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WO2023184688A1 - 功能性β-半乳糖苷酶变体、AAV介导的人β-半乳糖苷酶表达载体及其用途 - Google Patents

功能性β-半乳糖苷酶变体、AAV介导的人β-半乳糖苷酶表达载体及其用途 Download PDF

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WO2023184688A1
WO2023184688A1 PCT/CN2022/095638 CN2022095638W WO2023184688A1 WO 2023184688 A1 WO2023184688 A1 WO 2023184688A1 CN 2022095638 W CN2022095638 W CN 2022095638W WO 2023184688 A1 WO2023184688 A1 WO 2023184688A1
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galactosidase
sequence
expression cassette
gene
seq
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French (fr)
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吴小兵
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北京锦篮基因科技有限公司
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    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
    • C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
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    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
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Definitions

  • the present invention relates to the field of biotechnology. Specifically, the present invention relates to a recombinant adeno-associated virus vector carrying an optimized human ⁇ -galactosidase gene expression cassette. By administering the recombinant adeno-associated virus vector of the present invention, specific expression of ⁇ -galactosidase can be obtained, thereby preventing and/or treating GM1 gangliosidosis.
  • GM1 gangliosidosis also known as GM1 gangliosidosis, is an autosomal recessive disease caused by mutations in the galactosidase beta-1 (GLB1) gene that affects GLB activity. GLB1 gene mutations cause lysozyme Body ⁇ -galactosidase ( ⁇ -gal) activity decreases or disappears. The lack of ⁇ -gal leads to the accumulation of GM1 ganglioside and its sialylated derivative GA1 in the central nervous system (CNS). In addition, oligosaccharides and keratan sulfate accumulate in internal organs.
  • GLB1 gene mutations cause lysozyme Body ⁇ -galactosidase ( ⁇ -gal) activity decreases or disappears.
  • ⁇ -gal lysozyme Body ⁇ -galactosidase
  • GA1 central nervous system
  • oligosaccharides and keratan sulfate accumulate in internal organs
  • GM1-gangliosidosis The incidence of GM1-gangliosidosis is estimated to be 1:100 000-200 000 live births, but the incidence is higher in certain ethnic groups and countries. Based on the age of onset and the residual activity of the corresponding mutant enzyme variant, GM1 patients are divided into three categories, with either a very aggressive or a relatively slow-progressing course. In infant patients, because there is almost no residual enzyme activity, the nervous system rapidly declines and the ability to control voluntary movements is lost, leading to general paralysis, extreme weight loss, and death. Other organs and tissues are also affected, such as characteristic hepatosplenomegaly and skeletal dysplasia. The disease in late-stage children and adults is relatively mild and tends to become chronic.
  • the present invention first provides a GLB1 gene expression cassette, which is suitable for construction into a gene therapy vector and contains at least a promoter sequence and a GLB1 gene sequence, wherein the promoter can drive or direct the GLB1 gene to human ⁇ -galactosidase. Encoded to express active human beta-galactosidase in human cells.
  • the invention also provides an AAV virus vector carrying the GLB1 gene expression cassette.
  • the AAV viral vector only retains the two ITR sequences or variants thereof required for packaging viruses in the wild-type AAV viral genome, and does not contain the protein-coding genes in the wild-type AAV viral genome. This makes the AAV viral vector useful in administration.
  • the immunogenicity after being administered to patients is low; in addition, AAV viral vectors usually achieve sustained and stable expression of the foreign gene reading frame in the form of non-integrated extrachromosomal genetic material, avoiding the random integration of foreign genes introduced into the organism. security issues.
  • the AAV viral vector of the present invention can effectively diffuse and distribute throughout the body, especially the central nervous system, heart, liver, spleen, lungs, kidneys, muscles, serum and other tissues, organs and parts, ensuring GLB1 gene expression
  • the frame can efficiently express active ⁇ -galactosidase throughout the body, especially in the central nervous system, heart, liver, spleen, lungs, kidneys, muscles, serum and other tissues, organs and parts, thereby compensating/compensating
  • the previously missing enzyme activity thus provides a new genetic drug for the prevention and/or treatment of GM1 gangliosidosis.
  • the invention also provides functional beta-galactosidase enzymes, such as human beta-galactosidase variant enzymes.
  • the human ⁇ -galactosidase encoded by the GLB1 gene sequence contained in the expression cassette has the same or substantially the same amino acid sequence as the naturally occurring human wild-type ⁇ -galactosidase, That is, it has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher, or 100% sequence identity with the latter, and has a sequence identity that is not significantly lower than that of the latter. In vivo activity, thereby being able to effectively degrade GM1 ganglioside substrates to avoid and eliminate their accumulation, and alleviate or completely eliminate disease symptoms.
  • the amino acid sequence of the naturally occurring human wild-type ⁇ -galactosidase is, for example, the sequence shown in SEQ ID NO: 10.
  • the human ⁇ -galactosidase encoded by the GLB1 gene sequence contained in the expression cassette has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher, or 100% sequence identity, and having at least the amino acid sequence point mutations I353L, G245D, R299 (L/A/F /Q), and has an in vivo activity not significantly lower than the latter, thereby being able to effectively degrade the GM1 ganglioside substrate to avoid and eliminate its accumulation, and alleviate or completely eliminate disease symptoms.
  • the amino acid sequence of the naturally occurring human wild-type ⁇ -galactosidase is, for example, the sequence shown in SEQ ID NO: 10.
  • the amino acid sequence of human ⁇ -galactosidase encoded by the GLB1 gene sequence contained in the expression cassette is based on the sequence shown in SEQ ID NO: 10 and also has point mutations I353L, One of G245D, R299 (L/A/F/Q).
  • amino acid sequence of human ⁇ -galactosidase encoded by the GLB1 gene sequence contained in the expression cassette is the sequence shown in any one of SEQ ID NO: 11-16.
  • amino acid sequence of human ⁇ -galactosidase encoded by the GLB1 gene sequence contained in the expression cassette is the sequence shown in SEQ ID NO: 10.
  • the nucleotide sequence encoding ⁇ -galactosidase (i.e., GLB1 gene sequence) contained in the expression cassette is the same or substantially the same as the naturally occurring wild-type GLB1 gene, i.e., the latter Be at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater, or 100% identical.
  • the sequence of the naturally occurring wild-type GLB1 gene is, for example, the sequence shown in SEQ ID NO: 9.
  • the GLB1 gene sequence contained in the expression cassette is codon-optimized, expressing the same amino acid sequence while containing more of the most common or more common in humans than the naturally occurring wild-type GLB1 gene. codon form, thereby improving expression speed and expression volume.
  • the codon-optimized sequence is the sequence shown in SEQ ID NO:2.
  • the GLB1 gene sequence contained in the expression cassette is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence shown in SEQ ID NO:2 Or higher, or 100% identical.
  • the coding sequence at positions 895-897 of the GLB1 gene is modified.
  • the codons for amino acids are changed to CTG/GCT/TTT/CAA (with the start codon ATG at positions 1-3).
  • the codon encoding glycine e.g., GGC
  • GAC codon encoding glycine
  • the codon encoding isoleucine at positions 1057-1059 in the GLB1 gene coding sequence (for example, ATC) changed to CTG.
  • the GLB1 gene sequence contained in the expression cassette is the sequence shown in SEQ ID NO: 17, 19, 21, 23, 25 or 27.
  • the GLB1 gene sequence contained in the expression cassette is the sequence shown in SEQ ID NO: 2 or 9.
  • the present invention provides an optimized human GLB1 gene expression cassette.
  • the expression cassette includes:
  • a highly active promoter sequence that can efficiently initiate the transcription of the target gene in both the central nervous system and peripheral tissues and avoid being silenced;
  • a nucleotide sequence encoding a functional ⁇ -galactosidase such as the human GLB1 gene sequence, or its codon-optimized coding sequence; and optionally
  • Optional regulatory sequences such as polyA signal sequence, etc.
  • the functional ⁇ -galactosidase has an in vivo activity that is not significantly lower than that of naturally occurring human wild-type ⁇ -galactosidase, thereby being able to effectively degrade the GM1 ganglioside substrate to avoid and eliminate its accumulation, and Relieve or completely eliminate disease symptoms.
  • the expression cassette includes:
  • nucleotide sequences encoding beta-galactosidase variant enzymes e.g., including mutations at position 299, 245, or 353 of the amino acid sequence, such as SEQ ID NO: 17, 19, 21, The nucleotide sequence shown in 23, 25 or 27;
  • the expression cassette comprises (3), and (3) is a BGH polyA sequence as set forth in SEQ ID NO: 4 or having at least about 90% identity thereto.
  • the expression cassette further comprises:
  • the optimized human GLB1 gene expression cassette of the present invention has a nucleotide sequence as shown in SEQ ID NO: 5, 7, 18, 20, 22, 24, 26 or 28 or is the same as SEQ ID NO : 5, 7, 18, 20, 22, 24, 26 or 28 sequences having at least about 90% identity.
  • both ends of the optimized human GLB1 gene expression cassette of the present invention are connected to the ITR sequence of the AAV viral vector.
  • the expression product of the optimized human GLB1 gene expression cassette of the present invention is a functional ⁇ -galactosidase, which can degrade GM1 ganglioside; in addition, optionally, the expression cassette can also be Suppresses, at least partially, the immune response of an organism.
  • the present invention provides a viral vector comprising the human GLB1 gene expression cassette of the first aspect of the present invention.
  • the viral vector comprises the sequence set forth in SEQ ID NO: 2, 9, 17, 19, 21, 23, 25 or 27.
  • the viral vector comprises the sequence set forth in SEQ ID NO: 5, 7, 18, 20, 22, 24, 26 or 28.
  • the viral vector contains the human GLB1 gene expression cassette of the present invention, and both ends of the human GLB1 gene expression cassette are connected to the ITR sequence of the AAV viral vector.
  • the viral vector comprises, for example, a sequence set forth in SEQ ID NO: 6 or 8.
  • the viral vector of the invention is a recombinant adeno-associated virus vector AAV, including but not limited to, selected from AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, or combinations thereof
  • Recombinant adeno-associated virus vectors of serotypes are preferably recombinant AAV5, AAV3B, AAV8, and AAV9 vectors.
  • the viral vectors of the invention are AAV9 viral vectors.
  • the AAV9 is recombinant AAV9.
  • the genome of a viral vector of the invention can self-complementary to form a double-stranded DNA molecule.
  • the invention provides a functional ⁇ -galactosidase variant enzyme having the same or substantially the same amino acid sequence as a naturally occurring human wild-type ⁇ -galactosidase, i.e. having at least the same amino acid sequence as the latter. 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher, or 100% sequence identity and having in vivo activity not significantly lower than the latter, thereby being effective Degrade GM1 ganglioside substrates to avoid and eliminate their accumulation and alleviate or completely eliminate disease symptoms.
  • the functional ⁇ -galactosidase variant enzyme comprises amino acid sequence position 299 or 245 or 353 relative to naturally occurring human wild-type ⁇ -galactosidase. mutation. In a preferred embodiment, the functional ⁇ -galactosidase variant enzyme comprises a mutation at position 299 relative to naturally occurring human wild-type ⁇ -galactosidase. In some more specific embodiments, the functional ⁇ -galactosidase variant enzyme comprises the following amino acid sequence mutations (i)-(iii) relative to naturally occurring human wild-type ⁇ -galactosidase.
