least 2 hours at 4°C. The LNP solution was ultrafiltered using an Amicon Ultra-15 unit (MWCO 100 kDa, Millipore). The size and polydispersity of the LNPs were measured using a Zetasizer Nano ZS ZEN3600 instrument (Malvern Instruments, Worchestershire, UK). The encapsulation efficiency and total concentration of mRNA were determined by Ribogreen assay.

[0071] (8) In Vivo LNP Administration All animal care and experimental procedures were approved by the Animal Experiment Ethics Committee of the University of Tokyo. Male Jcl:ICR (CLEA Japan) mice aged 3-4 weeks were used. LNPs and saline as a negative control were administered via the tail vein. Blood was collected in serum separator tubes (MiniCollect® II, Greiner Bio-One) from the median lobe of the liver for quantification of circulating TTR by ELISA.

[0072] (9) TTR ELISA Analysis. Total TTR serum levels were determined in Jcl:ICR mice using a Mouse Pre-albumin (Transthyretin) ELISA kit (Aviva Systems Biology, cat. OKIA00111). Serum was finally diluted 1,000-10,000 times. Plates were read at an absorbance of 450 nm using a SYNERGY LX multimode reader (BioTek). Serum TTR levels were calculated using Gen5 software (v3.09). Final serum values ​​were adjusted for assay dilution.

[0073] (10) TTR IHC Mice were anesthetized with isoflurane. Liver samples were fixed in 4% PFA and embedded in paraffin. Liver sections (4 μm thick) were stained with anti-TTR antibody (LSBio, LS-C407961) according to a previously described method (Finn, J. D. et al. Cell Rep 22, 2227-2235 (2018)).

[0074] (11) Cas3 Western Blot Mouse liver tissue samples were snap-frozen in liquid nitrogen and disrupted using a TissueLyser II (Qiagen, Hilden, Germany) in cell lysis buffer (Cell Signaling, Danvers, MA, USA) supplemented with protease inhibitors (Sigma-Aldrich). The disrupted samples were incubated on ice for 30 minutes and then centrifuged at 10,000 rpm for 30 minutes at 4°C. The supernatant was used as the lysate sample. Total protein samples (30 μg) were separated using Bolt™ Bis-Tris Plus Mini Protein Gels, 4-12% (Invitrogen) and transferred to a polyvinyl difluoride membrane (Invitrogen, Carlsbad, CA, USA). Cas3 was detected with rat polyclonal anti-Cas3 (1:1000, provided by C4U Corporation) followed by treatment with HRP-conjugated anti-rat IgG antibody (1:2000, Cell Signaling, #7077). α-Tubulin was detected with α-tubulin antibody (1:2000, Cell Signaling, #2144) followed by HRP-conjugated anti-rabbit IgG antibody (1:2000, Cell Signaling, #7074). Signals were detected with SuperSignal WestPico Plus Chemiluminescence Substrate (Thermo Fisher, Waltham, MA, USA), and quantitative analysis was performed using an iBright CL 1500 (Thermo Fisher).

[0075] (12) Cytokine Analysis. The tail vein was incised, and 50-100 μL of blood was collected. Serum cytokine measurements were performed 4 hours, 24 hours, 72 hours, and 1 week after administration. Serum cytokine analysis was performed using an ECL multiplex assay (U-PLEX Custom Biomarker Group 1 (Mouse) Assay, Catalog No. K15069M-1) on samples collected at the Research Center for Immunological Analysis, Inc. (Japan). Serum was diluted 3-fold using Sample Diluent 41. Data were analyzed using DISCOVERY WORKBENCH 4.0 (Meso Scale Discovery).

[0076] (13) Amplicon Sequencing. The proportion of in-frame mutations generated by Cas9 was analyzed by NGS of PCR amplicons. Amplicons from PCR were purified by column purification with the NucleoSpin® Gel and PCR Clean-up Kit (Macherey-Nagel GmbH & Co. KG, Germany) and sequenced at the NGS core facility using MiSeq (2 × 250 bp) according to standard procedures. The raw data for each sample was used to calculate ratios using CRISPRESSO2 (http: / / crispresso.pinellolab.partners.org / ) according to the developer's protocol (Clement, K. et al. Nat Biotechnol 37, 224-226 (2019)).

