WO2021190226A1 - 单碱基编辑介导的剪接修复在制备治疗脊髓性肌萎缩症中的应用 - Google Patents

单碱基编辑介导的剪接修复在制备治疗脊髓性肌萎缩症中的应用 Download PDF

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WO2021190226A1
WO2021190226A1 PCT/CN2021/077622 CN2021077622W WO2021190226A1 WO 2021190226 A1 WO2021190226 A1 WO 2021190226A1 CN 2021077622 W CN2021077622 W CN 2021077622W WO 2021190226 A1 WO2021190226 A1 WO 2021190226A1
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exon
smn2
cas9n
tada
base
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杨辉
陈万金
左二伟
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中国科学院脑科学与智能技术卓越创新中心
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Definitions

  • the present invention belongs to the field of biotechnology. More specifically, the present invention relates to the application of single-base editing-mediated splicing repair in the preparation and treatment of spinal muscular atrophy.
  • the splicing repair is preferably aimed at SMN2 gene No. 7 The repair of the child.
  • SMA Spinal muscular atrophy
  • the pathogenic gene of SMA is the Survival Motor Neuron 1 (SMN1) gene.
  • Humans have two highly homologous SMN genes, namely SMN1 on the telomere side and SMN2 on the centromere side.
  • SMN1 encodes the whole
  • the long SMN protein is a functional SMN protein, and SMN2 mainly encodes a truncated SMN protein.
  • 95% of SMA patients have homozygous deletion of the SMN1 gene, resulting in the loss of functional full-length SMN (SMN-FL) protein, causing SMA-related clinical symptoms.
  • the two genes SMN1 and SMN2 are highly similar.
  • C6T 6th base located on exon 7, which converts an exon splicing enhancer ESE into an exon splicing Cut the silencer ESS, which will cause about 95% of the transcription product of the SMN2 gene to be encoded as a truncated, non-functional SMN2- ⁇ 7 protein. This protein is inefficient and is quickly degraded.
  • ASO antisense oligonucleotides
  • the purpose of the present invention is to provide the application of single-base editing-mediated splicing correction in preparation and treatment of spinal muscular atrophy.
  • a method for increasing the expression of functional SMN2 protein comprising: targeting exon 7 of the SMN2 gene and mutating one or more bases; preferably, the one or more The bases are the 4th, 5th, 6th, 36th, 38th and/or 45th bases of the 7th exon.
  • the 4th, 5th, or 6th base of exon 7 is mutated to base C; or the 36th, 38th, or 45th base of exon 7 is mutated to G .
  • the 36th position of exon 7 is mutated to G, and the amino acid at the corresponding position in the SMN2 protein (the 12th amino acid (Ser) of exon 7) is unchanged.
  • the 45th position of exon 7 is mutated to G, and the amino acid at the corresponding position in the SMN2 protein (the 15th amino acid (Leu) of exon 7) is unchanged.
  • the 36th and 38th positions of exon 7 are changed to G, and the amino acids at the corresponding positions in the SMN2 protein (the 12th and 13th amino acids of exon 7) are changed from Ser-Ser to Ser-Arg.
  • the 4th and 5th positions of the exon 7 are changed to C, and the amino acid at the corresponding position in the SMN2 protein (the second amino acid of exon 7) is changed from Phe to Pro.
  • the 5th position of exon 7 is changed to C, and the amino acid at the corresponding position in the SMN2 protein (the 2nd amino acid of exon 7) is changed from Phe to Ser.
  • the sixth position of exon 7 is changed to C, and the amino acid at the corresponding position in the SMN2 protein (the second amino acid (Phe) of exon 7) is unchanged.
  • gene editing site-directed mutagenesis or homologous recombination is used to carry out the mutation of exon 7 of SMN2 gene; preferably, single-base editing-mediated splicing repair is used for gene editing.
  • the single-base editing-mediated splicing repair is performed using a DNA single-base editor; preferably, the DNA single-base editor is an adenine base editor (ABE) or its Variants; more preferably, the adenine base editor (ABE) includes the following operatively linked elements: TadA and/or TadA*, Cas9n.
  • ABE adenine base editor
  • the adenine base editor or a variant thereof includes the following operatively connected elements:
  • ABE TadA-TadA*, Cas9n or Cas9n-KKH;
  • ABEmax F148A TadA F148A -TadA* F148A , Cas9n or Cas9n-KKH;
  • MiniABEmax TadA*, Cas9n or Cas9n-KKH;
  • V82G TadA* V82G , Cas9n/Cas9n-KKH;
  • a nuclear localization sequence is also included at the amino terminal and/or carboxy terminal.
  • the carboxyl end of the construct formed by the operatively connected elements further includes a PolyA sequence.
  • the nuclear localization sequence is bpNLS.
  • the Cas9n is SpCas9n.
  • the Cas9n-KKH is SaCas9n-KKH.
  • the functional SMN2 protein is a full-length SMN2 protein.
  • the sgRNA of the sequence shown in SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 8 is used for base editing.
  • the method is applied to induced pluripotent stem cells (iPSCs), embryonic stem cells (mESCs), germ cells (such as oocytes or fertilized eggs) or somatic (tissue) cells, in which the SMN2 gene Encoding a truncated SMN2 protein; the method includes: providing sgRNA and a DNA single-base editor directed against exon 7 of the SMN2 gene, and introducing it into the cell.
  • iPSCs induced pluripotent stem cells
  • mESCs embryonic stem cells
  • germ cells such as oocytes or fertilized eggs
  • somatic (tissue) cells in which the SMN2 gene Encoding a truncated SMN2 protein
  • the method for increasing the expression of functional SMN2 protein is a non-therapeutic and non-diagnostic method.
  • the method for increasing the expression of functional SMN2 protein is an ex vivo method.
  • the induced pluripotent stem cells are commercialized SMA hiPSC cell lines: GM24468 and hiPSC derived from SMA patients.
  • the embryonic cells are commercialized or established embryonic cells.
  • SMN2 gene mutant in which one or more base mutations occur in exon 7 of the nucleotide sequence, and the mutation exists in the 4th exon of the 7th exon. , 5, 6, 36, 38 and/or 45 bases.
  • the 4th, 5th, or 6th base of exon 7 is mutated to base C; or the 36th, 38th, or 45th base of exon 7 is mutated to G .
  • the 36th position of exon 7 is mutated to G, and the amino acid at the corresponding position in the SMN2 protein (the 12th amino acid (Ser) of exon 7) No change; the 45th position of exon 7 is mutated to G, the amino acid at the corresponding position in the SMN2 protein (the 15th amino acid (Leu) of exon 7) remains unchanged; the 36th position of exon 7 And the 38th position is changed to G, the amino acids at the corresponding positions in the SMN2 protein (the 12th and 13th amino acids of exon 7) are changed from Ser-Ser to Ser-Arg; the 4th and 5th positions of the 7th exon
  • the position mutation is C, the amino acid at the corresponding position in the SMN2 protein (the second amino acid in exon 7) is mutated from Phe to Pro; the fifth position of the exon 7 is mutated to C, the amino acid at the corresponding position in the SMN2 protein (
  • sequence of exon 7 of the SMN2 gene is as SEQ ID NO: 9 (54 bp).
  • a cell in which the functional SMN2 protein is normally expressed or highly expressed contains any of the SMN2 gene variants; preferably, the The cells include (but are not limited to): induced pluripotent stem cells (iPSCs), embryonic stem cells (mESCs), germ cells (such as oocytes or fertilized eggs) or somatic cells.
  • iPSCs induced pluripotent stem cells
  • mESCs embryonic stem cells
  • germ cells such as oocytes or fertilized eggs
  • the use of the SMN2 gene variant is provided for preparing constructs or reagents for increasing the expression of functional SMN2 protein.
  • the cells include (but not limited to): induced pluripotent stem cells (iPSCs), embryonic stem cells (mESCs) , Germ cells (such as oocytes or fertilized eggs) or somatic cells.
  • iPSCs induced pluripotent stem cells
  • mESCs embryonic stem cells
  • Germ cells such as oocytes or fertilized eggs
  • an agent for increasing the expression of functional SMN2 protein in the preparation of a medicament for alleviating or treating spinal muscular atrophy is targeting SMN2 gene No. 7
  • a reagent that mutates one or more bases preferably, the one or more bases are the 4th, 5th, 6th, 36th, 38th and/or 45th bases of the 7th exon ;
  • the reagents include: gene editing reagents, site-directed mutagenesis reagents or homologous recombination reagents; preferably, the reagents include DNA single-base editor and sgRNA; more preferably, the DNA
  • the single base editor is the adenine base editor (ABE) or a variant thereof.
  • the adenine base editor includes the following operatively connected elements: TadA and/or TadA*, Cas9n; more preferably, includes: ABE: TadA-TadA*, Cas9n or Cas9n-KKH; ABEmax F148A : TadA F148A -TadA* F148A , Cas9n or Cas9n-KKH; MiniABEmax: TadA*, Cas9n or Cas9n-KKH; miniABEmax V82G : TadA* V82G , Cas9n/Cas9n-KKH; The amino terminus and/or carboxy terminus also include a nuclear localization sequence.
  • the sgRNA is an sgRNA of the sequence shown in SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 8.
  • a method for preparing cells in which the functional SMN2 protein is highly expressed comprises: introducing into the cell an agent that increases SMN2 protein expression, and the agent is a target.
  • the method for preparing cells is a method for "non-therapeutic purposes".
  • a pharmaceutical composition or kit for alleviating or treating spinal muscular atrophy which contains an agent for increasing the expression of SMN2 protein, and the agent is targeted to exon 7 of SMN2 gene
  • a reagent for mutating one or more bases preferably, the one or more bases are the 4th, 5th, 6th, 36th, 38th and/or 45th bases of the 7th exon; More preferably, the reagent for increasing SMN2 protein expression includes: DNA single base editor, sgRNA.
  • the DNA single-base editor is an adenine base editor (ABE) or a variant thereof; more preferably, the adenine base
  • the editor (ABE) includes the following operatively connected elements: TadA and/or TadA*, Cas9n; more preferably, the adenine base editor or its variants include: ABE: TadA-TadA*, Cas9n or Cas9n -KKH; ABEmax F148A : TadA F148A -TadA* F148A , Cas9n or Cas9n-KKH; MiniABEmax: TadA*, Cas9n or Cas9n-KKH; or miniABEmax V82G : TadA* V82G , Cas9n/Cas9n-KKH.
  • the sgRNA has a sequence as shown in SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 8.
  • Single-base editing-mediated exon splicing silencer ESS transition can treat SMA.
  • a Schematic diagram of ESS-A and ESS-B transformation of SMN2 exon 7 mediated by single-base editing.
  • the black arrow indicates the difference between SMN1 and SMN2 in a single nucleotide (C6T).
  • the horizontal bars indicate sgRNA.
  • e, f TaqMan qPCR analysis of the full-length SMN2 (SMN2-FL) mRNA (e) and the truncated SMN ⁇ 7 mRNA (f).
  • SMA iPSC full-length SMN2
  • f truncated SMN ⁇ 7 mRNA
  • SMN protein complex (white arrow) is located in 4 genotypes of iPSC derived motor neuron MNs: SC-SM A36.38G , SC-SMA T5C , SMA and WT, and SMN (green) and HB9 ( Red) Co-staining with antibodies. Scale bar: 10 ⁇ m.
  • SC-SMA T5C primary mice On the 9th day after birth, the size of SC-SMA T5C primary mice is similar to that of litter heterozygotes (Smn +/- ; SMN 2TG/0 ) (referred to as HET).
  • k Timeline of base editing in MNs after mitosis of SMA iPSC differentiation. From left to right: the morphology of MNs on the 15th day after mitosis, and the treated MNs on the 20th day after mitosis were co-stained with ChAT (red), GFP (green) and DAPI (blue). Scale bar, 50 ⁇ m.
  • m A heat map showing the editing efficiency of MNs after mitosis of SMA iPSC edited by miniABEmax-sgRNA6.
  • n High-throughput sequencing analysis of SMA iPSC-derived untransfected (far left) and miniABEmax-sgRNA6 transfected MNs after mitosis and SMN2-FL transcriptional expression levels.
  • the line in the scatter chart represents the median.
  • p Flow cytometric analysis of SMN ⁇ 7 SMA mouse neuron cells after successful transfection.
  • q Heat map showing the editing efficiency of miniABEmax-sgRNA6 in SMN ⁇ 7 SMA mouse neurons.
  • Figure 2 The transition of exon splicing silencer mediated by single-base editing in SMA-induced pluripotent stem cells.
  • a Schematic diagram of SMN2 exon 7, indicating the sgRNA target sites of ESS-A and ESS-B.
  • Uppercase letters indicate the 54 nt sequence of SMN2 exon 7, and lowercase letters indicate partial sequences of intron 6 and intron 7.
  • the sequences of ESS-A (left end) and ESS-B (right end) are highlighted.
  • the dark blue line represents the target sequence of the DNA single-base editor SaCas9n-KKH sgRNAs; the light blue line represents the target sequence of the DNA single-base editor SpCas9n sgRNAs; the red line represents the lateral adjacent motif (PAM) sequence (SaCas9n- KKH is NNNRRT, SpCas9n is NGG).
  • PAM lateral adjacent motif
  • iPSC induced pluripotent stem cell
  • f-h Adenine Single Base Editor (ABE) sequencing results and analysis.
  • the heat map shows that three sgRNAs (sgRNA1, f; sgRNA2, g; sgRNA3, h) target ESS-A and ESS-B of SMN2 exon 7.
  • i, j In the #2-SC-SMA T4C, T5C and #5-SC-SMA T6C iPSC clones, Taqman-based qPCR analysis of full-length SMN2 (SMN2-FL) mRNA (i) and truncated SMN2- ⁇ 7 mRNA(j).
  • the data from WT and SMA are the same as panels e and f in Figure 1.
  • the data represents the mean ⁇ standard deviation. (***)P ⁇ 0.001;(****)P ⁇ 0.0001; one-way analysis of variance.
  • a, c Immunostaining of #1-SC-SMA T5C (a) and #2-SC-SMA A36G, A38G (c) iPSC showed pluripotency markers: NANOG, SSEA4 and TRA1-60.