  • the invention also provides nucleic acid molecules encoding the above-mentioned variant enzymes.
  • the present invention provides a medicine for preventing and/or treating GM1 gangliosidosis, such as genetic medicine, etc., wherein the medicine contains the functional ⁇ -galactosidase of the present invention.
  • Viral vector of variant enzyme and optimized human GLB1 gene expression cassette By intravenously injecting the drug of the present invention, such as a gene drug, functional human ⁇ -galactosidase and/or complementing ⁇ -galactosidase can be specifically expressed in the required tissue, organ, site (for example, central nervous system).
  • Galactosidase activity exerted to prevent and/or treat GM1 gangliosidosis or diseases caused by or associated with it.
  • the GM1 gangliosidosis is caused by a defect in the GLB1 gene.
  • the present invention provides the use of the functional ⁇ -galactosidase variant enzyme of the present invention, the optimized human GLB1 gene expression cassette and the viral vector of the present invention, for the preparation of prevention and/or treatment of GM1 neuron Drugs for arthrosidosis, for example, are used to prepare drugs for preventing and/or treating GLB1 gene defects.
  • the preventive and/or therapeutic drugs of the present invention are administered by single or multiple intracerebroventricular injections and/or single or multiple intravenous injections.
  • the viral vector of the present invention is administered by intracerebroventricular injection or intravenous injection, preferably by one-time intracerebroventricular injection or intravenous injection, which reduces the pain to the patient caused by repeated administration of the drug.
  • the viral vector of the present invention is administered by dual intracerebroventricular injection and intravenous injection, for example, a one-time administration.
  • the viral vector of the present invention can stably and continuously express ⁇ -galactosidase in cells after entering the body.
  • the viral vector of the present invention after being administered by intravenous injection, continues to express ⁇ -hemipeptide throughout the body, especially in tissues, organs and parts such as the heart, liver, spleen, lungs, kidneys, muscles, and peripheral blood. Lactosidase, exhibits persistent high levels of enzymatic activity and efficiently degrades the GM1 ganglioside substrate to avoid and eliminate its accumulation.
  • the viral vector of the present invention after being administered by intracerebroventricular injection, continues to express ⁇ -galactosidase throughout the body, especially in tissues, organs and parts such as the central nervous system, showing a sustained high level of levels of enzyme activity and effectively degrades GM1 ganglioside substrates to avoid and eliminate their accumulation.
  • the viral vector of the present invention is found throughout the body, especially in the central nervous system, heart, liver, spleen, lungs, kidneys, muscles, serum and other tissues, organs, and ⁇ -galactosidase is continuously expressed in the site, showing persistent high levels of enzyme activity and effectively degrading the GM1 ganglioside substrate to avoid and eliminate its accumulation.
  • the viral vector of the present invention continues to express ⁇ -galactosidase in tissues, organs and parts of the central nervous system after administration by intracerebroventricular injection and/or intravenous injection, showing sustained The presence of a high level of enzyme activity that meets or exceeds the enzyme activity in the same tissues, organs and sites in normal individuals and effectively degrades the GM1 ganglioside substrate to avoid and eliminate its accumulation and alleviate or completely eliminate the disease symptom.
  • the viral vector of the present invention continues to express ⁇ -galactosidase in tissues, organs and parts of the central nervous system after administration by intracerebroventricular injection and/or intravenous injection, showing sustained The presence of high levels of enzyme activity, which is lower than the enzyme activity in the same tissues, organs and sites in normal individuals, but is still able to effectively degrade the GM1 ganglioside substrate to avoid and eliminate its accumulation, and is sufficient to alleviate or Complete elimination of disease symptoms.
  • the viral vector of the present invention continues to express ⁇ -galactosidase in peripheral tissues, organs and sites after administration by intracerebroventricular injection and/or intravenous injection, showing sustained high levels of The enzyme activity reaches or exceeds the enzyme activity of the same tissues, organs and parts in normal individuals, and effectively degrades the GM1 ganglioside substrate to avoid and eliminate its accumulation, and alleviate or completely eliminate disease symptoms.
  • the viral vector of the present invention continues to express ⁇ -galactosidase in peripheral tissues, organs and sites after administration by intracerebroventricular injection and/or intravenous injection, showing sustained high levels of A level of enzyme activity that is lower than the enzyme activity of the same tissue, organ, and site in normal individuals, but is still able to effectively degrade the GM1 ganglioside substrate to avoid and eliminate its accumulation, and is sufficient to alleviate or completely eliminate the disease symptom.
  • the construct of the present invention contains one or more miR-142-3P target sequence fragments, such that the entire viral construct retains the expression level of ⁇ -galactosidase while greatly reducing the Transgene expression in phagocytes, thereby reducing immunogenicity and improving therapeutic effect.
  • ⁇ -galactosidase e.g., wild-type enzyme or variant enzyme
  • Efficient and specific expression in the gene while reducing the possible immune response, improves the effectiveness and safety of viral vectors as gene drugs.
  • Figure 1 shows a structural diagram of an example of the rAAV9-CAR-coGLB1 plasmid vector.
  • Figure 2 shows validation experiments of in vitro expression of plasmid rAAV9-CAR-coGLB1 by X-gal cell staining.
  • Figure 3 shows the SDS-PAGE results of rAAV9-CAR-coGLB1 ultrafiltration concentration. Obvious and clear electrophoretic bands of capsid proteins VP1, VP2 and VP3 of specific AAV viruses were visible.
  • Figure 4 shows the activity measurement of ⁇ -galactosidase protein in cells transfected in vitro.
  • the enzyme activity of cells transfected with pRDAAV-CAR-coGLB1 was measured as 430 nmol/mg ⁇ h, while the control group was 100.
  • Figure 5 shows the ⁇ -galactosidase activity assay of HEK-293, U-87MG, and RAW264.7 cells infected in vitro.
  • Figure 6 shows the detection results of ⁇ -Gal enzyme activity in brain nuclei of 20-week-old homozygous mutant model mice and control mice treated with neonatal IV IV.
  • Figure 7 shows the detection results of ⁇ -Gal enzyme activity in the brain nuclei of neonatal mouse ICV-treated 8-week-old homozygous mutant model mice and control mice.
  • Figure 8 shows the detection results of ⁇ -Gal enzyme activity in the brain nuclei of 8-week-old homozygous mutant model mice and control mice treated with ICV+IV dual route in newborn mice.
  • Figure 9 shows the detection results of ⁇ -Gal enzyme activity in peripheral tissues of 20-week-old homozygous mutant model mice and control mice treated with neonatal IV IV.
  • Figure 10 shows the detection results of ⁇ -Gal enzyme activity in peripheral tissues of 8-week-old homozygous mutant model mice and control mice treated with neonatal ICV.
  • Figure 11 shows the tissue ⁇ -Gal enzyme activity detection results of neonatal mouse ICV-treated 16-week-old homozygous mutant model mice and control mice.
  • Figure 12 shows the detection results of ⁇ -Gal enzyme activity in peripheral tissues of 8-week-old homozygous mutant model mice and control mice treated with ICV+IV dual pathway in neonatal mice.
  • Figure 13 shows the detection results of serum ⁇ -Gal enzyme activity in 20-week-old homozygous mutant model mice and control mice treated with neonatal IV IV.
  • Figure 14 shows the serum ⁇ -Gal enzyme activity detection results of neonatal mouse ICV-treated 8-week-old homozygous mutant model mice and control mice.
  • Figure 15 shows the detection results of serum ⁇ -Gal enzyme activity in 16-week-old homozygous mutant model mice and control mice treated with neonatal ICV.
  • Figure 16 shows the detection results of serum ⁇ -Gal enzyme activity in 8-week-old homozygous mutant model mice and control mice treated with ICV+IV dual pathway in neonatal mice.
  • Figure 17 shows the behavioral test results of tail vein injection treatment model mice.
  • the picture on the left shows the results of the upper limb suspension experiment. It can be seen that rAAV9-CAR-coGLB1 virus has a dose-dependent improvement in suspension time.
  • the picture on the right shows the rotarod fatigue experiment. It can be seen that the rAAV9-CAR-coGLB1 virus has a dose-dependent improvement in the residence time of mice on the rod. The above improvement was most obvious in the 8th week.
  • Figure 18 shows the Luxol Fast Blue (LFB) staining of the brain tissue of mice treated with tail vein injection.
  • the first row of images is from the microscopic view of the cerebral cortex tissue, and the second and third rows are from the thalamus and hippocampus tissue respectively.
  • the leftmost column shows GM1 mice given high-dose rAAV9-CAR-coGLB1 virus.
  • the second to fourth columns show GM1 mice given low-dose rAAV9-CAR-coGLB1 virus, GM1 mice given saline control, and wild-type mice. Contrast. There is basically no blue dye in the first and fourth columns, a trace amount of blue dye can be seen in the second column (pointed by the arrow), and a large amount of blue dye accumulation can be seen in the third column (pointed by the arrow).
  • Figure 19 shows the immunofluorescence of the cerebral cortex of mice treated with tail vein injection (green: GM1 ganglioside; blue: DAPI). It can be seen that the immunofluorescence staining of the uninjected model mice showed obvious accumulation of green signal, compared with the weak GM1 signal intensity of the mice in the HD group and LD group.
  • Figure 20(a) shows the results of in vitro transfection of cells with nucleic acids encoding ⁇ -galactosidase proteins containing different point mutations, followed by activity assay.
  • the untransfected control group of HEK-293 cells was 64 ⁇ 11nmol/mg ⁇ h.
  • the enzyme activity of cells transfected with rAAV9-CAR-coGLB1 was measured to be 509 ⁇ 129nmol/mg ⁇ h.
  • the enzyme activity of cells transfected with rAAV9-CAR-coGLB1 R299L was measured as 509 ⁇ 129nmol/mg ⁇ h.
  • the enzyme activity of cells transfected with rAAV9-CAR-coGLB1 G245D was measured as 718 ⁇ 113nmol/mg ⁇ h.
  • the enzyme activity of cells transfected with rAAV9-CAR-coGLB1 G245D was measured as 588 ⁇ 167nmol/mg ⁇ h.
  • the enzyme activity of cells transfected with rAAV9-CAR-coGLB1 I353L was measured. It is 280 ⁇ 83nmol/mg ⁇ h.
  • Figure 20(b) shows the plasmid concentration gradient cell transfection enzyme activity assay.
  • the two groups of transfected plasmids were rAAV9-CAR-coGLB1 R299L and rAAV9-CAR-coGLB1.
  • Figure 21(a) shows the results of in situ detection of ⁇ -gal enzyme activity by X-Gal cell staining.
  • the scale bar is 100 ⁇ m.
  • the cells were treated with plasmid vectors encoding ⁇ -gal enzymes carrying R299L, R299A, R299Q, and R299F mutations, and encoding wild-type ⁇ -gal enzymes.
  • Type ⁇ -gal enzyme plasmid vector transfection set up a set of non-transfected controls.