[0077] B. Results (1) Screening of crRNAs for genome editing of the mouse Ttr gene in Hepa1-6 cells. Five CRISPR RNAs (crRNAs #1-5) targeting exons 1 and 2 of the mouse Ttr gene were designed (Figure 1A). To efficiently screen for crRNAs that knock out the Ttr gene, we constructed an all-in-one plasmid (pRB-EF1a-Cas3Cascade-U6v2-BbsI-GFP: pRB-Cas) expressing crRNA, Cas3 / Cascade, and EGFP. The pRB-Cas plasmids containing different crRNAs were introduced into Hepa1-6 cells, a mouse hepatocellular carcinoma cell line, by electroporation. Forty-eight hours after electroporation, EGFP-positive cells were isolated by fluorescence-activated cell sorting (FACS) (BD FACS Sorparia, BD Biosciences). After PCR amplification of a 3.9-kb region, the genome-edited fragment by Cas3 was observed by agarose gel electrophoresis. The editing efficiency of Cas3 was quantified using droplet digital PCR (ddPCR). The results showed that crRNA#2 showed a 59% genome editing efficiency, while the remaining four crRNAs showed genome editing efficiencies of less than 50% (Figure 1B).

[0078] (2) On- and Off-Target Mutation Analysis. For off-target analysis, in silico analysis of potential off-target (POT) sites was performed using the GGGenome computer program (https: / / GGGenome.dbcls.jp / ) and Cas-OFFinfer (Bae, S. et al., Bioinformatics 30, 1473-1475 (2014)). 189 POT sites with 16-19 consecutive nucleotide matches to the crRNA#2 spacer sequence and 166 POT sites that tolerated 5-7 nucleotide mismatches within the 32-nucleotide #2 sequence were identified. Meanwhile, 653 POT sites that tolerated 3-5 nucleotide mismatches within the 20-nucleotide sequence of the Cas9 sgRNA G211 sequence were identified. We created custom microarrays covering 5 kb or 2 kb around each Cas3 or Cas9 POT site and performed capture sequencing analysis with 7540-9870x coverage. No significant off-target mutations were detected at the Cas3 crRNA#2 POT site. Surprisingly, various Cas9-edited indel mutations were identified at two off-target sites at the Cas9 sgRNA G211 POT site.

[0079] (3) Modified mRNA and modified crRNA for CRISPR-Cas3 in vivo editing. The Cas3 / Cascade system includes Cas3, 5, 6, 7, 8, and 11. For each Cas, N1-Me-Pseudo UTP-modified Cap1-mRNA was used. Three mRNA introduction patterns (all-in-one (AiO), separated (Sep), and divide-in-two (DiT)) were compared. AiO used a series of mRNAs linking Cas3, 5, 6, 7, 8, and 11; Sep used independent Cas mRNAs; and DiT used mRNAs linking Cas3, 5, 6, 7, 8, and 11 with mRNAs linking Cas6, 7, and 8. We introduced both 2'-O-methyl-modified crRNA #2 and the mRNAs for Cas3 / Cascade into Hepa1-6 cells by lipofection. DDPCR revealed that the N1-Me-Pseudo UTP-modified CAP1 mRNAs of the Sep and DiT types exhibited higher genome editing efficiency (Figure 2).

[0080] Next, five modification patterns of crRNA were designed and applied to the selected crRNA #2 targeting the Ttr gene (Figures 3A-C).

[0081] ddPCR analysis in Hepa1-6 cells revealed that a crRNA modified at both ends and at the stem-loop structure (TTR#2 Stem1 ver2) was the most efficient for genome editing (Fig. 3D), likely due to the RNA stability provided by the 2'-O-methyl modification and the inhibition of endonuclease activity by the thiophosphate modification.

[0082] (4) Cas3 in vivo editing in ICR mice. Modified crRNA (TTR#2 Stem1 ver2) and Cas3 / cascade were encapsulated in lipid nanoparticles (LNPs) and administered intravenously to Jcl:ICR mice as Sep-Cas3-LNP or DiT-Cas3-LNP (Figure 4A). Serum TTR levels were measured by ELISA 1, 2, 3, and 4 weeks after administration. One week after administration, serum TTR levels in mice treated with 6 mg / kg Sep-Cas3-LNP and 6 mg / kg DiT-Cas3-LNP were reduced by approximately 70% and 75%, respectively, compared to those in the saline-treated group (Figure 4B). This decrease in serum Ttr levels was observed continuously at 2, 3, and 4 weeks after administration and was dose-dependent (Fig. 4C).

[0083] PCR amplification of the Cas3 target region in genomic DNA (gDNA) extracted from liver cells 4 weeks after administration of 6 mg / kg of DiT-Cas3-LNP detected DNA fragments characteristic of Cas3-mediated genome editing (Figure 5A). ddPCR analysis demonstrated genome editing efficiency in the liver of approximately 50%. Similar results were obtained by immunohistochemistry of liver tissue (Figure 5B).