  • pluripotent stem cells have normal karyotypes.
  • DAPT treatment induces a gradual transition from motor neuron progenitor cells to MNs.
  • f, g WT, SMA, #1-SC-SMA T5C , #2-SC-SMA A36G, A38G iPSC differentiated to form OLIG2-positive motor neuron precursors on day 10 (f), and on day 13 (g ) Differentiate to form HB9-positive MNs.
  • h,i In all DAPI-labeled cells (h), the cell count of OLIG2-positive motor neuron precursors was quantified, and in all MAP2-labeled cells (i), the cell count of HB9-positive MNs was quantified.
  • the data represents the mean ⁇ SD. ns; not significant; one-way analysis of variance. Scale bar, 50 ⁇ m.
  • HB9 is used to label surviving MNs
  • DAPI is used to label the total number of cells.
  • n 3.
  • Figure 7 Screening of effective sgRNAs targeting ESS-A and ESS-B at position 7 of SMN2 exon.
  • Figure 8 Summary of base editing activities of four DNA single-base editors in three different cell types.
  • ac Sanger sequencing chromatogram confirms that the four DNA single-base editors ABE, ABEmax F148A , miniABEmax and miniABEmax V82G target the SMN2 exons in HEK293T cells (a), SMA mESCs (b) and SMA iPSCs (c), respectively 7 Editing activity of sgRNAs 1-6 of ESS-A and ESS-B.
  • the purple arrow indicates the editing site.
  • the inventors used single-base editing-mediated splicing repair technology for the first time to edit the exon 7 of the SMN2 gene, and successfully improved the functionality by mutating part of the base of the exon. Expression of SMN2 protein.
  • the "element” refers to a series of functional nucleic acid/protein sequences useful for protein expression, and the “element” is systematically constructed to form an expression construct.
  • the sequence of the "element” may be those provided in the present invention, as well as their variants, as long as these variants basically retain the function of the "element” by inserting or deleting some bases (such as 1-50 bp; preferably 1-30 bp, more preferably 1-20 bp, more preferably 1-10 bp), or obtained by random or site-directed mutagenesis.
  • operably linked refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences.
  • the promoter region is placed at a specific position relative to the nucleic acid sequence of the target gene, so that the transcription of the nucleic acid sequence is guided by the promoter region, so that the promoter region is "operably linked” to the nucleic acid sequence.
  • the "construct (or construct)” refers to a single-stranded or double-stranded DNA molecule that has been made through human intervention to contain DNA fragments assembled and arranged according to sequences that do not exist in nature .
  • the "construct” includes a "plasmid”, an "in vitro transcription product” or a viral vector, etc.; or, the “construct” is contained in an expression vector as a part of the expression vector.
  • the “sgRNA” refers to “Single-guide RNA (Single-guide RNA, sgRNA)”, which is designed based on the “target site on the target gene” and contains sufficient sequences to be compatible with the endonuclease Cas9 Synergistic action, leading to Cas9-mediated DNA double-strand breaks at the target site.
  • the "sgRNA” includes sgRNA in the form of RNA (for example, in the form of mRNA), as well as sequences in the form of DNA corresponding to the sgRNA sequence or constructs containing the sequence, as long as they can be processed in the cell Or converted into active "sgRNA”.
  • the "mammal” is not particularly limited, as long as its cells have a genome in a general sense, and the gene editing system is active in the cells.
  • the animals may be mammals, including humans, non-human primates (monkeys, orangutans), domestic and agricultural animals (for example, pigs, sheep, cattle), rats (mice), and rodents. Animals (e.g., mice, rats, rabbits) and so on.
  • the cells of the animal express SMN protein.
  • the "cell” refers to a cell capable of expressing SMN protein, and the gene editing system is active in it.
  • the “cells” include but are not limited to: somatic (tissue) cells, induced pluripotent stem cells, germ cells (e.g., fertilized egg cells, oocytes).
  • the "off-target effect” refers to the failure of the modification of a specific position in the genome to achieve the preset goal, the deviation of the modification or the failure of the modification.
  • the reasons for the occurrence of “off-target effects” include, but are not limited to: inaccurate binding to the target, inaccurate cleavage operations after the recognition sequence, and insufficient accuracy of editing the cleavage site.
  • SMN2 is a gene highly similar to SMN1, but Due to the mutation of C6T on exon 7 of SMN2 gene, exon 7 was skipped during the splicing process, and a transcript lacking exon 7 was formed, and the translation produced a truncated SMN protein. Theoretically speaking, it is enough to correct the truncated expression to the normal expression. There are many theoretical operation methods, but there are few truly effective operating tools to achieve this successfully, stably, and error-free. Express corrections. Therefore, the present inventors are dedicated to optimizing research on genome editing strategies for SMN.
  • the present invention provides a method for increasing the expression of functional SMN2 protein, the method comprising: targeting SMN2 gene exon 7 and mutating one or more bases; preferably, the one or Multiple bases are the 4th, 5th, 6th, 36th, 38th and/or 45th bases of the 7th exon.
  • the method is in vitro (ex vivo), that is, an operation performed on cells in vitro.
  • the present invention provides a method for preparing cells with high expression of SMN protein, the method comprising: targeting SMN2 gene exon 7 and mutating one or more bases; preferably, said One or more bases are the 4th, 5th, 6th, 36th, 38th and/or 45th bases of the 7th exon.
  • the method is in vitro (ex vivo), that is, an operation performed on cells in vitro.
  • the method includes: (1) introducing an agent that increases SMN protein expression into an animal germ cell such as a fertilized egg, and said increasing SMN2 protein
  • the expressed reagent is a reagent that targets exon 7 of SMN2 gene and mutates one or more bases; preferably, the one or more bases are the 4th, 5th, and 6th of exon 7 , 36, 38 and/or 45 bases; (2) Develop the fertilized egg of the animal of (1) to obtain an animal whose genome is edited in the gene region to be edited.
  • the method is for research purposes rather than for treatment purposes.
  • the 4th, 5th or 6th base of exon 7 is changed to base C, or the 36th, 38th or 45th base of exon 7 is changed to G.
  • Part of the mutation makes the nucleotide sequence change, but also makes the corresponding position in the amino acid sequence of the protein mutate, that is, non-synonymous mutation (non-synonymous mutation); while the other part of the mutation does not change the amino acid Sequence, that is, synonymous mutation (synonymous mutation) occurs.
  • some sites where synonymous mutations are generated are: the 36th position of exon 7 is changed to G, the amino acid at the corresponding position in the SMN2 protein (the 12th amino acid of exon 7 ( Ser)) unchanged; the 45th position of exon 7 is mutated into G, and the amino acid at the corresponding position in the SMN2 protein (the 15th amino acid (Leu) of exon 7) remains unchanged; or, the 7th exon The 6th position of the son is changed to C, and the amino acid at the corresponding position in the SMN2 protein (the 2nd amino acid (Phe) of exon 7) remains unchanged.
  • some sites that produce non-synonymous mutations are: the 36th and 38th positions of exon 7 are changed to G, and the amino acid at the corresponding position in the SMN2 protein (exon 7) The 12th and 13th amino acids) were changed from Ser-Ser to Ser-Arg; the 4th and 5th positions of the 7th exon were changed to C, the amino acid at the corresponding position in the SMN2 protein (the 2nd position of exon 7) The amino acid) is changed from Phe to Pro; or, the 5th position of exon 7 is changed to C, and the amino acid at the corresponding position in the SMN2 protein (the 2nd amino acid of exon 7) is changed from Phe to Ser.
  • SMN2 gene exon 7 After knowing the base positions of some SMN2 gene exon 7 that have been disclosed in the present invention, those skilled in the art can use a variety of known techniques to carry out mutations at these positions, so as to improve the expression of SMN protein.
  • the said technology is for example but not limited to: site-directed mutagenesis technology, RNA interference technology, homologous recombination technology or gene editing technology.
  • Some gene editing reagents, site-directed mutagenesis reagents or homologous recombination reagents can also be designed by those skilled in the art.
  • the gene editing technology is preferred, and the mutation of the site is performed by single-base editing. For this reason, the present invention also provides a suitable DNA single-base editor.
  • the DNA single-base editor is an adenine base editor (ABE) or a mutant thereof; more preferably, the adenine base editor includes the following operatively linked Components: TadA and/or TadA*, Cas9n.
  • ABE adenine base editor
  • the adenine base editor includes the following operatively linked elements: TadA and/or TadA*, Cas9n; more preferably, it includes:
  • ABE TadA-TadA*, Cas9n or Cas9n-KKH;
  • ABEmax F148A TadA F148A -TadA* F148A , Cas9n or Cas9n-KKH;
  • MiniABEmax TadA*, Cas9n or Cas9n-KKH; or
  • V82G TadA* V82G , Cas9n/Cas9n-KKH.
  • the amino terminal and/or carboxy terminal of the adenine base editor further includes a nuclear localization sequence.
  • the present invention also provides some preferred sgRNAs.
  • the sgRNA is an sgRNA of the sequence shown in SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 8.
  • a synonymous mutation (synonymous serine) of A36G targeting ESS-B of SMN2 exon 7 is provided.
  • This mutation was obtained by using a combination of miniABEmax and sgRNA6.
  • the excellent features of this mutation are: on the one hand, it can achieve synonymous mutations, that is, without changing the amino acid sequence of SMN2; on the other hand, its ability to increase the expression level of SMN2 is very significant.
  • the embodiments of the present invention, particularly This is shown in Figure 1e, which shows an extremely effective increase in the expression level of SMN2; in the third aspect, in an experiment to investigate the DNA off-target effect, the inventors found that there is no detectable off-target effect for this site. Undoubtedly, this is more conducive to clinical research and clinical application.
  • a SMN2 gene mutant is also provided.
  • the SMN2 gene mutant has one or more bases in exon 7 of its nucleotide sequence.
  • Base variation said variation exists in the 4th, 5th, 6, 36th, 38th and/or 45th bases of the 7th exon.
  • the 4th, 5th or 6th base of exon 7 is mutated to base C; or the 36th, 38th or 45th base of exon 7 is mutated to G.
  • the wild-type SMN2 nucleotide sequence is known to those skilled in the art, for example, the sequence may be as shown in SEQ ID NO: 2.
  • the sequence may be as shown in SEQ ID NO: 2.
  • other positions on the SMN2 nucleotide sequence may also have partial bases in different individuals or from different sources. Changes, these changes may not affect the expression of SMN2 protein, they should also be covered in the present invention.
  • SMN2 sequences that are more than 80%, 85%, 90%, 95%, 98%, 99% or higher with the sequence shown in SEQ ID NO: 2, or also covers SEQ ID NO: 2 Fragments of the sequence shown, they can also encode the full-length SMN2 protein or its active part.
  • the SMN2 gene mutant of the present invention may be in the form of DNA or RNA.
  • the form of DNA includes cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the sequence may be a coding sequence that only encodes the mature protein; the coding sequence of the mature protein and various additional coding sequences; the coding sequence (and optional additional coding sequence) and non-coding sequences of the mature protein.
  • its full-length sequence or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • the present invention also relates to a vector containing the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the modified enzyme coding sequence, and a method for producing the protein of the present invention through recombinant technology.
  • the polynucleotide sequence of the present invention can be used to recombinantly express or produce recombinant SMN2 protein.
  • the present invention also provides a cell with high expression of SMN2 protein, which contains the SMN2 gene variant of the present invention; preferably, the cell includes (but is not limited to): Induced pluripotent stem cells (iPSCs), embryonic stem cells (mESCs), germ cells (such as oocytes or fertilized eggs), somatic cells; or, it is a genetically engineered host cell (for example, for recombinant expression of SMN2 protein) .
  • iPSCs Induced pluripotent stem cells
  • mESCs embryonic stem cells
  • germ cells such as oocytes or fertilized eggs
  • somatic cells or, it is a genetically engineered host cell (for example, for recombinant expression of SMN2 protein) .
  • the cells with high SMN2 protein expression of the present invention may contain exogenous nucleic acids (nucleic acid constructs or reagents) that increase SMN2 protein expression, and the reagents for increasing SMN2 protein expression target exon 7 of SMN2 gene.
  • exogenous nucleic acids nucleic acid constructs or reagents
  • the reagents for increasing SMN2 protein expression target exon 7 of SMN2 gene may contain exogenous nucleic acids (nucleic acid constructs or reagents) that increase SMN2 protein expression, and the reagents for increasing SMN2 protein expression target exon 7 of SMN2 gene.
  • One or more base reagents preferably, the one or more bases are the 4th, 5th, 6th, 36th, 38th and/or 45th bases of the 7th exon.
  • the reagents for increasing SMN2 protein expression may include, but are not limited to: gene editing reagents, site-directed mutagenesis reagents or homologous recombination reagents.
  • the reagent includes a DNA single-base editor and sgRNA; more preferably, the DNA single-base editor is adenine base editor (ABE) or a mutant thereof.
  • ABE adenine base editor
  • the present invention provides a splicing repair technology mediated by single-base editing, a technology for editing exon 7 of the SMN2 gene, and a series of methods and tools for performing the repair.
  • the technical scheme of the present invention can be applied to some aspects including but not limited to the following: (1) applied to gene editing of hiPSC and other cells, selecting modified cell lines, and verifying the expression level of SMN2 and functional verification; (2) Applied to gene editing of germ cells such as fertilized eggs, verification of SMN2 expression level and functional verification of obtained cells or individuals; (3) Application to injections of organisms such as mice, such as intracerebroventricular and intrathecal injection Inject, observe the improvement of the disease, the survival of the organism, and the function of SMN2.
  • SMA Spinal muscular atrophy
  • the present invention provides a pharmaceutical composition or kit for alleviating or treating SMA, which contains an agent for increasing the expression of SMN2 protein, and the agent is targeted at one or more of exon 7 of SMN2 gene and variants.
  • One base reagent preferably, the one or more bases are the 4th, 5th, 6th, 36th, 38th and/or 45th bases of the 7th exon; more preferably, the The reagents to increase SMN2 protein expression include: DNA single base editor, sgRNA.