  • Figure 21(b) shows the results of Western blot detection of ⁇ -galactosidase protein expression in the above six groups of cells in parallel.
  • Figure 21(c) shows the results of in vitro enzyme activity assay of the above six groups of cells in parallel.
  • Figure 22(a) shows the virus dot hybridization titer detection of rAAV9-coGLB1 and rAAV9-GLB1 R299L.
  • Figure 22(b) shows the results of full protease activity after rAAV9-coGLB1 and rAAV9-GLB1 R299L were infected with HEK-293 cells at different MOIs. .
  • the invention discloses a drug for preventing and/or treating GM1 gangliosidosis, for example, a genetic drug, and involves the design, small-scale preparation and functional verification of the drug.
  • encoding refers to the inherent properties of a specific nucleotide sequence in a nucleic acid used for the synthesis of a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence and derived therefrom in a biological process. Templates for other polymers and macromolecules with biological properties.
  • a gene, cDNA, or RNA encodes a protein if transcription and translation of the mRNA corresponding to the gene produce a protein in a cell or other biological system.
  • Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing) and the non-coding strand (used as a template for transcription of a gene or cDNA) can be said to encode the protein or other product of the gene or cDNA.
  • protein and “polypeptide” are used interchangeably herein to refer to a polymer sequence containing amino acid residues. Unless otherwise stated, the one-letter and three-letter codes for amino acids defined by the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) are used in this article. The single letter X refers to any one of twenty amino acids. It is also understood that due to the degeneracy of the genetic code, a polypeptide may be encoded by more than one nucleotide sequence. Mutations in an amino acid sequence may be named as follows: the one-letter code for the parent amino acid, followed by the position number, followed by the one-letter code for the variant amino acid.
  • mutation of arginine (R) at position 299 into leucine (L) is represented by "R299L”.
  • R299L mutation of arginine (R) at position 299 into leucine (L)
  • a slash (/) is used to limit the multiple alternative options.
  • R299(L/A/F/Q) means that the arginine (R) at position 299 can be mutated into leucine.
  • L or alanine (A) or phenylalanine (F) or glutamine (Q).
  • expression refers to the transcription and/or translation of a specific nucleotide sequence driven by a promoter.
  • ⁇ -galactosidase lipoprotein lipase is a glycoside hydrolase (EC3.2.1.23), belonging to the glycoside hydrolase protein 35 family, which can hydrolyze galactose and its organic structure The ⁇ -glycosidic bond formed between them catalyzes the hydrolysis of ⁇ -galactoside into monosaccharides. It can also cleave fucoside and arabinoside, but with low efficiency.
  • substrates for ⁇ -galactosidase include ganglioside GM1, lactosylceramide, lactose, and various glycoproteins. It is encoded by the human GLB1 gene, which is located on chromosome 3 p22 and contains 16 exons with a full length of 62.5kb.
  • the CDS sequence of the wild-type GLB1 gene is shown in SEQ ID NO:9.
  • the term "functional beta-galactosidase” refers to enzymes with full-length wild-type (native) human beta-galactosidase (e.g., NCBI Reference Sequence: NP_000395 .3) enzyme, its variants (for example, variants with conservative amino acid substitutions, or variants with equivalent or improved functions), and fragments thereof, the variants or fragments provide at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, or about The same, or greater than 100%, biological activity level of full-length wild-type (native) beta-galactosidase.
  • the invention provides a functional ⁇ -galactosidase variant enzyme that is relative to naturally occurring human wild-type ⁇ -galactosidase.
  • ( ⁇ -gal) contains a mutation at position 299, 245, or 353 of the amino acid sequence, and preferably has a mutation at position 299.
  • the ⁇ -galactosidase variant enzyme comprises the following amino acid sequence mutations (i)-(iii) relative to naturally occurring human wild-type ⁇ -galactosidase (GLB1) Any of: (i) at position 299 of the amino acid sequence, from arginine to leucine, alanine, phenylalanine or glutamine; (ii) at position 245 of the amino acid sequence, from Glycine is mutated to aspartic acid; (iii) mutation at position 353 of the amino acid sequence is mutated from isoleucine to leucine.
  • the present invention also provides nucleic acid molecules encoding the functional ⁇ -galactosidase variant enzymes described above.
  • the term “conservative amino acid substitution” or “conservative amino acid substitution” refers to changing, substituting, or substituting an amino acid into a different amino acid with similar biochemical properties (such as charge, hydrophobicity, and size), as is the skill in the art personnel are publicly known.
  • a protein with such sequence is different relative to the wild-type protein. They are called "variants” or “mutants”.
  • the effect of the mutation on the function and activity of the protein may be improved (i.e., for example, increased activity), equivalent (i.e., for example, activity is substantially similar), or reduced or eliminated (i.e., for example, abnormality or loss of function ).
  • variant proteins can be used to replace the natural wild-type protein for gene therapy, as long as the activity of the variant protein is sufficient to achieve the therapeutic effect, for example, using ⁇ -galactopyranoside Enzyme variants replace wild-type ⁇ -galactosidase for gene therapy in GM1 gangliosidosis.
  • a designed nucleotide substitution is introduced into the coding region of the resulting GLB1 gene sequence by performing PCR amplification using a mutation construction primer pair with a nucleic acid sequence point mutation, thereby passing ⁇ -galactosidase variants with point mutations in the amino acid sequence are expressed by translating the mutated codons.
  • Enzyme variants may have reduced, equivalent, or improved activity compared to wild-type beta-galactosidase.
  • Codon-optimization is a technology to improve the expression level of target proteins.
  • the original guiding idea is that codons are degenerate, but the abundance of tRNA corresponding to multiple synonymous codons in cells The degrees are different, so by replacing synonymous codons to better avoid rare codons and using preferred codons, you can improve translation efficiency and thereby increase expression levels.
  • factors such as simplifying the secondary structure of mRNA, optimizing repetitive sequences, eliminating enzyme cutting sites, adjusting GC content, etc. as much as possible to comprehensively redesign the gene encoding. sequence.
  • codon optimization techniques may be preferred in situations where coding sequences from other species are expressed in host cells, this strategy is in fact beneficial for the vast majority of genetic engineering.
  • a “signal sequence” is a sequence of amino acids linked to the N-terminal portion of a protein that facilitates secretion of the protein out of the cell.
  • the mature form of the extracellular protein does not have a signal sequence, which is cleaved during the secretion process.
  • N-terminus refers to the last amino acid at the N-terminus
  • C-terminus refers to the last amino acid at the C-terminus
  • operably linked means that the specified components are in a relationship that allows them to function in an intended manner.
  • Sequence identity between sequences is calculated as follows: To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., they can be Gaps are introduced into one or both of the first and second amino acid sequences or nucleic acid sequences or non-homologous sequences may be discarded for comparison purposes).
  • the length of the aligned reference sequences is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. The molecules are identical when a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence.
  • Mathematical algorithms can be used to perform sequence comparison and calculation of percent identity between two sequences.
  • the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm is used which has been integrated into the GAP program of the GCG software package (available at http://www.gcg.com available), determine the distance between two amino acid sequences using the Blossum 62 matrix or the PAM250 matrix with gap weights 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4, 5, or 6 Percent identity.
  • the GAP program in the GCG software package (available at http://www.gcg.com) is used, using the NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 80 and A length weight of 1, 2, 3, 4, 5, or 6 determines the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • nucleic acid sequences and protein sequences described herein may further be used as "query sequences" to perform searches against public databases, for example to identify other family member sequences or related sequences.
  • regulatory sequence refers to a nucleic acid sequence that induces, inhibits, or otherwise controls protein transcription of an encoding nucleic acid sequence to which it is operably linked. Regulatory sequences may be, for example, initiation sequences, enhancer sequences, intron sequences, promoter sequences, and the like.
  • CAR promoter refers to such an element, which is obtained by the inventor by structurally optimizing the enhancer sequence of the human CMV virus. It avoids the silencing characteristics of the traditional CMV promoter in the liver and can be used throughout the body, including the liver. Widely expressed in various parts and tissues.
  • the nucleic acid sequence of the CAR promoter is as shown in sequence SEQ ID No: 1.
  • exogenous nucleic acid or protein means that the nucleic acid or protein is not naturally present in the chromosome or host cell location in which it is found. Exogenous nucleic acid sequences also refer to sequences derived from and inserted into the same host cell or subject but present in a non-native state, for example, in a different copy number, or under the control of different regulatory elements.
  • AAV Addeno-associated virus
  • AAV is a dependent virus that requires other viruses such as adenovirus, herpes simplex virus, human papillomavirus, or cofactors to provide auxiliary functional proteins to replicate.
  • AAV2 AAV serotype 2
  • ITRs inverted terminal repeats
  • ORFs open reading frames
  • the full-length genome of AAV2 has been cloned into an E. coli plasmid (Samulski RJ et al., Proc Natl Acad Sci USA. 1982; 79:2077-2081. Laughlin CA et al., Gene. 1983; 23:65-73).
  • ITR is a cis-acting element of the AAV vector genome and plays an important role in the integration, rescue, replication and genome packaging of AAV viruses.
  • the ITR sequence contains Rep protein binding site (Rep binding site, RBS) and terminal melting site trs (terminal resolution site), which can be recognized by Rep protein and generate a nick at trs.
  • the ITR sequence can also form a unique "T" letter-shaped secondary structure, which plays an important role in the life cycle of the AAV virus.
  • the rest of the AAV2 genome can be divided into 2 functional regions, the Rep gene region and the Cap gene region.
  • Rep gene region encodes four Rep proteins: Rep78, Rep68, Rep52 and Rep40.
  • Rep protein plays an important role in the replication, integration, rescue and packaging of AAV viruses.
  • Rep78 and Rep68 specifically bind to the terminal melting site trs and the GAGY repeat motif in the ITR, initiating the replication process of the AAV genome from single strand to double strand.
  • the trs and GAGC repeat motifs and/or GAGY repeat motifs in the ITR are the center of AAV genome replication. Therefore, although the ITR sequences in various serotypes of AAV viruses are different, they can all form a hairpin structure and have Rep binding. site.
  • Rep52 and Rep40 have ATP-dependent DNA helicase activity but have no DNA-binding function.
  • the Cap gene encodes the capsid proteins VP1, VP2 and VP3 of the AAV virus.
  • VP3 has the smallest molecular weight but the largest number.
  • the ratio of VP1, VP2, and VP3 in mature AAV particles is roughly 1:1:10.
  • VP1 is required for the formation of infectious AAV;
  • VP2 assists VP3 in entering the nucleus;
  • VP3 is the main protein that makes up AAV particles.
  • AAV vector refers to an efficient foreign gene transfer tool that has been transformed from wild-type AAV virus with the understanding of the life cycle of AAV virus and its related molecular biological mechanisms, that is, AAV vector .
  • the modified AAV vector genome only contains the ITR sequence of the AAV virus and the expression cassette carrying the foreign gene to be transported.
  • the Rep and Cap proteins required for AAV virus packaging are provided in trans by other exogenous plasmids, thus reducing the possible harm caused by Rep and Cap gene packaging into AAV vectors.
  • the AAV virus itself is not pathogenic, which makes the AAV vector recognized as one of the safest viral vectors.