[0084] (5) Evaluation of Cytotoxicity and Immunogenicity The potential safety risk of LNPs due to bioaccumulation in the liver has become a major concern. To evaluate this, cytokine stimulation was monitored at 4, 6, 24 hours, and 1 week after administration. PCR amplification of the target region suggested that Cas3-mediated genome editing was initiated within 4 hours after LNP administration (Figure 6).

[0085] Cytokine levels, including IFN-α, TNF-α, IL-6, and MCP-1, were transiently increased 4 hours after administration but returned to levels comparable to saline controls within 1 week of administration (Figure 7). Western blot analysis confirmed that Cas3 was cleared from the liver within 24 hours of administration (Figure 8).

[0086] Furthermore, RT-PCR analysis of cascade RNAs showed that they were rapidly degraded and did not sustain significant expression beyond one week after administration (FIG. 9).

[0087] (6) Screening of crRNAs for human TTR gene genome editing in HepG2 cells. Aiming for the clinical application of Cas3 therapy to humans, a systematic crRNA screening was conducted using HepG2 cells, a cell line derived from human hepatocellular carcinoma, to identify crRNAs that exhibit high efficacy in human TTR gene editing. The crRNA was modified in the same way as the "TTR#2 Stem1 ver2" (Figure 3A) described above in (3). A total of 10 candidate crRNAs (Figure 10A) were incorporated into the pCas3-Cascade-GFP plasmid (Figure 10B) and introduced into HepG2 cells. Genome editing efficiency was evaluated using ddPCR and RT-PCR. As a result, the three types of crRNA, UT-05, UT-06, and UT-07, demonstrated highly effective genome editing (Figure 10C) and were confirmed to reduce Ttr mRNA expression levels (Figure 10D). All of these showed genome editing efficiencies comparable to those of NTLA-2001, which is undergoing clinical trials for the treatment of hereditary transthyretin amyloidosis (ATTRv) and wild-type transthyretin amyloidosis (ATTRwt) using the CRISPR-Cas9 system.

[0088] Similarly to (4) above, modified crRNA (UT-05) was encapsulated in lipid nanoparticles (LNPs) together with Cas3 / cascade (mRNA linked to Cas3-5-11 and mRNA linked to Cas6-7-8) and intravenously administered to TTR-humanized mice (Figure 11A). One week later, serum TTR concentrations were measured by ELISA and found to be reduced by 90% compared to controls (Figure 11B).

[0089] The CRISPR-Cas3 system of the present invention can be used in a variety of fields as a highly efficient and specific genome editing system. For example, in the medical field, it is expected to be used as a system for effective and safe in vivo gene therapy.

Claims

1. A crRNA of the CRISPR-Cas3 system, having a 5' repeat sequence-spacer sequence-3' repeat sequence structure, in which the ribose of the nucleotide is 2'-O-methyl modified and the internucleotide phosphodiester bond is thiophosphate modified in at least a portion of each of the following regions (a) and (b): (a) the region constituting the 5' repeat sequence loop and the region 5' of the region constituting the 3' repeat sequence loop and the region 3' of the region constituting the 3' repeat sequence loop.

2. The crRNA of claim 1, wherein the riboses of at least 50% of the nucleotides in each of regions (a) and (b) are 2'-O-methyl modified.

3. The crRNA of claim 1, wherein at least four phosphodiester bonds from the 5' end of the region (a) and at least four phosphodiester bonds from the 3' end of the base (b) are thiophosphate modified.

4. A CRISPR-Cas3 system comprising the crRNA according to any one of claims 1 to 3 and mRNA encoding the proteins that constitute the CRISPR-Cas3 system.

5. The CRISPR-Cas3 system of claim 4, wherein the mRNA has a Cap1 structure.

6. The CRISPR-Cas3 system according to claim 4, wherein the proteins constituting the CRISPR-Cas3 system are divided into two groups, and the proteins of each group are encoded by a single mRNA.

7. A lipid nanoparticle encapsulating the CRISPR-Cas3 system according to any one of claims 4 to 6.

8. The lipid nanoparticle of claim 7, wherein the crRNA targets the transthyretin gene.

9. The lipid nanoparticle of claim 8, wherein the crRNA targets the 5'UTR or exon 1 of the transthyretin gene.

10. A therapeutic agent for transthyretin amyloidosis, comprising the lipid nanoparticles according to claim 8 or 9.

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