  • the sgRNA can be in the form of mRNA, which can be injected into the cell by injection (such as microinjection), or it can be constructed on a construct such as an expression plasmid and then introduced into the cell.
  • the construct can be an expression plasmid, including: a viral plasmid or a non-viral plasmid.
  • it is a viral plasmid, such as but not limited to: adeno-associated virus plasmid (AAV), lentivirus plasmid (Lentivirus) and the like.
  • the DNA single-base editor can be in the form of mRNA and can be injected into the cell by injection (such as microinjection), or it can be constructed on a construct such as an expression plasmid and then introduced into the cell.
  • the construct can be an expression plasmid, including: a viral plasmid or a non-viral plasmid.
  • it is a viral plasmid, such as but not limited to: adeno-associated virus plasmid (AAV), lentivirus plasmid (Lentivirus) and the like.
  • the sgRNA and the DNA base editor can be introduced into the cell separately or together.
  • the said pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration, and the pharmaceutical composition of the present invention can be made into an injection form, for example, with physiological saline or an aqueous solution containing glucose and other adjuvants for preparation by conventional methods.
  • the pharmaceutical composition should be manufactured under aseptic conditions.
  • the amount of active ingredient administered is a therapeutically effective amount.
  • the pharmaceutical preparation of the present invention can also be made into a sustained-release preparation.
  • the present invention also provides a kit for alleviating or treating spinal muscular atrophy, said kit comprising the pharmaceutical composition of the invention; or comprising the sgRNA and base editor of the invention; Or include the expression constructs of the present invention.
  • the present inventors used mice transferred into the human SMN gene as SMA model mice. While observing a significant increase in the full-length SMN2 transcript and SMN protein, the present inventors also observed The edited SMA mice have significantly increased weight, motor function and life span. At the same time, the present inventors unexpectedly discovered that the effect of targeting mutations at positions other than position 6 (position 6) of exon 7 is better. The results observed by the inventors confirm that base editing-mediated splicing can be used to correct SMN2 gene, and single-base editing technology can be used to treat SMA patients or prepare SMA drugs, which is a new therapeutic approach.
  • SMN1 nucleotide sequence (SEQ ID NO: 1) (underlined in exon 7):
  • SMN2 nucleotide sequence (SEQ ID NO: 2) (location of the mutation underlined):
  • sgRNA sequence (the target position is shown in Figure 1a and Figure 2a):
  • sgRNA1 5'-TTTGTCTAAAACCCTGTAAG-3' (SEQ ID NO: 3);
  • sgRNA2 5’-TAGACAAAATCAAAAAGAAG-3’ (SEQ ID NO: 4);
  • sgRNA3 5’-CACATTCCTTAAATTAAGGA-3’ (SEQ ID NO: 5);
  • sgRNA4 5’-TTTAGACAAAATCAAAAAGA-3’ (SEQ ID NO: 6);
  • sgRNA5 5'-GACAAAATCAAAAAGAAGGA-3' (SEQ ID NO: 7);
  • sgRNA6 5'-CACATTCCTTAAATTAAGGA-3' (SEQ ID NO: 8).
  • ABE from N-terminal to C-terminal: bpNLS (nuclear localization sequence), TadA, TadA*, SpCas9n/SaCas9n-KKH, bpNLS, polyA.
  • ABEmax F148A (from N end to C end): bpNLS, TadA F148A -TadA* F148A , SpCas9n/SaCas9n-KKH, bpNLS, polyA.
  • MiniABEmax (from N end to C end): bpNLS, TadA*, SpCas9n/SaCas9n-KKH, bpNLS, PolyA.
  • V82G (from N end to C end): bpNLS, TadA* V82G , SpCas9n/SaCas9n-KKH, bpNLS, PolyA.
  • sequence of bpNLS is: N-terminal: 5'-atgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtc-3' (SEQ ID NO: 10); C-terminal: 5'-aaaagaaccgccgacggcagcgaattcgagcccaagaagaagaggaaagtc-3' (SEQ ID).
  • TadA adenosine deaminase sequence
  • LWKoblan,JLDoman,C.Wilson,et al Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction[J],Nat Biotechnol,2018,36(9): 843-846.
  • TadA* mutant adenosine deaminase sequence: LWKoblan,JLDoman,C.Wilson,et al,Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction[J],Nat Biotechnol,2018,36(9 ): 843-846.
  • saCas9-KKH sequence see literature BPKleinstiver, MSPrew, SQTsai, et al, Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition[J], Nat Biotechnol, 2015, 33(12): 1293-1298 .
  • SaCas9n-KKH (SaCas9-KKH mutant, D10A mutation occurs on the basis of SaCas9-KKH) sequence: see literature BPKleinstiver, MSPrew, SQTsai, et al, Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition[J], Nat Biotechnol, 2015, 33(12): 1293-1298.
  • TadA* V82G refers to the mutation of V at position 82 to G on the basis of TadA*.
  • TadA F148A TadA* F148A mutation location: see literature C. Zhou, Y. Sun, R. Yan, et al, Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis[J],Nature,2019,571 (7764):275-27.
  • the SMN Exon7 sequence is (54bp) (SEQ ID NO: 9):
  • T4C T ⁇ C transition at position 4 (base 4 of Exon7) on exon 7 of SMN2 gene; corresponding to the nucleotide sequence of SMN2 (SEQ ID NO: 2), referring to the occurrence of position 838 change.
  • T5C The T ⁇ C transition at position 5 (the 5th base of Exon7) on the 7th exon of SMN2 gene; corresponds to the SMN2 nucleotide sequence (SEQ ID NO: 2), referring to the 839th position change.
  • T6C The T ⁇ C transition at position 6 (the 6th base of Exon7) on the 7th exon of the SMN2 gene; corresponds to the SMN2 nucleotide sequence (SEQ ID NO: 2), referring to the 840th position change.
  • A45G The A ⁇ G transition at position 45 (the 45th base of Exon7) on the exon 7 of the SMN2 gene; corresponds to the SMN2 nucleotide sequence (SEQ ID NO: 2), referring to the 879th position change.
  • A36G The A ⁇ G transition at position 36 (the 36th base of Exon7) on the exon 7 of the SMN2 gene; corresponds to the SMN2 nucleotide sequence (SEQ ID NO: 2), referring to the 870th position change.
  • A38G The A ⁇ G transition at position 38 (the 38th base of Exon7) on the exon 7 of the SMN2 gene; corresponds to the SMN2 nucleotide sequence (SEQ ID NO: 2), referring to the occurrence at position 872 change.
  • the method steps for transfecting cells with ABE or modified ABE together with sgRNA: HEK293T or heavy SMA mouse embryonic stem cells are digested and plated on a 6-well plate with a cell density of ⁇ 60%, and the cells are transfected with Lipofectamine 3000 (Thermo Fisher Scientific), The amount of plasmid transfection per well was 4ug, and fluorescent positive cells were obtained by flow sorting 72 hours after transfection.
  • iPSC culture method see the literature Lin X, Li JJ, Qian WJ, et al. Modeling the differential phenotypes of spinal muscular atrophy with high-yield generation of motor neurons from . human induced pluripotent stem cells [J ], Oncotarget, 2017,8:. 42030-42042 be cell density of ⁇ 80%, were transfected using nuclear instrument (Lonza 2B) electroporation cells, 1X10 7 cells in an amount corresponding to the amount of plasmid It is 10ug, 72 hours after transfection to obtain fluorescence positive cells by flow sorting.
  • motor neuron differentiation method see the literature Lin X, Li JJ, Qian WJ, et al. Modeling the differential phenotypes of spinal muscular atrophy with high-yield generation of motor neurons from .
  • Steps of in vivo neuron electroporation and tissue staining The SMN ⁇ 7 SMA mice on day 0 after birth are anesthetized on ice for 30-60 seconds, 1.5 ⁇ g plasmid is mixed with 0.05% Fast Green dye, and a glass needle is used for intracerebroventricular injection . Subsequently, the ECM-830 instrument was used for in-vivo electroporation, the electric pulse duration was 60ms, the interval was 950ms, and the voltage was 80V. The mice were sacrificed on the 7th day after birth. The brain tissue was digested with trypsin, and GFP-positive neurons were separated by flow cytometry for subsequent steps. Mice prepared for immunohistochemical staining were perfused with PFA and 30% sucrose and then sliced brain tissues with a thickness of 40 ⁇ m. DAPI was then used for staining for 30 minutes.
  • Fertilized egg injection and embryo transfer steps light SMA mice (Smn -/- , SMN2 2TG/2TG ) female mice were superovulated with heterozygous Smn knockout mice (Smn +/- ). Subsequently, the fertilized egg was taken out, and 50ng/ ⁇ l ABE mRNA and 100ng/ ⁇ l sgRNA1 in vitro transcripts were injected into the cytoplasm of the fertilized egg by microinjection, and then incubated in KSOM overnight to the two-cell stage, and then transferred to pseudo-pregnant ICR female mice In the fallopian tube.
  • TaqMan probe method qPCR analysis steps use TaqMan Universal PCR Master Mix kit and FAM-based TaqMan assays custom probe kit according to the operating instructions to perform the reaction in the LightCycler480 real-time quantitative PCR system and determine the SMN2-FL and SMN2- ⁇ of the cells 7 Transcript level.
  • the specific primers and probe sequences used above are shown in Table 1.
  • Immunostaining steps cells were fixed with 4% formaldehyde solution and then punched and blocked with 0.2% Triton-X-100 and 10% donkey serum. Incubate overnight at 4°C with the primary antibody. Then use fluorescent secondary antibody (dilution ratio 1:1000) and DAPI (dilution ratio 1:1000) to incubate at room temperature for two hours. The specific information and dilution ratio of the primary antibody are shown in Table 2.
  • Antibody Dilution ratio Antibody Dilution ratio OLIG2 1:300 SSEA4 1:400 HB9 1:100 SOX2 1:1000 MAP2 1:5000 TRA-1-60 1:1000 SMN 1:75 TUNEL 1:10 NANOG 1:1000 To To To
  • mice used for immunofluorescence staining were sacrificed at 9 days after birth, and the spinal cord was taken out and fixed in 4% PFA at room temperature for 1-3 hours, and then dehydrated with 30% sucrose at 4°C overnight.
  • the lumbar spinal cord (L1-L2) was sliced with a thickness of 20 ⁇ m.
  • Motor neurons and nuclear gemini were incubated with ChAT antibody (dilution ratio 1:500) and SMN antibody (dilution ratio 1:300) with TritonX-100, BSA, and NaN3 at 4°C overnight. Then incubate overnight with fluorescent secondary antibody and DAPI at 4°C.
  • Use Leica TCS SP8 for image acquisition, and then use Image-J software to count the cells.
  • High-throughput sequencing database construction and data processing steps flow sorting positive cell population lysis, nested PCR amplification (see Table 3 below, nested primers), single neuron through reverse transcription and specific amplification (single cell primers) ). Construct amplicons based on Illumina HiSeq X-Ten platform for sequencing. The data was preprocessed using fastp (v0.19.6), the FASTQ file was separated by barcodes sequence, and matched with the reference genome by Smith-Waterman algorithm, and the number of mutation sequences was extracted and counted.
  • SMN2 is a gene highly similar to SMN1, but due to the difference in C6T in exon 7 of SMN2, exon 7 is skipped during the splicing process, as shown in Figure 1a.
  • Hua et al. reported in 2007 that targeting the ESS in the 7th exon of SMN2 through ASO can enhance the inclusion of the 7th exon of SMN2 (the splicing product contains exon7). On this basis, the inventors tested whether this strategy can also be achieved through genome editing.
  • NHEJ non-homologous end joining
  • the inventor chose the single-base editing scheme, and designed three ABE (producing A-to-G or T-to-C conversion) KKH variants (Staphylococcus aureus) for this purpose.
  • Cas9 single nickase (Cas9n), sgRNA (sgRNA1-3) with recognition sequence NNNRRT) acts on ESS-A or ESS-B at the 7th exon site of SMN2 ( Figure 2a) and used in subsequent experiments.
  • the map of the expression construct of the ABE gene editor designed by the present invention is shown in Fig. 1b.
  • the ABE gene editor can produce a precise nucleotide transition at a specific gene site without double-strand breaks.
  • the inventors calculated the AG editing frequency in the 20bp region upstream of the PAM site, and used a heat map to show that three sgRNAs (sgRNA1; sgRNA2; sgRNA3) target ESS-A and ESS-B of SMN2 exon 7 (Figure 2f ⁇ h) the situation. High-throughput sequencing showed that sgRNA1 and sgRNA3 produced effective edits.
  • FIG. 1d The heat map of the editing efficiency of ABE-sgRNA1 editing SMA iPSC and ABE-sgRNA3 editing SMA iPSC is shown in Figure 1d.
  • the results of TaqMan qPCR analysis of full-length SMN2 (SMN2-FL) mRNA and truncated SMN ⁇ 7 mRNA are shown in Figures 1e ⁇ f and Figures 2i ⁇ j. It can be seen that #1-SC-SMA T5C , #1-SC-SMA T6C , # The full-length SMN2 in 1-SC-SMA A45G , #1-SC-SMA T4C, T5C has a significant increase, while the expression of SMN ⁇ 7 has a significant decrease. Sanger sequencing to confirm the editing results are shown in Figure 3a ⁇ b.
  • the inventors differentiated spinal cord MNs from three genotypes of iPSCs: #1-SC-SMA T5C , SMA and WT.
  • the timeline of induced pluripotent stem cells to differentiate into spinal cord MNs and the factors used for induction are shown in Figure 4e.
  • WT, SMA, #1-SC-SMA T5C iPSCs differentiated to form OLIG2 + motor neuron precursors on day 10 ( Figure 4f), and differentiated to form HB9 + MNs on day 13 ( Figure 4g).
  • the results showed that SC-SMA T5C, SMA and WT, these cells all showed typical differentiation (Figure 4e ⁇ i).
  • the present inventors co-injected mRNA encoding ABE and sgRNA1 into the zygotes obtained by crossing light SMA mice (Smn -/- , SMN2 2TG/2TG ) and heterozygous Smn knockout mice (Smn +/- )
  • a heavy SMA mouse Smn -/- , SMN2 2TG/0
  • T5C editing called: SC-SMA T5C , Smn -/- , SMN2 T5C/0
  • SC-SMA T5C primary mice were significantly healthier than unedited SMA mice of the same age ( Figure 1i).