  • Deleting the D sequence and trs sequence in the ITR sequence on one side of the AAV virus can also make the genome carried by the packaged recombinant AAV virus vector self-complementary and form a double strand, significantly improving the in vivo and in vitro transduction efficiency of the AAV vector (Wang Z et al. , Gene Ther. 2003; 10(26):2105-2111; McCarty DM et al., Gene Ther. 2003; 10(26): 2112-2118).
  • the packaged virus becomes scAAV (self-complementary AAV) virus, the so-called double-stranded AAV virus. It is different from ssAAV (single-stranded AAV), which is a traditional AAV virus that has no mutations in both ITRs.
  • the packaging capacity of scAAV viral vector is smaller, only half of that of ssAAV viral vector, about 2.2kb-2.5kb, but the transduction efficiency is higher after infecting cells.
  • AAV virus serotypes There are many AAV virus serotypes, and different serotypes have different tissue infection tropisms. Therefore, the use of AAV vectors can transport foreign genes to specific organs and tissues (Wu Z et al., Mol Ther. 2006; 14(3): 316-327). Certain serotype AAV vectors can also cross the blood-brain barrier and introduce foreign genes into brain neurons, providing the possibility of brain-targeted gene transduction (Samaranch L et al., Hum Gene Ther. 2012; 23(4) ):382-389).
  • AAV vectors have stable physical and chemical properties and show strong tolerance to acids, alkalis and high temperatures (Gruntman AM et al., Hum Gene Ther Methods. 2015; 26(2):71-76), making it easy to develop stable Biological products with higher toxicity.
  • the existing technology has a relatively mature packaging system for AAV vectors, which facilitates large-scale production of AAV vectors.
  • AAV vector packaging systems mainly include a three-plasmid co-transfection system, a system in which adenovirus is used as a helper virus, a packaging system in which Herpes simplex virus type 1 (HSV1) is used as a helper virus, and a packaging system based on baculovirus. system.
  • HSV1 Herpes simplex virus type 1
  • the three-plasmid transfection and packaging system does not require helper viruses and is highly safe. It is the most widely used AAV vector packaging system and is currently the mainstream production system in the world. A slight drawback is that the lack of efficient large-scale transfection methods limits the application of the three-plasmid transfection system in large-scale preparation of AAV vectors.
  • Yuan et al. established a large-scale AAV packaging system using adenovirus as a helper virus (Yuan Z et al., Hum Gene Ther. 2011; 22(5):613-624). This system has high production efficiency, but the adenovirus in the packaging system is Finally, trace amounts exist in the finished AAV products, which affects the safety of the finished AAV products.
  • HSV1 as a helper virus packaging system is another type of AAV vector packaging system that is widely used.
  • Wu Zhijian and Conway et al. proposed an AAV2 vector packaging strategy using HSV1 as a helper virus internationally at almost the same time (Wu Zhijian, Wu Xiaobing et al., Science Bulletin, 1999, 44(5): 506-509; Conway JE et al., Gene Ther. 1999, 6: 986-993).
  • Wustner et al. proposed an AAV5 vector packaging strategy using HSV1 as a helper virus (Wustner JT et al., Mol Ther. 2002, 6(4): 510-518).
  • HSV1 viruses used two HSV1 viruses to carry the Rep/Cap gene of AAV and the inverted terminal repeat (ITR)/foreign gene expression cassette of AAV, and then co-infected them with these two recombinant HSV1 viruses.
  • Production cells are used to package and produce AAV viruses (Booth MJ et al., Gene Ther. 2004; 11:829-837). Thomas et al. further established a suspension cell system for the production of dual HSV1 virus AAV (Thomas DL et al., Gene Ther. 2009; 20:861-870), making larger-scale AAV virus production possible.
  • Urabe et al. used three baculoviruses to carry the structural genes, non-structural genes and ITR/exogenous gene expression cassettes of AAV respectively to construct a baculovirus packaging system for AAV vectors. Taking into account the instability of baculovirus carrying foreign genes, the number of baculoviruses required in the production system was subsequently reduced, gradually from the initial need for three baculoviruses to the need for two or one baculovirus ( Chen H., Mol Ther. 2008, 16(5): 924-930; Galibert L. et al., J Invertebr Pathol.
  • AAV vectors Due to the above characteristics, AAV vectors have gradually become a foreign gene delivery tool widely used in gene therapy, especially gene therapy for genetic diseases. As of August 2016, there are 173 approved AAV vector-based gene therapy clinical trial protocols in the world (http://www.abedia.com/wiley/vectors.php). More importantly, the AAV vector-based lipoprotein lipase gene therapy drug Glybera was approved for marketing by the European Medicines Agency in 2012, becoming the first gene therapy drug approved in the Western world ( S., Mol Ther. 2012; 20(10):1831-1832); hemophilia B (Kay MA et al., Nat Genet.
  • AAV vector gene therapy drugs have achieved good clinical trial results and are expected to be marketed in the near future to benefit the majority of patients. .
  • vector genome refers to the nucleic acid sequence packaged within the rAAV capsid to form the rAAV vector.
  • the vector genome contains at least the 5' to 3' AAV2 5' ITR, a nucleic acid sequence encoding a functional GLB1, and the AAV2 3' ITR. It is also possible to select ITRs from different source AAVs other than AAV2.
  • the vector genome may contain regulatory sequences that direct expression of functional GLB1.
  • microRNA refers to single-stranded non-coding RNA with a length of 18 to 25 nucleotides (nt) that is widely present in humans and animals.
  • miRNA was first discovered in Caenorhabditis elegans (C. elegans).
  • the lin-4 gene in Caenorhabditis elegans can downregulate the expression of the lin-14 gene, but the product encoded by the lin-4 gene is not a protein, but a small RNA molecule, which indicates that the small RNA molecule encoded by itself can regulate gene expression.
  • a variety of similar small RNA molecules were discovered in different species and cells, and miRNA began to become the collective name for this type of small RNA.
  • miRNA genes are typically located in exons, introns, and intergenic regions of the genome (Olena AF et al., J Cell Physiol. 2010; 222:540-545; Kim VN et al., Trends Genet. 2006; 22:165- 173).
  • the production process of miRNA is as follows. First, in the nucleus, the miRNA gene is transcribed by RNA polymerase II or III to produce the initial product pri-microRNA; pri-microRNA self-folds part of the sequence to form a stem-loop structure.
  • the processing complex composed of ribonuclease III Drosha and DGCR8 molecules acts on pri-microRNA, cutting off excess sequences, leaving a stem-loop structure of about 60nt, which is the precursor miRNA molecule pre-microRNA.
  • pre-microRNA enters the cytoplasm from the nucleus and is processed by Dicer enzyme to remove the loop part of its stem-loop structure and become a double-stranded RNA molecule.
  • double-stranded RNA molecules are bound by protein factors such as AGO2, one strand is degraded, and the other strand forms an RNA-induced silencing complex (RISC) with protein factors.
  • RISC RNA-induced silencing complex
  • RISC recognizes target sequences in mRNA, reduces the expression level of mRNA by degrading the mRNA molecule, promoting deadenylation at the 3' end of the mRNA molecule and inhibiting translation, and regulates gene expression at the post-transcriptional level (Fabian MR et al., Annu Rev Biochem .2010;79:351-379). Therefore, using highly expressed miRNA in cells and inserting the target sequence of the miRNA into the 3’UTR (untranslated region) of the exogenous gene can effectively inhibit the expression of the exogenous gene in the introduced cells.
  • miR-142-3p is a miRNA that is highly expressed in cells derived from hematopoietic stem cell lines.
  • Immune cells are homogeneously differentiated from hematopoietic stem cell lines, so the principle of miRNA inhibition of gene expression is used (Kim VN. Nat Rev Mol Cell Biol. 2005; 6(5): 376-385), genes carrying the miR-142-3p target sequence Expression will be significantly inhibited in immune cells, thereby reducing the probability that the body will generate an immune response against the gene expression product (Dismuke DJ et al., Curr Gene Ther. 2013; 13(6): 434-452).
  • ⁇ -galactosidase dysfunction is usually caused by GLB1 gene mutations, resulting in the inability to express the ⁇ -galactosidase precursor protein of the normal sequence, which in turn causes a significant decrease or even loss of ⁇ -galactosidase activity in the cells, leading to Its substrate cannot be degraded and is abnormally deposited, such as GM1 ganglioside deposition, which in turn causes severe metabolic and functional disorders in the body, manifesting in serious diseases, such as GM1 gangliosidose disease.
  • treatment refers to a clinical intervention intended to alter the natural course of a disease in the individual being treated. Desired therapeutic effects include, but are not limited to, preventing the emergence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of disease, reducing the rate of disease progression, ameliorating or alleviating disease status, and alleviating or improving prognosis.
  • Desired therapeutic effects include, but are not limited to, preventing the emergence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of disease, reducing the rate of disease progression, ameliorating or alleviating disease status, and alleviating or improving prognosis.
  • GM1 gangliosidosis the severity of the disease is positively correlated with the total amount of abnormally deposited GM1 ganglioside and inversely correlated with the residual activity of abnormally functioning ⁇ -gal in the body.
  • a vector (such as, but not limited to, the AAV vector of the present invention) containing the ⁇ -galactosidase variant enzyme of the present invention, and/or the expression cassette of the present invention is transferred into the exogenous ⁇ -galactosidase expressed in vivo.
  • Glycosidase will play a compensatory role and degrade the abnormally deposited GM1 ganglioside, thereby reducing the severity of the disease from one or more aspects, that is, playing a therapeutic role.
  • the abnormally deposited GM1 gangliosides are effectively degraded, which may be manifested by one or more of the following: increased pharmacodynamics and biological activity of GLB1 in cerebrospinal fluid (CSF); increased GLB1 pharmacodynamics and biological activity in serum; Increased GLB1 pharmacodynamics and biological activity; increased mean lifespan (survival) of patients; delayed disease progression of GM1 gangliosidosis (by age at achievement, age at loss, and maintenance or acquisition of age-appropriate development and motor milestones) and changes in age-equivalent cognitive, gross motor, fine motor, receptive and expressive communication scores, text based on the Bailey Scales of Infant and Toddler Development Improvement in neurocognitive development in one or more of the changes in standard scores for each domain of the Lan Adaptive Behavior Scale; improvement in dysphagia, gait function, motor skills, language, and/or respiratory function; reduction in seizure frequency and/or increased age of seizure onset; increased likelihood of
  • CSF and serum beta-galactosidase activity CSF GM1 concentration
  • serum and urine Liquid keratan sulfate
  • liver and spleen volumes EEG and visual evoked potentials (VEP).
  • abnormally deposited GM1 gangliosides are effectively degraded, which may be manifested by one or more of the following: increased lifespan (survival); reduced need for feeding tubes; incidence of epilepsy, Decreased frequency and duration and delayed seizure onset; improved quality of life, e.g., as measured by PedsQL; slowed progression of neurocognitive decline and/or improved neurocognitive development, e.g., adaptive behavior, cognition, language (receptive sexual and expressive communication) and motor function (gross motor, fine motor) development improved or improved; motor milestones achieved earlier and motor milestones lost later; brain tissue volume (cerebral cortex and other smaller structures) and Delayed increase in ventricular volume, delayed size reduction of brain structures including the corpus callosum, caudate and putamen, and cerebellar cortex, and stabilization of brain atrophy and volume changes; delayed progression of abnormal T1/T2 signal intensity in the thalamus and basal ganglia; Increased beta-gal enzyme
  • the activity of exogenous ⁇ -galactosidase transferred into a patient and expressed through a vector containing the expression cassette of the invention can be higher than, It is equal to or lower than the activity of ⁇ -galactosidase in normal individuals, and plays a compensatory role to degrade abnormally deposited GM1 ganglioside.