  • SC-SMA T5C mice showed a longer lifespan (>400 days), a significant increase in body weight, and an increase in motor function (righting reflex) (Figure 6d ⁇ f).
  • Example 2 Single-base editing of ESSA and ESSB using a modified base editor
  • the inventors confirmed that the ABE used initially does not produce detectable DNA off-target effects, however, it can produce thousands of off-target RNA mononucleotide mutations. Therefore, in order to minimize RNA off-target and any biological effects in the T5C editing process, the inventors used three newly developed high-fidelity single-base editors: TadA F148A -TadA* F148A -ABEmax (abbreviated as ABEmax F148A ), TadA*-ABEmax (abbreviated as miniABEmax) and TadA* V82G- ABEmax (abbreviated as miniABEmax V82G ), and further designed sgRNAs 4-6 (Figure 1a ⁇ b, Figure 2a).
  • mESCs embryonic stem cells
  • iPSC mouse induced pluripotent stem cells
  • miniABEmax and sgRNA6 miniABEmax-sgRNA6
  • Figure 1k miniABEmax-sgRNA6
  • the high-throughput sequencing results of a single MNs cell after transfection showed that the expression of SMN2-FL gene of edited MNs was significantly increased, and the conversion and editing efficiency of A36G was higher than that of A38G ( Figure 1n).
  • the in vivo neurodissection of SMN ⁇ 7SMA mice edited by miniABEmax-sgRNA6 showed that the editing efficiency of its base conversion (A36G, A38G) was tested as shown in Figure 1o ⁇ q.
  • the present inventors successfully used the single-base editing system to edit ESS-A and ESS-B of SMN2 exon 7, thereby realizing efficient synonymous A36G transformation and achieving abnormal splicing of SMN2 gene. Repair.
  • In vitro apoptosis experiments confirmed that the SMN protein induced by the simultaneous conversion of A36G and A38G has a normal function.
  • the research of the present invention shows that the single-base editing system can be used to repair the abnormal splicing of SMN2 gene, and the single-base editing technology can be used to treat SMA patients, and the effect of targeting mutations other than the 6th position of exon 7 Even better, this is a new way of treatment.

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Abstract

提供了一种利用单碱基编辑介导的剪接修复技术，针对SMN2基因的7号外显子进行编辑，通过对该外显子部分碱基的变异，成功地提高了功能性SMN2蛋白的表达。

Description

单碱基编辑介导的剪接修复在制备治疗脊髓性肌萎缩症中的应用 技术领域
本发明属于生物技术领域，更具体地，本发明涉及单碱基编辑介导的剪接修复在制备治疗脊髓性肌萎缩症中的应用，所述的剪接修复较佳地是针对SMN2基因7号外显子的修复。
背景技术
脊髓性肌萎缩症(Spinal Muscular Atrophy；SMA)是一种致死性的常染色体隐性运动神经元疾病，主要累及脊髓前角，临床上主要表现为四肢进行性萎缩及无力，死亡率及致残率极高。在婴幼儿致死性遗传病中排名第一位，每6000名新生儿中就有一位发病。在缺乏有效干预的情况下，SMA I型(严重型)患儿会在2岁前死亡。
SMA的致病基因是运动神经元生存1(Survival Motor Neuron 1，SMN1)基因，人类有两种高度同源的SMN基因，分别为端粒侧的SMN1和着丝粒侧的SMN2，SMN1编码全长的SMN蛋白为功能性SMN蛋白，而SMN2主要编码截短的SMN蛋白。95％的SMA病人纯合缺失SMN1基因，从而导致功能性全长SMN(SMN-FL)蛋白缺失，引起SMA相关的临床症状。SMN1与SMN2这两个基因高度相似，最重要的差异点是位于7号外显子上的第6个碱基(C6T)，从而将一个外显子剪切增强子ESE转换成一个外显子剪切沉默子ESS，这会造成SMN2基因约95％的转录产物编码成截短的，无功能性的SMN2-△7蛋白质。这种蛋白是低效能的，且很快被降解。
2011年，美国冷泉港实验室的研究人员设计了一种反义寡核苷酸(Antisense oligonucleotides，ASO)SMN-Rx来掩蔽这个ISS-NI位点，从而能够调节SMN2的剪接并提高SMN蛋白的表达，临床上称为Spinraz(nusinersen，诺西那生纳注射液)，已被FDA批准为儿童SMA治疗药物。然而，其制备的ASO在细胞内会降解，功能性SMN蛋白的表达是短期的。以临床SMA治疗为例，维持SMN蛋白表达需要定期不断补充ASO，这需要通过腰椎穿刺鞘内注射给药，病人难以接受，且成本非常高昂，限制了该方法的相关应用。Zolgensma也已被FDA批准为儿童SMA治疗药物，其是一种单剂量的SMA基因替代疗法。然而，不幸的是，它在维持高水平、稳定的基因表达方面并不可靠。种种问题限制了上述两种已批准的SMA药物的治疗效果及可及性。
综上，尽管本领域中已经对于SMA的致病基因有所了解，然而仍然缺少有效的药物。在疾病靶点清楚的情况下，药物的研究仍然是极为困难的。本领域亟需探究安全有效、经济实用的增加SMN蛋白表达的方法，并探索其在SMN治疗中的应用。
发明内容
本发明的目的在于提供单碱基编辑介导的剪接纠正在制备治疗脊髓性肌萎缩症 中的应用。
在本发明的第一方面，提供一种增加功能性SMN2蛋白表达的方法，包括：靶向SMN2基因7号外显子，变异其中一个或多个碱基；较佳地，所述的一个或多个碱基为该7号外显子的第4、5、6、36、38和/或45位碱基。
在一个优选方式中，所述7号外显子的第4、5或6位碱基被变异为碱基C；或所述7号外显子的第36、38或45位碱基被变异为G。
在另一优选方式中，所述7号外显子的第36位变异为G，SMN2蛋白中相应位置的氨基酸(7号外显子第12位氨基酸(Ser))不变。
在另一优选方式中，所述7号外显子的第45位变异为G，SMN2蛋白中相应位置的氨基酸(7号外显子第15位氨基酸(Leu))不变。
在另一优选方式中，所述7号外显子的第36位和第38位变异为G，SMN2蛋白中相应位置的氨基酸(7号外显子第12和13位氨基酸)由Ser-Ser变异为Ser-Arg。
在另一优选方式中，所述7号外显子的第4位和第5位变异为C，SMN2蛋白中相应位置的氨基酸(7号外显子第2位氨基酸)由Phe变异为Pro。
在另一优选方式中，所述7号外显子的第5位变异为C，SMN2蛋白中相应位置的氨基酸(7号外显子第2位氨基酸)由Phe变异为Ser。
在另一优选方式中，所述7号外显子的第6位变异为C，SMN2蛋白中相应位置的氨基酸(7号外显子第2位氨基酸(Phe))不变。
在另一优选方式中，利用基因编辑、定点突变或同源重组来进行SMN2基因7号外显子的变异；较佳地，利用单碱基编辑介导的剪接修复来进行基因编辑。
在另一优选方式中，所述单碱基编辑介导的剪接修复利用DNA单碱基编辑器进行；较佳地所述DNA单碱基编辑器为腺嘌呤碱基编辑器(ABE)或其变体；更佳地，所述腺嘌呤碱基编辑器(ABE)包括以下操作性连接的元件：TadA和/或TadA*，Cas9n。
在另一优选方式中，所述腺嘌呤碱基编辑器或其变体包括以下操作性连接的元件：
ABE：TadA-TadA*，Cas9n或Cas9n-KKH；
ABEmax F148A：TadA F148A-TadA* F148A，Cas9n或Cas9n-KKH；
MiniABEmax：TadA*，Cas9n或Cas9n-KKH；
miniABEmax V82G：TadA* V82G，Cas9n/Cas9n-KKH；
在另一优选方式中，在其氨基端和/或羧基端还包括核定位序列。
在另一优选方式中，在所述操作性连接的元件形成的构建体的羧基端还包括PolyA序列。
在另一优选方式中，所述核定位序列为bpNLS。
在另一优选方式中，所述Cas9n为SpCas9n。
在另一优选方式中，所述Cas9n-KKH为SaCas9n-KKH。
在另一优选方式中，所述功能性SMN2蛋白为全长SMN2蛋白。
在另一优选方式中，利用SEQ ID NO:3、SEQ ID NO:5或SEQ ID NO:8所示序列的sgRNA进行碱基编辑。
在另一优选方式中，所述方法应用于诱导性多能干细胞(iPSCs)、胚胎干细胞(mESCs)、生殖细胞(如卵母细胞或受精卵)或体(组织)细胞，该细胞中SMN2基因编码截短的SMN2蛋白；所述方法包括：提供针对SMN2基因7号外显子的sgRNA以及DNA单碱基编辑器，引入到所述细胞中。
在另一优选方式中，所述的增加功能性SMN2蛋白表达的方法为非治疗性、非诊断性的方法。
在另一优选方式中，所述的增加功能性SMN2蛋白表达的方法为离体的方法。
在另一优选方式中，所述诱导性多能干细胞如商品化的SMA hiPSC细胞系：GM24468和SMA病人来源的hiPSC。
在另一优选方式中，所述胚胎细胞如商品化或已建系的胚胎细胞。
在本发明的另一方面，提供一种SMN2基因突变体，其核苷酸序列的7号外显子上发生一个或多个碱基的变异，所述变异存在于该7号外显子的第4、5、6、36、38和/或45位碱基上。
在一个优选方式中，所述7号外显子的第4、5或6位碱基被变异为碱基C；或所述7号外显子的第36、38或45位碱基被变异为G。
在另一优选方式中，所述的SMN2基因变体中，所述7号外显子的第36位变异为G，SMN2蛋白中相应位置的氨基酸(7号外显子第12位氨基酸(Ser))不变；所述7号外显子的第45位变异为G，SMN2蛋白中相应位置的氨基酸(7号外显子第15位氨基酸(Leu))不变；所述7号外显子的第36位和第38位变异为G，SMN2蛋白中相应位置的氨基酸(7号外显子第12和13位氨基酸)由Ser-Ser变异为Ser-Arg；所述7号外显子的第4位和第5位变异为C，SMN2蛋白中相应位置的氨基酸(7号外显子第2位氨基酸)由Phe变异为Pro；所述7号外显子的第5位变异为C，SMN2蛋白中相应位置的氨基酸(7号外显子第2位氨基酸)由Phe变异为Ser；或所述7号外显子的第6位变异为C，SMN2蛋白中相应位置的氨基酸(7号外显子第2位氨基酸(Phe))不变。优选的，所述7号外显子的第36位发生突变，更优选变异为G，SMN2蛋白中相应位置的氨基酸(7号外显子第12位氨基酸(Ser))不变。
在另一优选方式中，所述SMN2基因7号外显子序列如SEQ ID NO:9(54bp)。
在本发明的另一方面，提供一种细胞，所述的细胞中功能性SMN2蛋白正常表达或高表达，所述的细胞中包含任一所述的SMN2基因变体；较佳地，所述的细胞包括(但不限于)：诱导性多能干细胞(iPSCs)、胚胎干细胞(mESCs)，生殖细胞(如卵母细胞或受精卵)或体细胞。
在本发明的另一方面，提供所述的SMN2基因变体的用途，用于制备增加功能性 SMN2蛋白表达的构建体或试剂。
在一个优选方式中，用于制备增加功能性SMN2蛋白表达的构建体或细胞；较佳地，所述的细胞包括(但不限于)：诱导性多能干细胞(iPSCs)、胚胎干细胞(mESCs)、生殖细胞(如卵母细胞或受精卵)或体细胞。
在本发明的另一方面，提供增加功能性SMN2蛋白表达的试剂在制备缓解或治疗脊髓性肌萎缩症的药物中的用途；所述的增加SMN2蛋白表达的试剂是靶向SMN2基因7号外显子，变异其中一个或多个碱基的试剂；较佳地，所述的一个或多个碱基为该7号外显子的第4、5、6、36、38和/或45位碱基；
在一个优选方式中，所述的试剂包括：基因编辑试剂、定点突变试剂或同源重组试剂；较佳地，所述的试剂包括DNA单碱基编辑器和sgRNA；更佳地，所述DNA单碱基编辑器为腺嘌呤碱基编辑器(ABE)或其变体。
在另一优选方式中，所述腺嘌呤碱基编辑器(ABE)包括以下操作性连接的元件：TadA和/或TadA*，Cas9n；更佳地，包括：ABE：TadA-TadA*，Cas9n或Cas9n-KKH；ABEmax F148A：TadA F148A-TadA* F148A，Cas9n或Cas9n-KKH；MiniABEmax：TadA*，Cas9n或Cas9n-KKH；miniABEmax V82G：TadA* V82G，Cas9n/Cas9n-KKH；较佳地，在其氨基端和/或羧基端还包括核定位序列。
在另一优选方式中，所述sgRNA为SEQ ID NO:3、SEQ ID NO:5或SEQ ID NO:8所示序列的sgRNA。
在本发明的另一方面，提供一种制备细胞的方法，所述的细胞中功能性SMN2蛋白高表达，所述方法包括：将增加SMN2蛋白表达的试剂引入到细胞中，所述试剂是靶向SMN2基因7号外显子、变异其中一个或多个碱基的试剂；较佳地，所述的一个或多个碱基为该7号外显子的第4、5、6、36、38和/或45位碱基。
在一个优选方式中，所述的制备细胞的方法为“非治疗目的”的方法。
在本发明的另一方面，提供一种用于缓解或治疗脊髓性肌萎缩症的药物组合物或药盒，其中含有增加SMN2蛋白表达的试剂，所述试剂是靶向SMN2基因7号外显子、变异其中一个或多个碱基的试剂；较佳地，所述的一个或多个碱基为该7号外显子的第4、5、6、36、38和/或45位碱基；更佳地，所述的增加SMN2蛋白表达的试剂包括：DNA单碱基编辑器，sgRNA。
在一个优选方式中，所述的药物组合物或药盒中，所述DNA单碱基编辑器为腺嘌呤碱基编辑器(ABE)或其变体；更佳地，所述腺嘌呤碱基编辑器(ABE)包括以下操作性连接的元件：TadA和/或TadA*，Cas9n；更佳地，所述腺嘌呤碱基编辑器或其变体包括：ABE：TadA-TadA*，Cas9n或Cas9n-KKH；ABEmax F148A：TadA F148A-TadA* F148A，Cas9n或Cas9n-KKH；MiniABEmax：TadA*，Cas9n或Cas9n-KKH；或miniABEmax V82G：TadA* V82G，Cas9n/Cas9n-KKH。
在另一优选方式中，所述sgRNA具有如SEQ ID NO:3、SEQ ID NO:5或SEQ ID  NO:8所示的序列。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、单碱基编辑介导的外显子剪接沉默子ESS的转变可治疗SMA。
a：单碱基编辑介导SMN2外显子7的ESS-A和ESS-B的转化示意图。黑色箭头表示单个核苷酸(C6T)在SMN1和SMN2之间的差异。横杠表示sgRNA。
b：本发明建立的质粒图谱。
c：针对ESS-A和ESS-B的单碱基编辑效率。
d：显示ABE-sgRNA1编辑SMA iPSC、ABE-sgRNA3编辑SMA iPSC、和2个miniABEmax-sgRNA6编辑SMA iPSC的编辑效率的热图。
e、f：对全长SMN2(SMN2-FL)mRNA(e)和截短的SMN△7 mRNA(f)进行TaqMan qPCR分析。基因表达归一化为SMA iPSC，值取1(n＝3)。数据表示±SD。
g：SMN蛋白复合体(白色箭头)定位于4个基因型的iPSC分化来源的运动神经元MNs：SC-SM A36.38G、SC-SMA T5C、SMA和WT，与SMN(绿色)和HB9(红色)抗体共染色。比例尺：10μm。
h：计数100个HB9阳性运动神经元中的SMN蛋白复合体(n＝3)。数据表示±SD。
i：在出生后的第9天，SC-SMA T5C初代小鼠的大小与同窝崽杂合子相似(Smn +/-；SMN 2TG/0)(称为HET)。
j：SC-SMA T5C后代小鼠与HET T5C小鼠相比没有明显的表型差异。
k：SMA iPSC分化的有丝分裂后MNs中碱基编辑的时间轴。从左至右：有丝分裂后第15天的MNs形态，以及经过处理的有丝分裂后第20天的MNs与ChAT(红色)、GFP(绿色)和DAPI(蓝色)共染色。比例尺，50μm。
l：成功转染SMA iPSC的有丝分裂后MNs的流式分析。
m：显示miniABEmax-sgRNA6编辑的SMA iPSC分化的有丝分裂后MNs编辑效率的热图。
n：高通量测序分析SMA iPSC来源的未转染(最左侧)和miniABEmax-sgRNA6转染的有丝分裂后MNs的SMN2-FL转录表达水平。散点图中线表示中位数。
o：在SMN△7 SMA小鼠体内进行单碱基编辑的时间轴。GFP(绿色)和DAPI(蓝色)共转染神经元的免疫组化。图像中的插图向右放大(原始放大倍数，x5.0)。比例尺：100μm.
p：成功转染后的SMN△7 SMA小鼠神经元细胞流式分析。
q：显示SMN△7 SMA小鼠神经元中miniABEmax-sgRNA6编辑效率的热图。
图2、单碱基编辑在SMA诱导多功能干细胞中介导的外显子剪接沉默子的转变。
a：SMN2 7号外显子的示意图，指示了ESS-A和ESS-B的sgRNA靶位点。大写字母表示SMN2 7号外显子的54nt序列，小写字母表示6号内含子和7号内含子的部分序列。ESS-A(左端)和ESS-B(右端)的序列突出显示。深蓝色线条表示DNA单碱基编辑器SaCas9n-KKH sgRNAs的目标序列；浅蓝色线条表示DNA单碱基编辑器SpCas9n sgRNAs的目标序列；红色线条表示旁侧邻近基序(PAM)序列(SaCas9n-KKH为NNNRRT，SpCas9n为NGG)。
b：人SMA诱导多功能干细胞(iPSC)克隆的形态图，比例尺，100um。
c：免疫荧光法检测SOX2在人SMA iPSCs中的表达，比例尺，10um。
d：使用微滴数字PCR试剂盒(Bio-Rad)检测WT和SMA iPSC中的SMN2拷贝数。
e：使用多重连接探针扩增技术分析测量SMN1和SMN2拷贝数。红色箭头“1”和“3”表示SMN1外显子7和外显子8，黑色箭头“2”和“4”表示SMN2外显子7和外显子8。
f-h：腺嘌呤单碱基编辑器(ABE)测序结果及分析。热图显示了三个sgRNA(sgRNA1，f；sgRNA2，g；sgRNA3，h)靶向SMN2外显子7的ESS-A和ESS-B。
i，j：在#2-SC-SMA T4C,T5C和#5-SC-SMA T6C iPSC克隆中，基于Taqman的qPCR分析全长SMN2(SMN2-FL)mRNA(i)和截短的SMN2-Δ7 mRNA(j)。来自WT和SMA的数据与图1中panel e和f相同。数据表示平均值±标准差。(***)P<0.001；(****)P<0.0001；单向方差分析。
图3、单克隆SC-SMA多能干细胞的基因型鉴定研究。
a-f：通过Sanger测序确认编辑结果。与未处理的细胞(a，c，e)相比，本发明人观察到使用ABE-sgRNA1(b)靶向ESS-A的T到C的转变，同时观察到使用miniABEmax-sgRNA6(d)和miniABEmax V82G-sgRNA6(f)靶向ESS-B的A到G的转变。紫色箭头表示编辑位点。
图4、#1-SC-SMA T5C和#2-SC-SMA A36G,A38G多能干细胞的特征鉴定。
a,c：#1-SC-SMA T5C(a)和#2-SC-SMA A36G,A38G(c)iPSC的免疫染色显示多能性标记：NANOG、SSEA4和TRA1-60。
b,d：#1-SC-SMA T5C(b)和#2-SC-SMA A36G,A38G(d)多能干细胞的核型正常。
e：诱导多能干细胞向脊髓MNs分化的时间轴。DAPT处理诱导运动神经元祖细胞向MNs的渐进过渡。
f,g：WT、SMA、#1-SC-SMA T5C、#2-SC-SMA A36G,A38G iPSC在第10天(f)分化形成OLIG2阳性的运动神经元前体，在第13天(g)分化形成HB9阳性MNs。
h,i：在所有DAPI标记的细胞(h)中，OLIG2阳性运动神经元前体的细胞计数定量，和所有MAP2标记的细胞(i)中，HB9阳性MNs的细胞计数定量。数据代表平均值±SD。ns；不显著；单向方差分析。比例尺，50μm。
图5、在iPSC分化的MNs中，对错义突变的SMN蛋白的功能验证。MNs添加 DMSO(40μM，38小时)，喜树碱(10μM，21小时)或衣霉素(40μM，38小时)。
a：HB9用于标记存活的MNs，DAPI标记细胞总量。
b：处理后的MNs也用TUNEL检测，以标记凋亡细胞。
c：喜树碱或衣霉素处理后，细胞计数细胞活力的量化(存活与细胞总数之比)；与SMA MNs相比，#1-SC-SMA T5C MNs和#2-SC-SMA A36G,A38G MNs的细胞存活率更高。n＝3。
d：用喜树碱或衣霉素处理后，细胞计数死亡的MNs占总细胞的比例。与SMA MNs相比，#1-SC-SMA T5C MNs和#2-SC-SMA A36G,A38G MNs的MNs细胞凋亡程度降低，n＝3。箱形图：中心线表示中间值，误差棒表示最大值和最小值。ns,不显著；(**)P<0.01；(****)P<0.0001；单向方差分析。比例尺，50μm。
图6、HET T5C、SMA和SC-SMA T5C小鼠的表型分析。
a：ABE与sgRNA1针对单只雄性SC-SMA T5C初代小鼠SMN2外显子7的ESS-A识别位点的编辑活性(Smn -/-，SMN2 T5C/0)。
b：SC-SMA T5C初代小鼠与杂合Smn敲除小鼠(Smn +/-)杂交产生的SC-SMA T5C后代小鼠的繁殖策略。
c：HET(Smn +/-，SMN2 2TG/0)和SMA(Smn -/-，SMN2 2TG/0)的生存效率。*3只HET和SMA小鼠于第9天处死，进行免疫组化分析。
d：HET T5C(n＝14)、SMA(n＝30)和SC-SMA T5C(n＝9)小鼠的Kaplan-Meier生存曲线。
e：HET T5C(n＝8)、SMA(n＝18)和SC-SMA T5C(n＝4)小鼠的体重从第5天到第55天进行评估。
f：HET T5C(n＝8)、SMA(n＝15)、SC-SMA T5C(n＝4)小鼠P5、P7、P9、P11的翻正反射测定。
g：在#2-SC-SMA T5C和SMA小鼠中，通过与SMN(绿色)和ChAT(红色)抗体联合染色，观察了SMN蛋白复合体在脊髓L1-L2运动神经元中的定位。比例尺，50μm。
h：HET T5C(n＝3)、SMA(n＝3)和SC-SMA T5C(n＝3)小鼠中，每100个运动神经元中SMN蛋白复合体的计数量化。数据表示平均值±标准差。(*)P<0.05；(***)P<0.001；(****)P<0.0001；单向方差分析。
图7、筛选针对SMN2外显子7位点ESS-A和ESS-B的有效sgRNAs。
a：HEK293T细胞、SMA mESCs和SMA iPSC中筛选sgRNA的实验设计。
b：表达GFP的转染细胞的代表性图像。比例尺：100μm。
c：流式细胞仪分析转染细胞。
图8、四种DNA单碱基编辑器在三种不同细胞类型中的碱基编辑活性综述。
a-c：Sanger测序层析图证实了四个DNA单碱基编辑器ABE、ABEmax F148A、miniABEmax和miniABEmax V82G分别针对HEK293T细胞(a)、SMA mESCs(b)和SMA  iPSCs(c)中SMN2外显子7 ESS-A和ESS-B的sgRNAs 1-6的编辑活性。紫色箭头表示编辑位点。
具体实施方式
本发明人经过深入的研究，首次利用单碱基编辑介导的剪接修复技术，针对SMN2基因的7号外显子进行编辑，通过对该外显子部分碱基的变异，成功地提高了功能性SMN2蛋白的表达。
术语
如本文所用，所述的“元件”是指一些对于蛋白的表达有用的一系列功能性的核酸/蛋白序列，所述的“元件”被系统地构建以形成一种表达构建体。所述的“元件”的序列可以是本发明中所提供的那些，也包括它们的变体，只要这些变体基本上保留了所述“元件”的功能，其通过插入或删除一些碱基(如1-50bp；较佳地1-30bp，更佳地1-20bp，更佳地1-10bp)，或进行随机或定点突变等来获得。
如本文所用，所述的“操作性连接”或“可操作性相连”是指两个或多个核酸区域或核酸序列的功能性的空间排列。例如：启动子区被置于相对于目的基因核酸序列的特定位置，使得核酸序列的转录受到该启动子区域的引导，从而，启动子区域被“可操作地连接”到该核酸序列上。
如本文所用，所述的“构建物(或称构建体)”指一种已经通过人为干预，使其含有按照自然界中不存在的序列所组合和排列的DNA片段的单链或者双链DNA分子。所述的“构建物”包括“质粒”、“体外转录产物”或病毒载体等；或者，所述的“构建物”被包含在表达载体中、作为表达载体的一部分。
如本文所用，所述的“sgRNA”即“单一引导RNA(Single-guide RNA，sgRNA)”，其是基于“目标基因上的靶位点”设计，其包含的序列足以与内切核酸酶Cas9协同作用，引导发生Cas9介导的靶位点上DNA双链断裂。本发明中，所述的“sgRNA”包括RNA形式(如mRNA形式)的sgRNA，也包括与该sgRNA序列相应的DNA形式的序列或含有所述序列的构建体，只要它们在细胞内能够被加工或转化为活性的“sgRNA”。
如本文所用，所述“动物(mammal)”没有特别的限制，只要其细胞具有一般意义上的基因组，且基因编辑体系在其细胞内具有活性。例如，所述的动物可以是哺乳纲的动物，包含人、非人灵长类动物(猴、猩猩)、家畜与农畜(例如，猪、绵羊、牛)，鼠(小鼠)，以及啮齿动物(例如，小鼠、大鼠、兔)等等。较佳地，所述动物的细胞表达SMN蛋白。
如本文所用，所述“细胞”是指能够表达SMN蛋白的细胞，且基因编辑体系在其内具有活性。所述“细胞”包括但不限于：体(组织)细胞，诱导性多能干细胞，生 殖细胞(如受精卵细胞，卵母细胞)。
如本文所用，所述“脱靶效应”是指对于基因组中特定位置的改造未能达到预先设定的目标，改造发生偏移或未能发生改造。发生“脱靶效应”的原因包括但不限于：对靶点的结合不精确、识别序列以后的切割操作不精确、对切割位点的编辑的精确程度不够等。
提高SMN蛋白表达的方法及试剂
尽管基因组编辑技术在近年来已经被较多地运用，但是其存在着脱靶效应等问题，如何运用这一技术来纠正细胞基因组，改变疾病状态，仍是一个不易于实现的课题。现有技术中绝大多数的疾病，尚没有发现有效的基于基因编辑技术的治疗方法和治疗药物，例如脊髓性肌萎缩症(Spinal Muscular Atrophy；SMA)，SMN2是和SMN1高度相似的基因，但是由于SMN2基因7号外显子上C6T的变异导致其在剪接过程当中7号外显子被跳过，形成了缺少7号外显子的转录本，翻译产生了截短的SMN蛋白。理论上而言，使得截短表达的情况更正为正常表达即可，理论上的操作方式也不少，但是却鲜少有真正有效的操作工具来成功地、稳定地、无差错地实现这种表达更正。因此，本发明人致力于优化研究针对SMN的基因组编辑策略。
一方面，本发明提供了一种增加功能性SMN2蛋白表达的方法，所述方法包括：靶向SMN2基因7号外显子，变异其中一个或多个碱基；较佳地，所述的一个或多个碱基为该7号外显子的第4、5、6、36、38和/或45位碱基。在具体的一些实施方式中，该方法是体外(离体)的，也即是针对离体的细胞进行的操作。
另一方面，本发明提供了一种制备SMN蛋白高表达的细胞的方法，所述方法包括：靶向SMN2基因7号外显子，变异其中一个或多个碱基；较佳地，所述的一个或多个碱基为该7号外显子的第4、5、6、36、38和/或45位碱基。在具体的一些实施方式中，该方法是体外(离体)的，也即是针对离体的细胞进行的操作。
其它方面，基于本发明的揭示，还可以制备基因组发生基因编辑的动物，所述方法包括：(1)将增加SMN蛋白表达的试剂引入到动物生殖细胞如受精卵中，所述的增加SMN2蛋白表达的试剂是靶向SMN2基因7号外显子，变异其中一个或多个碱基的试剂；较佳地，所述的一个或多个碱基为该7号外显子的第4、5、6、36、38和/或45位碱基；(2)使(1)的动物受精卵发育，获得基因组的待编辑基因区域发生编辑的动物。在具体的一些实施方式中，该方法是以研究为目的的而非以为治疗目的的。
作为本发明的优选方式，将所述7号外显子的第4、5或6位碱基变异为碱基C，或将所述7号外显子的第36、38或45位碱基变异为G。部分的变异，使得核苷酸序列发生变化的同时，也使得蛋白的氨基酸序列中相应位点产生变异，即发生非同义变异(非同义突变)；而另一部分的变异，则没有改变氨基酸序列，即发生同义变异(同义突变)。
在更为具体的实施方式中，一些产生同义突变的位点为：所述7号外显子的第36位变异为G，SMN2蛋白中相应位置的氨基酸(7号外显子第12位氨基酸(Ser))不变；所述7号外显子的第45位变异为G，SMN2蛋白中相应位置的氨基酸(7号外显子第15位氨基酸(Leu))不变；或，所述7号外显子的第6位变异为C，SMN2蛋白中相应位置的氨基酸(7号外显子第2位氨基酸(Phe))不变。
在更为具体的实施方式中，一些产生非同义突变的位点为：所述7号外显子的第36位和第38位变异为G，SMN2蛋白中相应位置的氨基酸(7号外显子第12和13位氨基酸)由Ser-Ser变异为Ser-Arg；所述7号外显子的第4位和第5位变异为C，SMN2蛋白中相应位置的氨基酸(7号外显子第2位氨基酸)由Phe变异为Pro；或，所述7号外显子的第5位变异为C，SMN2蛋白中相应位置的氨基酸(7号外显子第2位氨基酸)由Phe变异为Ser。
在本发明中，相对更为优选的是发生同义突变的情形。本领域技术人员可以预期：在这一情形下，仅在基因水平上改造个别碱基的类型，而不产生蛋白水平上的变化，可以预期这种改变在改变(提高或增加)SMN2蛋白的表达以外，对细胞其它方面的影响必然是微小的或不发生改变的。
在获知了本发明已经揭示的一些SMN2基因7号外显子的碱基位点后，本领域技术人员可以运用多种已知的技术来进行这些位点的变异，从而实现SMN蛋白表达的提高。所述的技术例如但不限于：定点突变技术，RNA干扰技术，同源重组技术或基因编辑技术等。一些基因编辑试剂、定点突变试剂或同源重组试剂也是可以由本领域技术人员设计获得的。在本发明的优选方式中，优选的是基因编辑技术，通过单碱基编辑进行所述位点的变异，为此本发明也提供了适当的DNA单碱基编辑器。
在本发明的优选实施方式中，所述DNA单碱基编辑器为腺嘌呤碱基编辑器(ABE)或其突变体；更佳地，所述腺嘌呤碱基编辑器包括以下操作性连接的元件：TadA和/或TadA*，Cas9n。本发明人建立了多种腺嘌呤碱基编辑器或其变体，从而实现了所述位点的靶向性变异。在更为具体的实施方式中，所述腺嘌呤碱基编辑器包括以下操作性连接的元件：TadA和/或TadA*，Cas9n；更佳地，包括：