  • the activity of the exogenous ⁇ -galactosidase transferred into the patient and expressed by a vector containing the expression cassette of the invention can be higher than,
  • the activity of ⁇ -galactosidase is equal to or lower than that in normal individuals, and plays a compensatory role to degrade abnormally deposited GM1 gangliosides, but does not reduce the level of GM1 gangliosides in the patient's body (e.g., central, peripheral)
  • the residual amount of lipid is as low as or equivalent to the content of GM1 ganglioside in normal individuals, but is still sufficient to eliminate or partially eliminate the clinical symptoms without treatment and to alleviate the condition.
  • prevention includes the prevention or inhibition of the onset or progression of a disease or symptoms of a particular disease.
  • a subject has a GLB1 genetic abnormality, or is at risk for high levels of such, or is at risk for high levels of abnormal deposition of GM1 gangliosides , or subjects who already have relatively high levels of abnormal deposition of GM1 gangliosides but have not yet shown a certain degree of clinical symptoms, are candidates for the implementation of preventive programs.
  • prevention refers to the administration of a drug before the development of GM1 gangliosidosis, particularly in subjects with GLB1 gene defects.
  • the invention provides an expression cassette for a functional human ⁇ -galactosidase, which includes a nucleotide sequence encoding a functional ⁇ -galactosidase, and a regulatory sequence that directs its expression, such as an upstream Regulatory sequences and downstream regulatory sequences.
  • the expression cassette comprises a nucleotide sequence encoding a functional beta-galactosidase as described herein and regulatory sequences that direct its expression.
  • the amino acid sequence of the functional ⁇ -galactosidase is the sequence shown in any one of SEQ ID NO: 10-16.
  • the nucleotide sequence encoding a functional beta-galactosidase has at least 70%, 80%, 90% identity with SEQ ID NO: 2 or 9, for example, has at least 95%, 96%, 97%, 98%, 99% or higher, or 100% identity.
  • the nucleotide sequence encoding a functional beta-galactosidase is at least 70%, 80%, 90% identical to SEQ ID NO: 17, 19, 21, 23, 25 or 27 , for example, having at least 95%, 96%, 97%, 98%, 99% or greater, or 100% identity.
  • nucleotide sequence encoding functional GLB1 is linked to the promoter sequence shown in SEQ ID NO: 1 at its 5' end, or is linked to the promoter sequence shown in SEQ ID NO: 1 at its 5' end.
  • nucleotide sequence encoding a functional beta-galactosidase is selected from
  • a nucleotide sequence encoding a beta-galactosidase variant enzyme (e.g., including a mutation at position 299, 245, or 353 of the amino acid sequence), such as SEQ ID NO: 17, 19, 21 , the nucleotide sequence shown in 23, 25, 27 or 29;
  • the regulatory sequences that direct expression of functional ⁇ -galactosidase comprise a promoter, eg, chicken ⁇ -actin promoter, CMV promoter, etc.
  • the regulatory sequence comprises or is at least about 90% identical to the CAR promoter sequence set forth in SEQ ID NO: 1 (e.g., has at least 95%, 96%, 97%, 98%, 99% or higher identity) promoter sequence.
  • the use of the CAR promoter can avoid the shortcomings of the CMV promoter entering the body (especially the liver) and being easily silenced by methylation; compared with other commonly used promoters, such as chicken ⁇ -actin promoter etc., the CAR promoter sequence is shorter, which is convenient for virus packaging, so the expression level is significantly improved.
  • the regulatory sequence further comprises a regulatory sequence that can reduce the expression of functional ⁇ -galactosidase in immune-related cells (such as antigen-presenting cells, macrophages), thereby reducing the expression of functional ⁇ -galactosidase.
  • immune-related cells such as antigen-presenting cells, macrophages
  • the sequence of immune response thereby significantly reducing the probability of immune response against exogenous ⁇ -galactosidase protein
  • the sequence is, for example, one or more (for example, 1-8, 2-7, 3 , 4, 5 or 6) target sequences complementary to miR-142-3p in tandem, whereby the expression cassette of the present invention is transcribed to obtain the untranslated region of the mRNA encoding functional ⁇ -galactosidase (for example, the 5' untranslated region and/or the 3' untranslated region) contains the human miR-142-3p target sequence, which can effectively inhibit the expression of functional GLB1 in immune-related cells (such as antigen-presenting cells) , suppress immune response.
  • immune-related cells such as antigen-presenting cells
  • the regulatory sequences comprise one or more expression enhancers, eg, TBG enhancer, CMV enhancer, etc.
  • the regulatory sequence comprises a polyadenylation signal (polyA), e.g., human growth hormone (hGH) polyadenylation sequence, SV40 polyA, BGH polyA.
  • polyA polyadenylation signal
  • the control sequence comprises or is at least about 90% identical to SEQ ID NO: 4 (e.g., at least 95%, 96%, 97%, 98%, 99% or more identical thereto). High identity) BGH polyA sequence.
  • polyA polyadenylation signal
  • tens to hundreds of adenosine residues are added to the tail (3' end) of a correctly transcribed mRNA.
  • at least two of the above elements are connected in sequence to form the expression frame of the present invention.
  • a spacer sequence between the sequences of the elements which can also be called a connecting sequence, a linker, a linker, etc. .
  • These sequences are added to facilitate clone construction and subsequent identification, improve gene transcription and translation efficiency, etc., such as Kozak sequences, endonuclease action sites, universal primer sequences, batch tags, etc., as long as they do not interfere or inhibit
  • spacer sequences are considered to be within the protection scope of the technical solution of the present invention and do not need to be deliberately removed.
  • the spacer sequence is 0-20 nucleotides in length.
  • the spacer sequence includes the KpnI restriction site GGTACC; in a preferred embodiment, the spacer sequence includes the Kozak sequence GCCACC; in a preferred embodiment, the spacer sequence includes EcoRI restriction site GAATTC; in a preferred embodiment, the spacer sequence includes a SalI restriction site GTCGA; in a preferred embodiment, the spacer sequence includes a combination of a KpnI restriction site and a Kozak sequence ; In a preferred embodiment, the spacer sequence includes a combination of an EcoRI restriction site and a SalI restriction site.
  • the invention provides a viral vector, which is an artificial recombinant viral particle, in which a replication-deficient viral genome sequence comprising an expression cassette encoding functional GLB1 is packaged in a viral capsid or envelope,
  • the recombinant viral particles are thus unable to produce progeny virions but retain the ability to infect target cells.
  • the genomic sequence of the viral vector does not contain genes encoding enzymes required for viral replication and, therefore, the use of viral vectors in gene therapy is considered safe because in the absence of the enzymes required for viral replication , replication and infection of progeny virions will not occur.
  • the recombinant viral vector of the present invention can preferably be a recombinant adeno-associated virus (AAV), but can also be adenovirus, Bocavirus, AAV/bocavirus hybrid, herpes simplex virus or lentivirus to deliver genes. Equally effective common vectors.
  • AAV adeno-associated virus
  • Packaging cell lines for the production of recombinant viral vectors can be prokaryotic or eukaryotic cells (e.g., human cells, insect cells, or yeast cells) containing cells produced by any means (e.g., electroporation, calcium phosphate precipitation, Microinjection, transformation, viral infection, transfection, and protoplast fusion) introduce foreign DNA into cells.
  • Packaging cell line cells include, but are not limited to, E. coli cells, yeast cells, human cells, non-human cells, mammalian cells, non-mammalian cells, insect cells, HEK293 cells, hepatocytes, kidney cells, glial cells, tumor cells or stem cells.
  • target cell refers to a target cell in which expression of functional beta-galactosidase is desired.
  • target cells include, but are not limited to, functional cells in the central nervous system, heart, liver, spleen, lungs, kidneys, muscles, supportive cells, stromal cells, etc., such as nerve cells, glial cells, cardiomyocytes, hepatocytes .
  • the vector is delivered to target cells in vivo.
  • the viral vector is a recombinant adeno-associated virus (rAAV) vector, which contains an AAV capsid and a vector genome packaged therein.
  • rAAV vectors are used to treat diseases caused by GLB1 gene defects such as GM1 gangliosidosis.
  • the vector genome contains the AAV 5’ inverted terminal repeat (ITR) or AAV 5’ ⁇ ITR, a nucleic acid sequence encoding functional GLB1, regulatory sequences that direct the expression of GLB1 in target cells, and the AAV 3’ITR or AAV 3’ ⁇ ITR.
  • the ⁇ ITR is an ITR in which the D sequence and the terminal melting site trs are deleted.
  • the ITR is the genetic element responsible for the replication and packaging of the genome during vector production and is the only viral cis-element required for rAAV production. It is possible to select ITRs from different sources of AAV. In one embodiment, the ITR is from a different AAV than the capsid of the viral particle.
  • both ends of the expression cassette are each connected to an ITR sequence.
  • the AAV capsid, ITR and other AAV components described herein can be readily selected from any AAV, including but not limited to those commonly identified as AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV of serotypes AAV8, AAV9, AAVrh.10, or combinations thereof.
  • the AAV capsid is an AAV5 capsid or a variant thereof.
  • the AAV capsid is an AAV3B capsid or a variant thereof.
  • the AAV capsid is an AAV8 capsid or a variant thereof.
  • the AAV capsid is an AAV9 capsid or a variant thereof.
  • the capsid protein is designated by a number or a combination of numbers and letters following the term "AAV" in the name of the rAAV vector.
  • the recombinant viral vector of the invention comprises the human GLB1 gene expression cassette of the invention.
  • a rAAV comprising a capsid selected from the group consisting of AAV serotype 5 (AAV5), serotype 3B (AAV3B), serotype 8 (AAV8), serotype 9 (AAV9); and Containing in its genome any one of SEQ ID NO:2, 5-9 and 17-28 or having at least about 90% identity with any one of SEQ ID NO:2, 5-9 and 17-28 sequence.
  • the recombinant viral vector of the invention expresses ⁇ -galactosidase upon infection of a host cell.
  • an AAV “variant” refers to any AAV sequence derived from a known AAV sequence, including those that have conservative amino acid substitutions, and that are at least 70%, at least 75% identical to the amino acid or nucleic acid sequence of an AAV , sequences with at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or greater sequence identity.
  • AAV capsids include variants that may comprise up to about 10% variation compared to any described or known AAV capsid sequence.
  • the AAV capsid is about 90% identical to about 99.9% identical, about 95% to about 99% identical, or about 97% to about 97% identical to AAV capsids provided herein and/or known in the art. About 98% identical. In one embodiment, the AAV capsid is at least 95%, 96%, 97%, 98%, 99% or higher identical to the AAV capsid variant.