ABE：TadA-TadA*，Cas9n或Cas9n-KKH；
ABEmax F148A：TadA F148A-TadA* F148A，Cas9n或Cas9n-KKH；
MiniABEmax：TadA*，Cas9n或Cas9n-KKH；或
miniABEmax V82G：TadA* V82G，Cas9n/Cas9n-KKH。
在更为优选的实施方式中，在所述腺嘌呤碱基编辑器的氨基端和/或羧基端还包括核定位序列。
与上述这些腺嘌呤碱基编辑器相配合地，本发明也提供了一些优选的sgRNA。在优选的实施例中，所述sgRNA为SEQ ID NO:3、SEQ ID NO:5或SEQ ID NO:8所示序列的sgRNA。
在本发明的最为优选的实施方式中，提供了一种靶向SMN2外显子7的ESS-B的A36G的同义突变(同义丝氨酸)。这一突变通过使用miniABEmax和sgRNA6的组合来获得。这一突变的优异特点在于：一方面，其可实现同义突变，也即不改变SMN2的氨基酸序列；另一方面，其提高SMN2表达水平的能力是非常显著的，本发明的实施例、特别是图1e中，显示了其对于SMN2表达水平的极为有效的提高；第三方面，在一项考察DNA脱靶效应的实验中，本发明人发现针对这一位点没有可检测到的脱靶效应，无疑这是更有利于临床研究以及临床应用的。
SMN2基因突变体及含有突变体的细胞
基于本发明所揭示的技术方案，还提供了一种SMN2基因突变体，所述SMN2基因突变体与野生型的序列相比，其核苷酸序列的7号外显子上发生一个或多个碱基的变异，所述变异存在于该7号外显子的第4、5、6、36、38和/或45位碱基上。在优选的实施方式中，所述7号外显子的第4、5或6位碱基被变异为碱基C；或所述7号外显子的第36、38或45位碱基被变异为G。
野生型的SMN2核苷酸序列是本领域技术人员已知的，例如其序列可以如SEQ ID NO:2所示。在野生型序列的基础上，除了本发明中所感兴趣的第7外显子上的位点，SMN2核苷酸序列上的其它位置，在不同个体或不同来源下，也可能存在部分碱基的变化，这些变化形式可能是不影响SMN2蛋白表达的，它们也应被涵盖在本发明中。例如，还涵盖与SEQ ID NO:2所示序列同源性高于80％、85％、90％、95％、98％、99％或更高的SMN2序列，或还涵盖SEQ ID NO:2所示序列的片段，它们也能编码全长的SMN2蛋白或其具有活性的部分。
本发明的SMN2基因突变体可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。其序列可以是只编码成熟蛋白的编码序列；成熟蛋白的编码序列和各种附加编码序列；成熟蛋白的编码序列(和任选的附加编码序列)以及非编码序列。当需要制备所述的基因突变体时，其全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或经改造的酶编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述蛋白的方法。
通过常规的重组DNA技术，可利用本发明的多聚核苷酸序列来重组表达或生产重组的SMN2蛋白。一般来说有以下步骤：(1).用本发明的编码经改造的酶的多核苷酸，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；(2).在合适的培养基中培养的宿主细胞；(3).从培养基或细胞中分离、纯化蛋白质。
基于本发明的披露，本发明还提供了一种SMN2蛋白高表达的细胞，所述细胞中包含本发明所述的SMN2基因变体；较佳地，所述的细胞包括(但不限于)：诱导性 多能干细胞(iPSCs)、胚胎干细胞(mESCs)，生殖细胞(如卵母细胞或受精卵)，体细胞；或，其为一种基因工程宿主细胞(例如，用于重组表达SMN2蛋白)。
本发明的SMN2蛋白高表达的细胞，其中可含有外源的增加SMN2蛋白表达的核酸(核酸构建体或试剂)，所述的增加SMN2蛋白表达的试剂是靶向SMN2基因7号外显子，变异其中一个或多个碱基的试剂；较佳地，所述的一个或多个碱基为该7号外显子的第4、5、6、36、38和/或45位碱基。
所述的增加SMN2蛋白表达的试剂可以包括但不限于：基因编辑试剂、定点突变试剂或同源重组试剂。作为本发明的优选实施方式，所述的试剂包括DNA单碱基编辑器和sgRNA；更佳地，所述DNA单碱基编辑器为腺嘌呤碱基编辑器(ABE)或其突变体。
应用
本发明提供了一种利用单碱基编辑介导的剪接修复技术，针对SMN2基因的7号外显子进行编辑的技术，以及提供了一系列进行所述修复的方法和工具。本发明的技术方案可以应用于包括但不限于以下的一些方面：(1)应用于对hiPSC等细胞进行基因编辑，挑选出修正的细胞株，进行SMN2表达水平的验证以及功能验证；(2)应用于对生殖细胞如受精卵进行基因编辑，对获得的细胞或个体进行SMN2表达水平的验证以及功能验证；(3)应用于对生物体如小鼠进行注射，如对侧脑室和鞘内进行注射，观察疾病改善情况、生物体生存情况和SMN2的功能等。
脊髓性肌萎缩症(SMA)是导致婴幼儿死亡的遗传性疾病，主要由于SMN1基因的突变或者缺失导致SMN蛋白缺失所致。基于本发明的新发现，本发明还提供了一种增加SMN2蛋白表达的试剂在制备缓解或治疗脊髓性肌萎缩症中的用途；所述的增加SMN2蛋白表达的试剂是靶向SMN2基因7号外显子，变异其中一个或多个碱基的试剂；较佳地，所述的一个或多个碱基为该7号外显子的第4、5、6、36、38和/或45位碱基。
进一步地，本发明提供了一种用于缓解或治疗SMA的药物组合物或药盒，其中含有增加SMN2蛋白表达的试剂，所述试剂是靶向SMN2基因7号外显子、变异其中一个或多个碱基的试剂；较佳地，所述的一个或多个碱基为该7号外显子的第4、5、6、36、38和/或45位碱基；更佳地，所述的增加SMN2蛋白表达的试剂包括：DNA单碱基编辑器，sgRNA。
所述的sgRNA可以为mRNA形式的，通过注射(如显微注射)进入到细胞中，也可以将之构建于构建体如表达质粒上、进而引入到细胞中。所述的构建物可以为表达质粒，包括：病毒质粒或非病毒质粒。较佳地，为病毒质粒，例如但不限于：腺相关病毒质粒(AAV)，慢病毒质粒(Lentivirus)等。
所述的DNA单碱基编辑器可以为mRNA形式的，通过注射(如显微注射)进入到 细胞中，也可以将之构建于构建体如表达质粒上、进而引入到细胞中。所述的构建物可以为表达质粒，包括：病毒质粒或非病毒质粒。较佳地，为病毒质粒，例如但不限于：腺相关病毒质粒(AAV)，慢病毒质粒(Lentivirus)等。
所述的sgRNA可以与DNA碱基编辑器被分别或共同引入到细胞中。
所述的药物组合物中，还可含有药学上可接受的载体。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。活性成分的给药量是治疗有效量。本发明的药物制剂还可制成缓释制剂。
本发明还提供了一种用于缓解或治疗脊髓性肌萎缩症的药盒，所述的药盒包括本发明所述的药物组合物；或包括本发明所述的sgRNA和碱基编辑器；或包括本发明所述的表达构建物。
在本发明的具体实施例中，本发明人利用了转入人SMN基因的小鼠作为SMA模型小鼠，在观测到全长SMN2转录本和SMN蛋白明显提高的同时，本发明人还观测到编辑过的SMA小鼠体重明显增加、运动功能显著提高、寿命明显延长。同时，本发明人出乎意料地发现，靶向变异第7外显子6号位置(第6位)以外的位置的碱基效果更佳。本发明人观测到的这些结果，证实了可以运用碱基编辑介导的剪接纠正SMN2基因，藉由单碱基编辑技术来治疗SMA患或制备SMA药物，这是一种新的治疗途径。
根据SMA疾病的特点，临床上所有的病人都携带有病变的SMN2基因，所以本发明的这个策略适合临床所有的SMA病人。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社中所述的条件，或按照制造厂商所建议的条件。
材料和方法
SMN1核苷酸序列(SEQ ID NO:1)(以 下划线标出7号外显子)：
Figure PCTCN2021077622-appb-000001
Figure PCTCN2021077622-appb-000002
SMN2核苷酸序列(SEQ ID NO:2)(以 下划线标出突变位置)：
Figure PCTCN2021077622-appb-000003
sgRNA序列(其靶向位置如图1a、图2a中所示)：
sgRNA1：5’-TTTGTCTAAAACCCTGTAAG-3’(SEQ ID NO:3)；
sgRNA2：5’-TAGACAAAATCAAAAAGAAG-3’(SEQ ID NO:4)；
sgRNA3：5’-CACATTCCTTAAATTAAGGA-3’(SEQ ID NO:5)；
sgRNA4：5’-TTTAGACAAAATCAAAAAGA-3’(SEQ ID NO:6)；
sgRNA5：5’-GACAAAATCAAAAAGAAGGA-3’(SEQ ID NO:7)；
sgRNA6：5’-CACATTCCTTAAATTAAGGA-3’(SEQ ID NO:8)。
ABE基因编辑器表达构建体的建立：
本发明人设计了多种ABE基因编辑器，具体如下(结构示意图见图1b)：
ABE(从N端至C端)：bpNLS(核定位序列)、TadA、TadA*、SpCas9n/SaCas9n-KKH、 bpNLS、polyA。
ABEmax F148A(从N端至C端)：bpNLS、TadA F148A-TadA* F148A、SpCas9n/SaCas9n-KKH、bpNLS、polyA。
MiniABEmax(从N端至C端)：bpNLS、TadA*、SpCas9n/SaCas9n-KKH、bpNLS、PolyA。
miniABEmax V82G(从N端至C端)：bpNLS、TadA* V82G、SpCas9n/SaCas9n-KKH、bpNLS、PolyA。
bpNLS序列为：N端：5’-atgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtc-3’(SEQ ID NO:10)；C端：5’-aaaagaaccgccgacggcagcgaattcgagcccaagaagaagaggaaagtc-3’(SEQ ID NO:11)。
TadA(腺苷脱氨酶)序列见：L.W.Koblan,J.L.Doman,C.Wilson,et al,Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction[J],Nat Biotechnol,2018,36(9):843-846。
TadA*(突变的腺苷脱氨酶)序列：L.W.Koblan,J.L.Doman,C.Wilson,et al,Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction[J],Nat Biotechnol,2018,36(9):843-846。
saCas9-KKH序列：参见文献B.P.Kleinstiver,M.S.Prew,S.Q.Tsai,et al,Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition[J],Nat Biotechnol,2015,33(12):1293-1298。
SaCas9n-KKH(为SaCas9-KKH突变体，在SaCas9-KKH基础上发生D10A的突变)序列：参见文献B.P.Kleinstiver,M.S.Prew,S.Q.Tsai,et al,Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition[J],Nat Biotechnol,2015,33(12):1293-1298。
TadA* V82G是指在TadA*基础上，将其第82位的V突变为G。
TadA F148A，TadA* F148A突变位置：参见文献C.Zhou,Y.Sun,R.Yan,et al,Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis[J],Nature,2019,571(7764):275-27。
SMN Exon7序列为(54bp)(SEQ ID NO:9)：
Figure PCTCN2021077622-appb-000004
T4C：SMN2基因7号外显子上的4号位置(Exon7的第4个碱基)产生的T→C的转变；对应于SMN2核苷酸序列(SEQ ID NO:2)，指第838位发生转变。
T5C：SMN2基因7号外显子上的5号位置(Exon7的第5个碱基)产生的T→C的转变；对应于SMN2核苷酸序列(SEQ ID NO:2)，指第839位发生转变。
T6C：SMN2基因7号外显子上的6号位置(Exon7的第6个碱基)产生的T→C的转变；对应于SMN2核苷酸序列(SEQ ID NO:2)，指第840位发生转变。
A45G：SMN2基因7号外显子上的45号位置(Exon7的第45个碱基)产生的A→G的转变；对应于SMN2核苷酸序列(SEQ ID NO:2)，指第879位发生转变。
A36G：SMN2基因7号外显子上的36号位置(Exon7的第36个碱基)产生的A→G的转变；对应于SMN2核苷酸序列(SEQ ID NO:2)，指第870位发生转变。
A38G：SMN2基因7号外显子上的38号位置(Exon7的第38个碱基)产生的A→G的转变；对应于SMN2核苷酸序列(SEQ ID NO:2)，指第872位发生转变。
轻型SMA小鼠(Smn -/-，SMN2 2TG/2TG)的构建：参见文献H.M.Hsieh-Li,J.G.Chang,Y.J.Jong,et al,A mouse model for spinal muscular atrophy[J],Nat Genet,2000,24(1):66-70.
杂合Smn敲除小鼠(Smn +/-)的构建：参见文献H.M.Hsieh-Li,J.G.Chang,Y.J.Jong,et al,A mouse model for spinal muscular atrophy[J],Nat Genet,2000,24(1):66-70.
重型SMA小鼠胚胎干细胞的诱导：将雌性轻度SMA小鼠(Smn -/-,SMN2 2TG/2TG)与雄性杂合Smn敲除小鼠(Smn +/-)交配后，至囊胚期时取出胚胎，后续诱导及扩增方法，参见文献E.W Zuo,X.N.Huo,X.Yao,et al.,CRISPR/Cas9-mediated Targeted Chromosome Elimination[J],Genome Biol,2017,18(1):224.基因型鉴定，Smn -/-,SMN2 2TG/0为阳性克隆。
将ABE或经改造的ABE连同sgRNA转染细胞的方法步骤：HEK293T或重型SMA小鼠胚胎干细胞消化后铺于6孔板，～60％细胞密度，采用Lipofectamine 3000(Thermo Fisher Scientific)转染细胞，每孔细胞的质粒转染量为4ug，转染后72小时流式分选获得荧光阳性细胞。
iPSC的培养与电穿孔转染及单克隆获取步骤：iPSC的培养方法，参见文献Lin X,Li JJ,Qian WJ,et al.Modeling the differential phenotypes of spinal muscular atrophy with high-yield generation of motor neurons from human induced pluripotent stem cells.[J],Oncotarget,2017,8:42030-42042.待细胞密度达～80％，采用核穿孔转染仪(Lonza 2B)电转细胞，1X10 7细胞量对应质粒转染量为10ug，转染后72小时流式分选获得荧光阳性细胞。单克隆采用机械法挑取获得，具体参见文献J.J.Li,X.Lin,C.Tang,et al.,Disruption of splicing-regulatory elements using CRISPR/Cas9 to rescue spinal muscular atrophy in human iPSCs and mice.[J]Natl Sci Rev,2019,7(1):92-101.
iPSC的运动神经元分化与电转染步骤：运动神经元的分化方法，参见文献Lin X,Li JJ,Qian WJ,et al.Modeling the differential phenotypes of spinal muscular atrophy with high-yield generation of motor neurons from human induced pluripotent stem cells.[J],Oncotarget,2017,8:42030-42042.培养至第15天后，采用核穿孔转染仪(Lonza 2B)电转细胞，1X10 7细胞量对应质粒转染量为10ug，转染后72小时流式分选获得荧光阳 性单细胞。
在体神经元电穿孔与组织染色的步骤：将出生后第0天的SMNΔ7 SMA小鼠在冰上麻醉30-60秒，将1.5μg质粒混合0.05％Fast Green染料，使用玻璃针进行侧脑室注射。随后使用ECM-830仪器进行在体电穿孔，电脉冲持续时间60ms，间隔950ms，电压80V。出生后第7天将小鼠处死。使用胰酶消化脑组织，通过流式细胞分选分离出GFP阳性神经元进行后续步骤。准备进行免疫组化染色的小鼠使用PFA及30％蔗糖进行灌注后进行脑组织切片，切片厚度为40μm。随后使用DAPI染色30分钟。
受精卵注射及胚胎移植步骤：将轻型SMA mice(Smn -/-,SMN2 2TG/2TG)雌性小鼠超数排卵后与杂合Smn敲除小鼠(Smn +/-)交配。随后取出受精卵，通过通过显微注射法将50ng/μl ABE mRNA and 100ng/μl sgRNA1体外转录产物注射入受精卵胞质中，在KSOM中孵育过夜至二细胞期后，移入假孕ICR雌性小鼠的输卵管中。
TaqMan探针法qPCR分析步骤：使用TaqMan Universal PCR Master Mix试剂盒及FAM-based TaqMan assays定制探针试剂盒依据操作说明书，在LightCycler480实时定量PCR系统进行反应并测定细胞的SMN2-FL以及SMN2-△7转录本水平。以上具体使用到的引物及探针序列如表1。
表1
名称 引物或探针(5’-3’)
Fwd_SMN2-FL GCTGATGCTTTGGGAAGTATGTTA(SEQ ID NO:12)
Rev_SMN2-FL GAGCACCTTCCTTCTTTTTGA(SEQ ID NO:13)
SMN2-FL_probe TACATGAGTGGCTATCATACT(SEQ ID NO:14)
Fwd_SMN2-Δ7 TATCATACTGGCTATTATATGGAA(SEQ ID NO:15)
Rev_SMN2-Δ7 TCCAGATCTGTCTGATCGTTTCTT(SEQ ID NO:16)
SMN2-Δ7 Probe CTGGCATAGAGCAGCACTAAATGACACCAC(SEQ ID NO:17)
免疫染色步骤：细胞使用4％甲醛溶液进行固定后用0.2％Triton-X-100和10％驴血清进行打孔及封闭。使用一抗在4℃下孵育过夜。随后使用荧光二抗(稀释比1：1000)及DAPI(稀释比1：1000)室温孵育两小时。一抗的具体信息及稀释比例如表2。
表2
抗体 稀释比 抗体 稀释比
OLIG2 1:300 SSEA4 1:400
HB9 1:100 SOX2 1:1000
MAP2 1:5000 TRA-1-60 1:1000
SMN 1:75 TUNEL 1:10
NANOG 1:1000    
用于免疫荧光染色的小鼠在出生后9天时处死，取出脊髓后在4％PFA中室温固定1-3小时，随后使用30％蔗糖4℃脱水过夜。将脊髓腰段(L1-L2)以20μm厚度切片。运动神经元及核双子星体分别使用ChAT抗体(稀释比1：500)和SMN抗体(稀释比1：300)在4℃下与TritonX-100、BSA、NaN3混合孵育过夜。随后使用荧光二抗及DAPI4℃孵育过夜。使用Leica TCS SP8进行图像采集，随后使用Image-J软件对细胞进行计 数。
高通量测序建库及数据处理步骤：流式分选阳性细胞群裂解、巢式PCR扩增(如下表3，巢式引物)，单个神经元通过逆转录及特异性扩增(单细胞引物)。依据Illumina HiSeq X-Ten平台构建扩增子进行测序。数据使用fastp(v0.19.6)进行预处理，FASTQ文件通过barcodes序列进行分离，并通过Smith-Waterman算法与参考基因组匹配，提取并计算突变序列数目。
表3
名称 引物(5’-3’)
巢式引物-外-F AGCCACTGCAAGAAAACCTTAACTG(SEQ ID NO:18)
巢式引物-外-R CCAGAGGCTTGACGAATTCCA(SEQ ID NO:19)
巢式引物-内-F TGGGATAACTTTTAAAGTAC(SEQ ID NO:20)
巢式引物-内-R GAATTC-barcode-GGATCCGTTTTCCACAAACCATAAAG(SEQ ID NO:21)
单细胞引物-F TCTCTTGATGATGCTGATGC(SEQ ID NO:22)
单细胞引物-R TCTGATCGTTTCTTTAGTGGTGTC(SEQ ID NO:23)
实施例1、利用碱基编辑器ABE靶向ESSA和ESSB的单碱基编辑
SMN2是和SMN1高度相似的基因，但是由于SMN2的7号外显子上C6T的差异导致其在剪接过程当中7号外显子被跳过，如图1a。
Hua等在2007年报道，通过ASO靶向SMN2第7外显子中的ESS可增强SMN2第7外显子的inclusion(剪接产物包含exon7)。在此基础上，本发明人检验了这种策略是否也可以通过基因组编辑来实现。
本发明人发现，NHEJ(非同源末端连接)会导致潜在的随机序列生成和CRISPR/Cas9介导的同源重组修复途径的编辑效率低下。在此基础上，本发明人选择单碱基编辑的方案，为此设计了三个ABE(产生A-to-G或T-to-C的转变)的KKH变体(金黄色酿脓葡萄球菌的Cas9单切口酶(Cas9n)，识别序列为NNNRRT)的sgRNA(sgRNA1-3)作用于SMN2第7外显子位点的ESS-A或ESS-B(图2a)，并用于后续实验。本发明设计的ABE基因编辑器的表达构建体的图谱如图1b，该ABE基因编辑器能够在特定的基因位点产生一个精确的核苷酸的转变且不产生双链断裂。
本发明人测试了这些sgRNAs在诱导多功能干细胞(iPSCs)系中恢复全长SMN(SMN-FL)表达的能力，获取存在7号外显子C6T突变的细胞系，该细胞系来源于携带3个SMN2拷贝的SMA患者(图2b～d)。本发明人还使用了健康个体来源的诱导多功能干细胞作为野生型对照(有2个SMN1拷贝和3个SMN2拷贝)(图2b、d)。
本发明人计算了PAM位点上游20bp区域的A-G编辑频率，利用热图显示了三个sgRNA(sgRNA1；sgRNA2；sgRNA3)靶向SMN2外显子7的ESS-A和ESS-B(图2f～h)的情况。高通量测序显示，sgRNA1和sgRNA3产生有效编辑。
进行测序和基于TaqMan的qPCR分析，如图1c，以碱基编辑器ABE-sgRNA1和ABE-sgRNA3靶向ESSA和ESSB的单碱基编辑效率。对于所分析的46个克隆(sgRNA1)和10个克隆(sgRNA3)，可分别获得6个针对ESS-A的剪接纠正型SMA(SC-SMA)克隆(例如：﹟1-SC-SMA T5C；Phe-to-Ser)和1个针对ESS-B的克隆(﹟1-SC-SMA A45G；Leu-to-Leu)。ABE-sgRNA1编辑SMA iPSC、ABE-sgRNA3编辑SMA iPSC的编辑效率的热图显示于图1d中。全长SMN2(SMN2-FL)mRNA和截断的SMN△7 mRNA的TaqMan qPCR分析结果如图1e～f和图2i～j，可见﹟1-SC-SMA T5C、﹟1-SC-SMA T6C、﹟1-SC-SMA A45G、﹟1-SC-SMA T4C,T5C中全长SMN2有了显著的提高，而SMN△7的表达有了显著的降低。Sanger测序确认编辑结果如图3a～b。
各个突变导致的氨基酸变化如下表4。
表4
核苷酸突变 氨基酸变异  
SC-SMA T4C,T5C Phe→Pro 非同义
SC-SMA T5C Phe→Ser 非同义
SC-SMA T6C Phe→Phe 同义
SC-SMA A45G Leu→Leu 同义
为了验证错义T5C转变(在SMN2基因7号外显子上的5号位置产生的T-to-C的转变)产生的影响，本发明人选择了﹟1-SC-SMA T5C克隆作进一步研究。该克隆可以显示特征表达的多能性标记和正常核型(图4a，b)。通过全基因组测序(WGS)进行的DNA脱靶分析显示没有检测到非目标效应即脱靶效应(表5)。
表5、WGS检测#1-SC-SMA T5C and #2-SC-SMAA 36G,A38G iPSCs脱靶情况
Figure PCTCN2021077622-appb-000005
为了测试剪接修复对运动神经元(Motor neurons，MNs)的影响，本发明人将脊髓MNs从三种基因型的iPSCs中进行了分化：#1-SC-SMA T5C、SMA和WT。诱导多能 干细胞向脊髓MNs分化的时间轴以及诱导所用的因子如图4e。WT、SMA、#1-SC-SMA T5C iPSCs在第10天(图4f)分化形成OLIG2 +运动神经元前体，在第13天(图4g)分化形成HB9 +MNs。结果说明，SC-SMA T5C、SMA和WT，这些细胞都表现出典型的分化(图4e～i)。
此外，免疫细胞化学分析显示，与SMA MNs相比，﹟1-SC-SMA T5C MNs具有更多的SMN蛋白复合体(P<0.001，增加了约1.6倍)(图1g、h)。由于SMN蛋白也发挥轻微的抗凋亡作用，本发明人发现﹟1-SC-SMA T5C MNs中增加的SMN蛋白对凋亡诱导物喜树碱(Camptothecin)和衣霉素(Tunicamucin)的敏感性低于SMA MNs(图5)。