  • any variant protein eg, vp1, vp2, or vp3 can be compared.
  • Recombinant adeno-associated virus (AAV) vectors of the invention can be produced using known techniques. Such methods involve culturing packaging cells comprising nucleic acid sequences encoding AAV capsids; a functional Rep gene; an expression cassette as described herein, flanked by AAV inverted terminal repeats (ITRs) or ⁇ ITRs; and sufficient helpers functionality to allow packaging of the expression cassette into the AAV capsid protein.
  • AAV adeno-associated virus
  • packaging cells comprising nucleic acid sequences encoding AAV capsids; a functional Rep gene; an expression cassette as described herein flanked by AAV inverted terminal repeats (ITRs) or ⁇ ITRs; and sufficient accessory functions , to allow packaging of the expression cassette into the AAV capsid protein.
  • the host cell is a HEK 293 cell.
  • Suitable methods may include, but are not limited to, baculovirus expression systems or production by yeast.
  • the viral vector of the present invention can be injected intravenously and/or intraventricularly, and is highly effective in tissues, organs and parts of the body, especially the central nervous system, heart, liver, spleen, lungs, kidneys, muscles, serum, etc. Consistent, sustained, and specific expression of functional GLB1 prevents and/or treats severe GM1 gangliosidosis.
  • exogenous functional ⁇ -galactosidase is expressed in the patient’s cells that cannot normally express the GLB1 gene, and the expressed functional ⁇ -galactosidase Glycosidase can sustainably and efficiently degrade GM1 ganglioside and eliminate its deposition, thereby alleviating or eliminating the symptoms of the disease in whole or in part. And it can continuously degrade GM1 ganglioside in the body after one administration.
  • viral vectors of the invention may be used to prevent and/or treat GM1 gangliosidosis or diseases caused or associated therewith.
  • the viral vector of the present invention can be used to prevent and/or treat GM1 gangliosidosis caused by GLB1 gene deficiency or diseases caused by or associated with it.
  • the expression cassette of the present invention can be used to prepare medicaments for preventing and/or treating GM1 gangliosidosis or diseases caused or associated with it, or for preventing and/or treatment of GM1 gangliosidosis caused by GLB1 gene deficiency or diseases caused by or associated with it.
  • the medicament is prepared for oral, intraperitoneal, intravenous and/or intracerebroventricular administration, more preferably intravenous and/or intracerebroventricular administration.
  • the viral vectors of the present invention can be used to prepare medicaments for preventing and/or treating GM1 gangliosidosis or diseases caused or associated with it, or for Prevention and/or treatment of GM1 gangliosidosis caused by GLB1 gene deficiency or diseases caused by or associated with it.
  • the medicament is prepared for oral, intraperitoneal, intravenous and/or intracerebroventricular administration, more preferably intravenous and/or intracerebroventricular administration.
  • the functional ⁇ -galactosidase and its variant enzymes and the nucleic acid molecules encoding the functional ⁇ -galactosidase and its variant enzymes of the present invention can be used to prepare medicines, Drugs used to prevent and/or treat GM1 gangliosidosis or diseases caused by or related to them, or used to prevent and/or treat GM1 gangliosidosis caused by GLB1 gene defects or diseases caused by them. disease or disease associated with it.
  • the present invention also provides the resulting pharmaceutical composition, which includes the ⁇ -galactosidase of the present invention and its functional variant enzymes, nucleic acid molecules, optimized human GLB1 gene expression cassettes, viral vectors, Gene drugs, or any combination of the above.
  • GM1 gangliosidosis using an AAV vector carrying a ⁇ -galactosidase encoding gene to express exogenous ⁇ -galactosidase in the patient’s body to replace/complement the patient’s body.
  • the self-deficient ⁇ -galactosidase plays a role in degrading GM1 ganglioside.
  • helper plasmid pRep2Cap9 carrying Rep and Cap protein expression genes were from Beijing Ruixi Rare Disease Gene Therapy Technology Institute; Escherichia coli DH5 ⁇ competent cells were purchased from Beijing Qingke Biotechnology Co., Ltd.
  • Lipofectamine2000 is a product of Invitrogen; 4-Methylumbelliferyl- ⁇ -D-galactopyranoside (4-MU- ⁇ -D-Gal) substrate was purchased from Carbo Sens Company; 4-Methylumbelliferone (4-MU) standard was purchased from Sigma-Aldrich; ⁇ -galactosidase antibody was purchased from Novus Company; ⁇ -Tublin antibody and horseradish enzyme labeled goats Anti-mouse IgG (H+L) was purchased from Zhongshan Jinqiao Company; 5-bromo-4-chloro-3-indole- ⁇ -D-galactopyranoside (X-gal) substrate, potassium ferricyanide solution and sub- Potassium ferricyanide solution is a product of Beijing Solebao Company.
  • rAAV9-CAR-coGLB1 contains the main elements as follows:
  • ITR the only cis-acting element in AAV vectors derived from the AAV genome, is an essential element for preparing AAV vectors.
  • CAR promoter is composed of the enhancer sequence of human CMV virus with optimized structure.
  • the nucleic acid sequence is as shown in sequence SEQ ID No: 1.
  • BGH polyA the polynucleotide tailing signal of bovine growth hormone, the nucleic acid sequence is shown in SEQ ID No: 4.
  • the GLB1 expression cassette containing the above elements (2), (3) and (4) is shown in SEQ ID No: 5, which contains the connecting sequence GGTACCGCCACC (KpnI restriction site) between elements (2) and (3) and Kozak sequence), including the connecting sequence GAATTCGTCGA (EcoRI and SalI restriction sites) between elements (3) and (4).
  • the expression cassette sequence in this construct is shown in SEQ ID No:7.
  • zebrafish NP_001017547.1
  • mouse NP_033882.1
  • cat NP_001009860.1
  • dog NP_001032730.1
  • pig XP_020927212.1
  • Clustal X software was used to conduct a multiple sequence alignment analysis between positions 149 to 522 of human ⁇ -gal and other species. After excluding the pathogenic mutation sites reported in the literature, 193, 194, 197, 208, 245, 246, 284, 299 and 353 located in the ⁇ -gal TIM barrel domain (residues 1-359) were finally selected. Site-directed mutagenesis was carried out at the 459 and 488 sites in ⁇ domain I (residues 397-514) to construct 12 GLB1 gene mutants.
  • the method was to use the mutation primers in Table 1 for PCR amplification, and use the gene homologous recombination method to replace the coGLB1 gene sequence in the rAAV9-CAR-coGLB1 plasmid with the gene fragment carrying the mutation site (mtGLB1).
  • the recombinant plasmid was identified through gene sequencing, and the sequencing results showed that the expected GLB1 mutant had been obtained (results omitted).
  • X-Gal staining was used to detect ⁇ -gal enzyme activity in post-transfected cells in situ.
  • HEK-293 was used as experimental cells to transfect rAAV9-CAR-coGLB1 plasmid in vitro.
  • HEK-293 cells were evenly spread into 6 wells of a six-well cell culture plate. When the cell density in each well reached 60-70%, 3 wells were transfected with plasmid using Lipofectamine 2000 (Invitrogen, USA). The cells in well 3 were not transfected and served as negative control wells. 48 hours after transfection, the cells were stained with X-Gal substrate to verify that the constructed plasmid carrying the optimized GLB1 gene could express ⁇ -galactosidase protein in in vitro transfection. Preparation of dyeing solution working solution: 1mg/mL
  • X-Gal turns blue after hydrolysis under the catalysis of ⁇ -galactosidase.
  • the cells were observed under a bright field microscope.
  • the blue spots in the transfected cells increased significantly (the most obvious place is as indicated by the arrow in the picture), proving that the rAAV9-CAR-coGLB1 plasmid It can effectively express active ⁇ -galactosidase protein in cells in vitro.
  • the method was as follows: Cells were transfected as previously described. 48 hours after transfection, cells were collected, and total cell protein was extracted by repeated freezing and thawing followed by centrifugation. The total protein concentration of transfected rAAV9-CAR-coGLB1 plasmid, rAAV9-CAR-coGLB1-2x142-3P plasmid and blank cells was measured using Pierce BCA Protein Aaasy Kit (ThermoFisher, USA). Based on the results, take the whole protein sample extracted from the cells, add 30 ⁇ L of 0.75mM 4-methylumbelliferone- ⁇ -D-galactopyranoside (4-MU- ⁇ -D-Gal) and mix well.
  • transfection of plasmids encoding ⁇ -gal enzyme mutants carrying F193Y, A194T, R208H, S246N, I353L, N459L, and Y488F mutations increased the ⁇ -galactosidase activity, but the increase amount was significantly lower than that of cells transfected with wild-type ⁇ -gal enzyme (all P ⁇ 0.05).
  • the cell enzyme activity was similar to that of the untransfected control group (P>0.05).
  • the activity of cells overexpressing the G245D mutant showed no significant change compared with cells transfected with pRDAAV-CAR-coGLB1 (P>0.05), suggesting that the G245D mutation had little effect on enzyme activity.
  • the ⁇ -galactosidase activity exhibited by cells overexpressing the I353L mutant was approximately 50%-60% of the enzyme activity of cells transfected with pRDAAV-CAR-coGLB1, but was still much higher than that of the untransfected control group.
  • the control mutant N488Q which is known to reduce enzyme activity
  • its overexpression resulted in lower cell enzyme activity than untransfected cells, which was consistent with expectations.
  • the most prominent mutant among the mutants is R299L.
  • the ⁇ -galactosidase activity in cells transfected with its corresponding plasmid increased by 30% to 40% compared to cells transfected with wild-type GLB1, and the difference was statistically significant ( p ⁇ 0.00005).
  • HEK-293 cells were transfected with a series of concentration gradients (100, 200, 400, 600, 800ng per well) of pRDAAV-CAR-coGLB1 and pRDAAV-CAR-coGLB1R299L plasmids. . After 48 hours of transfection, the detection was performed according to the aforementioned method.
  • constructs with mutations R299L, G245D and I353L constructed according to the aforementioned element design and cloning steps are pRDAAV-CAR-coGLB1 R299L, pRDAAV-CAR-coGLB1 G245D and pRDAAV-CAR-coGLB1 I353L respectively.
  • the gene sequences of the three are shown in SEQ ID No: 17, 25 and 27 respectively; the corresponding expression box sequences are shown in SEQ ID No: 18, 26 and 28 respectively.
  • the inventor further imagined that other mutation methods at this site are also promising to obtain highly active ⁇ -gal enzyme variants.
  • the R299A, R299Q, and R299F mutant plasmid vectors were constructed according to the method described in Example 1, and the primer sequences are shown in Table 2.
  • the same amount of plasmid encoding wild-type and variant GLB1 was transfected into HEK-293 cells, and ⁇ -gal enzyme activity and protein expression were compared 48 hours later.
  • the X-Gal staining method was used to detect the ⁇ -gal enzyme activity in the cells in situ (the method steps are the same as in Example 2).
  • the results are shown in Figure 21(a): the R299L plasmid transfected group had the highest rate of X-Gal positive cells and the deepest staining.
  • the staining of the R299A group was higher than that of the coGLB1 group but slightly lower than that of the R299L group, and the staining of the R299Q and R299F groups was lower than that of the coGLB1 group.
  • X-gal staining of cells in all plasmid transfection groups was stronger than that of untransfected HEK-293 negative control cells. All results show that among the mutants obtained by our transformation, the point mutation at position 299 is often beneficial to the improvement of ⁇ -Gal enzyme activity, among which R299L has the highest ⁇ -Gal enzyme activity.
  • the 4-MU standard of known concentration was gradient diluted with sodium carbonate stop solution, and a standard curve was drawn with the 4-MU content as the abscissa and the fluorescence value as the ordinate. Calculate the amount of 4-MU product generated by the sample based on the standard curve. ⁇ -gal enzyme activity is generated per milligram of protein per hour Product, gL; T: reaction time, h; 4-MU product concentration y (nmol/mL) after the reaction hole is terminated.
  • the enzyme activity results that when the 299 position is mutated into smaller diameter leucine and alanine, the ⁇ -gal enzyme activity level can be increased.
  • the amino acid at position 299 is mutated to glutamine with a polar side chain, its enzyme activity level is not significantly different from that of human ⁇ -galactosidase with the natural amino acid sequence.
  • the method is as follows: 48 hours after HEK-293 cells were transfected, total cell protein was extracted, and the samples were separated by 12.5% SDS-polyacrylamide gel electrophoresis. Each lane was loaded with 10 ⁇ g protein. After electrophoresis, the protein was transferred to a PVDF membrane and incubated with ⁇ -galactosidase antibody and ⁇ -Tublin internal control antibody. Horseradish peroxidase-labeled goat anti-mouse immunoglobulin G (IgG) antibody was used as the secondary antibody and hybridization was performed in 5% skim milk. React with bioluminescent reagents to detect the expression of the target protein.
  • IgG horseradish peroxidase-labeled goat anti-mouse immunoglobulin G
  • the increase in enzyme activity in cells overexpressing the R299L and R299A variants is not caused by higher protein expression, but the result of the increased catalytic activity of the mutants.
  • constructs with mutations R299A, R299F and R299Q constructed according to the aforementioned element design and cloning steps are pRDAAV-CAR-coGLB1 R299A, pRDAAV-CAR-coGLB1 R299F and pRDAAV-CAR-coGLB1 R299Q respectively.
  • the gene sequences of the three are shown in SEQ ID No: 19, 21 and 23 respectively; the corresponding expression box sequences are shown in SEQ ID No: 20, 22 and 24 respectively.
  • helper plasmid pRep2Cap9 carrying Rep and Cap protein expression genes
  • helper plasmid pADHelper and HEK-293 cells were from Beijing Ruixi Rare Disease Gene Therapy Technology Institute.
  • HEK-293 cells were expanded and cultured. When the cells grew to a density of about 80%, they were co-transfected with the constructed AAV9 vector plasmid rAAV9-CAR-coGLB1 carrying the optimized GLB1 gene and the helper plasmids pRep2Cap9 and pADHelper.
  • the cells and culture supernatant were harvested, and DRase at a concentration of 10 U/mL was added to digest the viral outer nucleic acid. Increase virus concentration through ultrafiltration and concentration.
  • the rAAV9-CAR-coGLB1 concentrated virus liquid was purified. SDS-PAGE was performed, and the results are shown in Figure 3. Obvious and clear electrophoretic bands of the capsid proteins VP1, VP2, and VP3 of the specific AAV virus were visible.
  • HEK-293 ATCC CRL-1573
  • U-87MG ATCC HTB-14
  • RAW264.7 ATCC TIB-71
  • 3 wells were infected with rAAV9-CAR-coGLB1 virus, another 3 wells were infected with rAAV9-CAR-coGLB1-2x142-3P virus, and the remaining 3 wells were not infected as negative control wells. 0.1% of the medium volume of sodium butyrate was added to the culture medium 6 hours after virus infection to enhance expression.
  • Cells were harvested 48 hours after infection, and total cell protein was extracted by repeated freezing and thawing followed by centrifugation. Use Pierce BCA Protein Aaasy Kit (ThermoFisher, USA) to measure the total protein solution concentration of infected cells and blank cells respectively, and use it for ⁇ -galactosidase enzyme activity detection experiment to evaluate the in vitro expression effect of virus ⁇ -galactosidase. .
  • the cell protein extraction method and enzyme activity detection experimental method are the same as in Example 2.
  • rAAV9-CAR-coGLB1 virus can greatly increase the enzyme activity level of intracellular ⁇ -galactosidase after infection with HEK-293, U-87MG, and RAW264.7, which is in line with expectations.
  • the rAAV9-CAR-coGLB1-2x142-3P virus infection of HEK-293 and U-87MG cells can also increase the level of ⁇ -galactosidase enzyme activity, and the results are comparable to those of rAAV9-CAR-coGLB1 virus, but in RAW264.7 small
  • the level of ⁇ -galactosidase enzyme activity in mouse macrophages was significantly reduced, indicating that the virus adding the miRNA 142-3P target sequence fragment during treatment can retain ⁇ -galactosidase activity in a wide range of somatic cells and neural cells.
  • Expression level which can significantly reduce transgene expression in macrophages, reduce immunogenicity, and improve therapeutic effect.
  • the physical virus titers (see Figure 22(a)) of rAAV9-coGLB1 (2.75 ⁇ 10 13 vg/mL) and rAAV9-coGLB1-R299L (1.83 ⁇ 10 13 vg/mL) were obtained using the dot hybridization method.
  • HEK-293 cells were infected with viruses with three MOI values (2 ⁇ 10 4 , 5 ⁇ 10 4 and 1 ⁇ 10 5 ).
  • p.G455R (NM_009752.2:c.1363g>a) corresponding to human coGLB1G453R on the mouse Glb1 locus was edited.
  • the oligonucleotide G455R (GGA ⁇ AGA) mutation site was introduced into exon 14 through homology-directed repair, and a GM1 gangliosidosis model mouse carrying a G455R point mutation was obtained (for this model, see Liu S, Feng Y, Huang Y, et al.
  • a GM1 gangliosidosis mutant mouse model exhibits activated microglia and disturbed autophagy[J].
  • Experimental biology and medicine (Maywood, N.J.), 2021, 246(11): 1330-1341.).
  • mice After the experiment is completed, all mice will be sacrificed, the eyeballs will be removed to collect blood to obtain the blood of each mouse, and the serum will be obtained after processing. Mice were dissected and protein samples were extracted from key tissues such as heart, liver, spleen, lung, kidney, muscle, and brain (which can be subdivided into cortex, thalamus, brainstem, and cerebellum).
  • Tissue protein extraction Add PBS 2+ and protease inhibitors to homogenize the tissue, freeze the suspension at -70°C for 10 minutes, take it out and thaw it in a 37°C water bath for 2 minutes, and repeat the freezing and thawing three times. Centrifuge at 4°C (12000rpm/10min) and take the supernatant as tissue protein solution.
  • Enzyme activity detection Take 30 ⁇ L of each tissue sample (containing approximately 5-10 ⁇ g protein), add 30 ⁇ L of 0.75mM 4-methylumbelliferone- ⁇ -D-galactopyranoside (4-MU- ⁇ -D -Gal) and mix well. Set up a negative control and replace 30 ⁇ L of the sample to be tested with 30 ⁇ L of PBS 2+ buffer; incubate the reaction at 37°C for 30 minutes; add 120 ⁇ L of NaCO 3 stop solution pre-cooled in the refrigerator to the reaction system to terminate the reaction. After mixing thoroughly, the excitation light is 360 nm.
  • the emission light is at 460nm
  • use a microplate reader to measure the fluorescence intensity of free 4-methylumbelliferone
  • Enzyme activity was calculated using 4-methylumbelliferone as the standard, and the experiment was repeated three times.
  • ⁇ -gal enzyme activity is expressed as the amount of 4-methylumbelliferone produced per milligram of protein per hour, that is, the unit is nmol/mg ⁇ h.
  • mice a total of 12 neonatal model mice (P2) homozygous for GLB1 gene point mutation were randomly divided into 2 groups.
  • Group 1 served as the experimental group and was injected with rAAV9-CAR-coGLB1 virus through the superficial temporal vein. The injection dose was 3.40 ⁇ 10 10 vg viral vector per animal.
  • Another group was used as the negative control group, with each mouse injected with 50 ⁇ L PBS; and another group of 6 C57BL/6N wild-type mice was used as the normal mouse control group 3.
  • mice 20 weeks after the virus injection, all mice were sacrificed, and the blood of each mouse was obtained by removing the eyeballs and processing to obtain serum.
  • the mice were dissected and protein samples were extracted from key tissues such as heart, liver, spleen, lung, kidney, and brain (which can be subdivided into cortex, thalamus, brainstem, and cerebellum). The whole protein of mouse tissues was extracted, and the concentration of protein extracts of each tissue was measured using Pierce BCA Protein Aaasy Kit (ThermoFisher, USA).
  • mice a total of 18 neonatal model mice (P2) homozygous for point mutations in the GLB1 gene were randomly divided into 3 groups.
  • Group 1 a total of 10 mice, was treated as a low-dose group by injecting rAAV9-CAR-coGLB1 virus into the lateral cerebral ventricle. The injection dose was 8.50 ⁇ 10 9 vg viral vector per animal.
  • Group 2 a total of 6 mice, was treated as a high-dose group by injecting rAAV9-CAR-coGLB1 virus into the lateral ventricle. The injection dose was 3.40 ⁇ 10 10 vg viral vector per animal.
  • a total of 9 mice in group 3 were used as the negative control group, each of which was injected with 5 ⁇ L PBS; and another group of 9 C57BL/6N wild-type mice were selected as the normal mouse control group 4.
  • mice in group 1 Eight weeks after virus injection, 4 mice in group 1, 3 mice in group 3, and 3 mice in group 4 were sacrificed, and the blood of each mouse was obtained by removing the eyeballs and processing to obtain serum.
  • the mice were dissected and protein samples were extracted from key tissues such as heart, liver, spleen, lung, kidney, and brain (which can be subdivided into cortex, thalamus, brainstem, and cerebellum). The specific method is the same as above.
  • mice a total of 12 neonatal model mice (P2) homozygous for point mutations in the GLB1 gene were randomly divided into 2 groups.
  • Group 1 was treated as the experimental group by injecting rAAV9-CAR-coGLB1 virus through the lateral ventricle and superficial temporal vein. The injection dose was 8.50 ⁇ 10 9 vg for each lateral ventricle and 2.55 ⁇ 10 10 vg for each superficial temporal vein. Total 3.40 ⁇ 10 10 vg/piece.
  • Group 2 served as the negative control group, and each mouse was injected with the same volume of PBS solution as the experimental mouse; and another group of 6 C57BL/6N wild-type mice was selected as the normal mouse control group 3.
  • mice Eight weeks after the virus injection, all mice were sacrificed, and the blood of each mouse was obtained by removing the eyeballs and processing to obtain serum.
  • the mice were dissected and protein samples were extracted from key tissues such as heart, liver, spleen, lung, kidney, and brain (which can be subdivided into cortex, thalamus, brainstem, and cerebellum). The specific method is the same as above.