接下来，本发明人通过将编码ABE和sgRNA1的mRNA共注射到轻型SMA小鼠(Smn -/-，SMN2 2TG/2TG)与杂合Smn敲除小鼠(Smn +/-)杂交获得的合子细胞质中，出生了一只携带T5C编辑(称为：SC-SMA T5C，Smn -/-，SMN2 T5C/0)的重型SMA小鼠(Smn -/-，SMN2 2TG/0)(图6a)。在出生后第9天，SC-SMA T5C初代小鼠明显比同年龄未编辑的SMA小鼠更健康(图1i)。雄性的SC-SMA T5C初代小鼠与雌性杂合子Smn +/-小鼠杂交，产生12只存活的SMA幼鼠，均携带T5C转换位点(SC-SMA T5C)(图6b、c)，与携带T5C转换的同窝杂合子(HET T5C，Smn +/-，SMN2 T5C/0)相比，没有任何明显的表型差异(图1j，图6c)。
然而，与未编辑的SMA小鼠(14天内死亡)相比，SC-SMA T5C小鼠显示出更长的寿命(>400天)，体重的明显增加，以及运动功能的提高(翻正反射)(图6d～f)。
此外，免疫细胞化学分析显示，与未编辑过的SMA小鼠相比，SC-SMA T5C小鼠脊髓MNs中出现的SMN蛋白复合体数量显著增加(P<0.001，图6g、h)。
实施例2、利用改造的碱基编辑器靶向ESSA和ESSB的单碱基编辑
本发明人的工作证实，最初使用的ABE不会产生可检测到的DNA脱靶效应，然而，它会产生成千上万的脱靶RNA单核甘酸变异。因此，为了最小化T5C编辑过程中的RNA脱靶和任何生物效应，本发明人使用了三个新开发的高保真的单碱基编辑器：TadA F148A-TadA* F148A-ABEmax(简称ABEmax F148A)、TadA*-ABEmax(简称miniABEmax)和TadA* V82G-ABEmax(简称miniABEmax V82G)，并且进一步设计了sgRNAs 4～6(图1a～b，图2a)。
将上述单碱基编辑器同sgRNAs 1-6转染细胞，并在HEK293T细胞、重型SMA模型小鼠来源的胚胎干细胞(mESCs)和SMA小鼠诱导多功能干细胞(iPSC)中识别任何剪接修复相关位点(图7a)；转染后细胞表达报告基因GFP的代表性图像如图7b；流式细胞仪分析转染细胞如图7c。4种DNA单碱基编辑器(ABE、ABEmax F148A、miniABEmax和miniABEmax V82G)在3个细胞系中使用6种不同的sgRNAs(sgRNA1到sgRNA6)识别的编辑位点的摘要如表6(靶向SMN2外显子7中的ESS-A和ESS-B)，表中“N”表示没有编辑。
Figure PCTCN2021077622-appb-000006
通过使用miniABEmax和sgRNA6的组合来靶向SMN2外显子7的ESS-B，这个方式有助于功获得一个新的A36G的同义突变(同义丝氨酸)(图1c、f，图3c～f和图8)。
比较各个剪接修复型SMA(SC-SMA)克隆(图1e～f，图2i～j)中全长SMN2和SMN△7的表达，﹟1-SC-SMA A36G克隆中呈现相对更高的全长SMN2表达以及相对更低的SMN△7表达，其次为﹟2-SC-SMA  A36G,A38G克隆、﹟1-SC-SMA T4C,T5C克隆。
值得注意的是，本发明人获得的﹟2-SC-SMA  A36G,A38G克隆，具有两个转变，即前面提到的A36G(Ser-to-Ser)和A38G(His-to-Arg)(图1c～d，图4c～d)，未检测到DNA脱靶效应(表5)。此外，本发明人发现iPSC分化的﹟2-SC-SMA A36G,A38G MNs对凋亡诱导物喜树碱和衣霉素的敏感性低于SMA MNs(图1g、h，图4e～i和图5)。因此，从蛋白水平检测表型的结果可见，﹟2-SC-SMA A36G,A38G增加的SMN蛋白具有正常功能。
为了评估神经元有丝分裂后期的单碱基编辑可行性，本发明人用miniABEmax和sgRNA6(miniABEmax-sgRNA6)(图1k)转染了有丝分裂后期的源自于SMA iPCSs的MNs。实现了A36G和A38G碱基转变的成功编辑(图1l、m)。转染后的单个MNs细胞的高通量测序结果显示编辑后的MNs的SMN2-FL基因的表达明显增加，同时A36G转变编辑效率高于A38G(图1n)。此外，miniABEmax-sgRNA6编辑后的SMN△7SMA小鼠的在体内的神经元电切显示，其碱基转变(A36G、A38G)的编辑效率的检测如图1o～q。
综上所述，本发明人成功地使用单碱基编辑系统对SMN2外显子7的ESS-A和ESS-B进行了编辑，从而实现了高效的同义A36G转变，实现了SMN2基因异常剪接的修复。体外凋亡实验证实了A36G和A38G同步转变诱导的SMN蛋白的具备正常的功能。
本发明的研究表明，可以运用单碱基编辑系统修复SMN2基因的异常剪接，藉由单碱基编辑技术来治疗SMA患者，而且，靶向变异第7外显子6号位置以外的碱基效果更佳，这是一种新的治疗途径。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (23)

  1. 一种增加功能性SMN2蛋白表达的方法，其特征在于，该方法包括：靶向SMN2基因7号外显子，变异其中一个或多个碱基；较佳地，所述的一个或多个碱基为该7号外显子的第4、5、6、36、38和/或45位碱基。
  2. 如权利要求1所述的方法，其特征在于，所述7号外显子的第4、5或6位碱基被变异为碱基C；或所述7号外显子的第36、38或45位碱基被变异为G。
  3. 如权利要求1～2任一所述的方法，其特征在于，
    所述7号外显子的第36位变异为G，SMN2蛋白中相应位置的氨基酸不变；
    所述7号外显子的第45位变异为G，SMN2蛋白中相应位置的氨基酸不变；
    所述7号外显子的第36位和第38位变异为G，SMN2蛋白中相应位置的氨基酸由Ser-Ser变异为Ser-Arg；
    所述7号外显子的第4位和第5位变异为C，SMN2蛋白中相应位置的氨基酸由Phe变异为Pro；
    所述7号外显子的第5位变异为C，SMN2蛋白中相应位置的氨基酸由Phe变异为Ser；或
    所述7号外显子的第6位变异为C，SMN2蛋白中相应位置的氨基酸不变。
  4. 如权利要求1所述的方法，其特征在于，利用基因编辑、定点突变或同源重组来进行SMN2基因7号外显子的变异；较佳地，利用单碱基编辑介导的剪接修复来进行基因编辑。
  5. 如权利要求4所述的方法，其特征在于，所述单碱基编辑介导的剪接修复利用DNA单碱基编辑器进行；较佳地所述DNA单碱基编辑器为腺嘌呤碱基编辑器或其变体；更佳地，所述腺嘌呤碱基编辑器包括以下操作性连接的元件：TadA和/或TadA*，Cas9n。
  6. 如权利要求5所述的方法，其特征在于，所述腺嘌呤碱基编辑器或其变体包括以下操作性连接的元件：
    ABE：TadA-TadA*，Cas9n或Cas9n-KKH；
    ABEmax F148A：TadA F148A-TadA* F148A，Cas9n或Cas9n-KKH；
    MiniABEmax：TadA*，Cas9n或Cas9n-KKH；
    miniABEmax V82G：TadA* V82G，Cas9n/Cas9n-KKH；
    较佳地，在其氨基端和/或羧基端还包括核定位序列。
  7. 如权利要求5所述的方法，其特征在于，利用SEQ ID NO:3、SEQ ID NO:5或SEQ ID NO:8所示序列的sgRNA进行碱基编辑。
  8. 如权利要求5～7任一所述的方法，其特征在于，所述方法应用于诱导性多能干细胞、胚胎干细胞、生殖细胞或体细胞，该细胞中SMN2基因编码截短的SMN2蛋白；所述方法包括：提供针对SMN2基因7号外显子的sgRNA以及DNA单碱基编辑器，引入到所述细胞中。
  9. 一种SMN2基因突变体，其核苷酸序列的7号外显子上发生一个或多个碱基的变异，所述变异存在于该7号外显子的第4、5、6、36、38和/或45位碱基上。
  10. 如权利要求9所述的SMN2基因突变体，其特征在于，所述7号外显子的第4、5或6位碱基被变异为碱基C；或所述7号外显子的第36、38或45位碱基被变异为G。
  11. 如权利要求10所述的SMN2基因变体，其特征在于，
    所述7号外显子的第36位变异为G，SMN2蛋白中相应位置的氨基酸不变；
    所述7号外显子的第45位变异为G，SMN2蛋白中相应位置的氨基酸不变；
    所述7号外显子的第36位和第38位变异为G，SMN2蛋白中相应位置的氨基酸由Ser-Ser变异为Ser-Arg；
    所述7号外显子的第4位和第5位变异为C，SMN2蛋白中相应位置的氨基酸由Phe变异为Pro；
    所述7号外显子的第5位变异为C，SMN2蛋白中相应位置的氨基酸由Phe变异为Ser；或
    所述7号外显子的第6位变异为C，SMN2蛋白中相应位置的氨基酸不变。
  12. 如权利要求9～11任一所述的SMN2基因变体，其特征在于，所述SMN2基因7号外显子序列如SEQ ID NO:9。
  13. 一种细胞，所述的细胞中功能性SMN2蛋白正常表达或高表达，其特征在于，所述的细胞中包含权利要求9～12任一所述的SMN2基因变体；较佳地，所述的细胞包括：诱导性多能干细胞、胚胎干细胞，生殖细胞或体细胞。
  14. 权利要求9～12任一所述的SMN2基因变体的用途，用于制备增加功能性SMN2蛋白表达的构建体或试剂。
  15. 如权利要求1～8任一所述的方法的用途，用于制备增加功能性SMN2蛋白表达的构建体或细胞；较佳地，所述的细胞包括：诱导性多能干细胞、胚胎干细胞、生殖细胞或体细胞。
  16. 增加功能性SMN2蛋白表达的试剂在制备缓解或治疗脊髓性肌萎缩症的药物中的用途；所述的增加SMN2蛋白表达的试剂是靶向SMN2基因7号外显子，变异其中一个或多个碱基的试剂；较佳地，所述的一个或多个碱基为该7号外显子的第4、5、6、36、38和/或45位碱基；
  17. 如权利要求16所述的用途，其特征在于，所述的试剂包括：基因编辑试剂、定点突变试剂或同源重组试剂；较佳地，所述的试剂包括DNA单碱基编辑器和sgRNA；更佳地，所述DNA单碱基编辑器为腺嘌呤碱基编辑器或其变体。
  18. 如权利要求17所述的用途，其特征在于，所述腺嘌呤碱基编辑器包括以下操作性连接的元件：TadA和/或TadA*，Cas9n；更佳地，包括：
    ABE：TadA-TadA*，Cas9n或Cas9n-KKH；
    ABEmax F148A：TadA F148A-TadA* F148A，Cas9n或Cas9n-KKH；
    MiniABEmax：TadA*，Cas9n或Cas9n-KKH；
    miniABEmax V82G：TadA* V82G，Cas9n/Cas9n-KKH；
    较佳地，在其氨基端和/或羧基端还包括核定位序列。
  19. 如权利要求17所述的用途，其特征在于，所述sgRNA为SEQ ID NO:3、SEQ ID NO:5或SEQ ID NO:8所示序列的sgRNA。
  20. 一种制备细胞的方法，所述的细胞中功能性SMN2蛋白高表达，其特征在于，所述方法包括：将增加SMN2蛋白表达的试剂引入到细胞中，所述试剂是靶向SMN2基因7号外显子、变异其中一个或多个碱基的试剂；较佳地，所述的一个或多个碱基为该7号外显子的第4、5、6、36、38和/或45位碱基。
  21. 一种用于缓解或治疗脊髓性肌萎缩症的药物组合物或药盒，其特征在于，其中含有增加SMN2蛋白表达的试剂，所述试剂是靶向SMN2基因7号外显子、变异其中一个或多个碱基的试剂；较佳地，所述的一个或多个碱基为该7号外显子的第4、5、 6、36、38和/或45位碱基；更佳地，所述的增加SMN2蛋白表达的试剂包括：DNA单碱基编辑器，sgRNA。
  22. 如权利要求21所述的药物组合物或药盒，其特征在于，所述DNA单碱基编辑器为腺嘌呤碱基编辑器或其变体；更佳地，所述腺嘌呤碱基编辑器包括以下操作性连接的元件：TadA和/或TadA*，Cas9n；更佳地，所述腺嘌呤碱基编辑器或其变体包括：
    ABE：TadA-TadA*，Cas9n或Cas9n-KKH；
    ABEmax F148A：TadA F148A-TadA* F148A，Cas9n或Cas9n-KKH；
    MiniABEmax：TadA*，Cas9n或Cas9n-KKH；
    miniABEmax V82G：TadA* V82G，Cas9n/Cas9n-KKH。
  23. 如权利要求21所述的药物组合物或药盒，其特征在于，所述sgRNA具有如SEQ ID NO:3、SEQ ID NO:5或SEQ ID NO:8所示的序列。
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