  • ⁇ -Gal enzyme activity that is much higher than the blank value can be detected in heart, liver, spleen, lung, kidney, brain tissue and serum, but the enzyme activity value in muscle is lower.
  • brain tissue of experimental mice was subdivided into cerebellum, brainstem, cortex, and thalamic nuclei and tested separately, it was found that the enzyme activity distribution among different nuclei was relatively even.
  • the activity of ⁇ -galactosidase in the serum was extremely low and almost completely lost.
  • the residual enzyme activities in the liver, spleen, lungs, and kidneys are also significantly lower than those in wild-type mice of the same age.
  • the enzyme activities in the lungs and kidneys are only about 1% of those in wild-type mice, and the enzyme activities in spleen are about 1% of those in wild-type mice.
  • the liver enzyme activity of model mice is about 4%, and the liver enzyme activity is about 17% of wild-type mice, but the heart enzyme activity of model mice is only slightly lower than that of wild-type mice.
  • the partial enzyme activities of the brainstem, cortex, cerebellum and thalamic nuclei of model mice were significantly lower than those of wild-type control mice.
  • the most improved areas were the cerebellum and thalamus, which exceeded the brain tissue enzyme activity level of wild-type mice. However, the enzyme activity distribution in the brain nuclei of treated mice was not as good as that of wild-type mice. Rats were evenly distributed, and the enzyme activity level in the cortex was the highest.
  • the two injection methods have different treatment focuses, but they can clarify the effectiveness of ICV treatment in improving brain enzyme activity, and also improve serum enzyme activity to a certain extent.
  • IV injection mainly improves some peripheral tissues and serum enzyme activities.
  • dual-channel injection into the lateral ventricle and vein is an optimization of the single-channel injection method.
  • ICV injection of AAV vector into newborn mice high transduction efficiency and high-intensity expression of the target gene in brain tissue can be observed.
  • the transduction efficiency and expression intensity of IV injection in the central nervous system are significantly lower than those of ICV injection, but the effect of increasing enzyme activity in serum and important tissues and organs such as heart, liver, kidney, and spleen is very significant.
  • mice Two months after dual-channel injection into the lateral ventricle and intravenously, the enzyme activity in the peripheral tissues of mice could be increased to a level close to that of wild-type mice, and the enzyme activity in heart tissue and brain tissue was even higher than that in wild-type control mice.
  • neurological symptoms are the main symptoms
  • patients with GM1 gangliosidosis have certain pathological manifestations in the nervous system and peripheral tissues and organs.
  • the use of dual-path injection method can take into account the effects on the nervous system and important peripheral organs.
  • the protection against accumulation of GM1 ganglioside substrate has a broader therapeutic effect than a single injection.
  • mice homozygous for point mutations in the GLB1 gene were randomly divided into 3 groups.
  • Group 1 was treated as a high-dose experimental group by injecting rAAV9-CAR-coGLB1 virus into the tail vein, and the injection dose was 3 ⁇ 10 13 vg/kg per animal.
  • Group 2 was treated as a low-dose experimental group by injecting rAAV9-CAR-coGLB1 virus into the tail vein, and the injection dose was 8 ⁇ 10 13 vg/kg per animal.
  • Group 3 served as the negative control group without virus injection; and another group of 8 C57BL/6N wild-type mice was selected as the normal mouse control group 4.
  • Upper limb suspension test (Forelimb suspension test): Place the mouse lightly on the elevated wire, ensure that the mouse's upper limbs hold the wire and start timing until they fall from the pole or until the maximum test time of 10 minutes is reached. Each mouse was measured three times per experiment, with at least 10 minutes between tests. The longest time of repeated experiments was recorded as the upper limb suspension time as the final test result for each rat. The results are shown in the left picture of Figure 17.
  • Rotarod test Use an accelerating rotator to test the exercise ability of each group of rats. In each experiment, mice were subjected to three rotations. The rotor speed will gradually accelerate from 5rpm to 40rpm. When the rat falls off the rotating rod or remains on the rod for more than 3 minutes, the test will be stopped. The interval between each test is 15 minutes. The longest time among three repeated tests was recorded as the final result for each rat. The results are shown on the right side of Figure 17.
  • PBS phosphate buffered saline
  • TDT enzyme dUTP and buffer TUNEL staining kit
  • TUNEL staining kit A solution containing TDT enzyme dUTP and buffer (TUNEL staining kit) was added to the tissue sample at a ratio of 1:5:50, and then incubated in a humidified chamber at 37°C for 1 h. Then a 1:200 mixture of streptavidin-horseradish peroxidase (HRP) and TBST was added to completely cover the tissue, and the sections were placed in a 37°C incubator for 30 min. Subsequently, the slides were placed in PBS (pH 7.4) and washed three times on a destaining shaker for 5 minutes each time. Slightly dry.
  • HRP streptavidin-horseradish peroxidase
  • DAB chromogen was then added to the tissue, and the nuclei were counterstained with hematoxylin staining solution for 1 minute. Finally, the sections were washed in pure water, dehydrated, and placed in xylene to make the tissue transparent.
  • rAAV9-CAR-coGLB1 virus can significantly and persistently reduce the accumulation of GM1 ganglioside in the brain of model mice.

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Abstract

提供了功能性β-半乳糖苷酶，例如人β-半乳糖苷酶的功能性变体，及其制药用途。还提供了优化的人β-半乳糖苷酶基因表达框，以及携带该表达框的重组腺相关病毒载体及其制备方法和制药用途。所述的表达框能够在体内高效地表达功能性人β-半乳糖苷酶，包含表达框或病毒载体的药物通过脑室内注射和静脉注射方式给药后，在全身持续表达β-半乳糖苷酶，有效降解异常积聚的GM1神经节苷脂底物，缓解和消除疾病症状。

Description

功能性β-半乳糖苷酶变体、AAV介导的人β-半乳糖苷酶表达载体及其用途
本申请是以CN申请号为202210343493.3，申请日为2022年3月31日的申请为基础，并主张其优先权，该CN申请的公开内容在此作为整体引入本申请中。
技术领域
本发明涉及生物技术领域。具体地，本发明涉及一种携带优化的人β-半乳糖苷酶基因表达框的重组腺相关病毒载体。通过施用本发明的重组腺相关病毒载体，能够获得β-半乳糖苷酶特异性表达，由此预防和/或治疗GM1神经节苷脂贮积症。
背景技术
GM1神经节苷脂贮积症又称GM1神经节苷脂病，是由半乳糖苷酶β-1(GLB1)基因变异影响GLB活性所致的常染色体隐性遗传病，GLB1基因突变导致溶酶体β-半乳糖苷酶(β-gal)活性降低或消失。而β-gal的缺乏导致GM1神经节苷脂及其唾液酸化衍生物GA1在中枢神经系统(CNS)的积累。此外还有低聚糖和硫酸角质素在内脏器官积累。GM1-神经节苷脂病的发病率估计为1:100 000-200 000活产，但在某些种族群体和国家发病率更高。根据发病年龄，和相应的突变酶变体的残留活性，GM1患者被分为三类，并分别具有极具侵略性或进展相对缓慢的病程。在婴儿期病人中，由于几乎没有残余的酶活性，神经系统迅速衰退，丧失自主运动控制能力，导致全身瘫痪，极度消瘦和死亡。其他器官和组织也受到影响，如特征性的肝脾肿大和骨骼发育不良。晚期儿童和成人发病者的病情则相对较轻，趋于慢性化。
目前，对GM1神经节苷脂病没有成熟的治疗方法。
一些基因治疗方法已经在GM1神经节苷脂病的动物模型中进行了测试，包括体内输注腺病毒、腺相关病毒构建体，以及用逆转录病毒载体对自体骨髓干细胞进行体外改造，等。这些策略的可行性是基于体内或体外转基因的内源性细胞能够过表达和释放大量的功能性溶酶体酶到细胞外环境中，而这种分泌使其通过细胞外液体，例如血流，进行扩散分布。而包括中枢神经系统在内的大多数细胞能够吸收这些酶。
本领域仍需要预防和/或治疗GM1神经节苷脂沉积病的基因疗法，并且通过多方优化，使这种备选基因疗法达到单次或多次施用后，能够表达足量功能性GLB1，并且使其有效扩散至代谢GM1神经节苷脂的组织或器官，发挥其活性。同时，作为对野生型β-半乳糖苷酶的替代或改进，带有氨基酸序列点突变的活性酶变体在治疗中的应用也有待于探索。
发明概述
本发明首先提供了一种GLB1基因表达框，其适合于构建到基因治疗载体中，并且至少包含启动子序列和GLB1基因序列，其中启动子能够驱动或指导GLB1基因对人β-半乳糖苷酶进行编码，从而在人体细胞内表达有活性的人β-半乳糖苷酶。
本发明还提供了一种携带GLB1基因表达框的AAV病毒载体。所述AAV病毒载体仅保留了野生型AAV病毒基因组中包装病毒所需要的两个ITR序列或其变体，不含有野生型AAV病毒基因组中的蛋白编码基因，这使得所述AAV病毒载体在施用于患者后的免疫原性低；此外，AAV病毒载体通常以不整合的染色体外遗传物质形式实现携带外源基因阅读框的持续稳定表达，避免了导入生物体的外源基因随机整合而带来的安全性问题。
本发明的AAV病毒载体在给药后，能够有效地扩散分布至全身，尤其是中枢神经系统、心脏、肝脏、脾脏、肺脏、肾脏、肌肉、血清等组织、器官和部位，保证了GLB1基因表达框能够在全身，尤其是中枢神经系统、心脏、肝脏、脾脏、肺脏、肾脏、肌肉、血清等组织、器官 和部位内高效地表达出有活性的β-半乳糖苷酶，从而代偿/弥补此前缺失的酶活性，由此，提供了一种新的预防和/或治疗GM1神经节苷脂贮积症的基因药物。
本发明还提供了功能性β-半乳糖苷酶，例如人β-半乳糖苷酶变体酶。
在本发明的一些实施方案中，表达框中所包含的GLB1基因序列所编码的人β-半乳糖苷酶具有与天然存在的人野生型β-半乳糖苷酶相同或基本相同的氨基酸序列，即与后者具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高、或100％的序列同一性，并且具有不显著低于后者的体内活性，从而能够有效降解GM1神经节苷脂底物以避免和消除其贮积，并缓解或完全消除疾病症状。其中所述天然存在的人野生型β-半乳糖苷酶的氨基酸序列例如是SEQ ID NO:10所示的序列。
在本发明的一些优选实施方案中，表达框中所包含的GLB1基因序列所编码的人β-半乳糖苷酶与天然存在的人野生型β-半乳糖苷酶相比，具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高、或100％的序列同一性，并且至少具有氨基酸序列点突变I353L、G245D、R299(L/A/F/Q)中的一个或多个，并且具有不显著低于后者的体内活性，从而能够有效降解GM1神经节苷脂底物以避免和消除其贮积，并缓解或完全消除疾病症状。其中所述天然存在的人野生型β-半乳糖苷酶的氨基酸序列例如是SEQ ID NO:10所示的序列。
在一个优选的实施方案中，表达框中所包含的GLB1基因序列所编码的人β-半乳糖苷酶的氨基酸序列是在SEQ ID NO:10所示的序列的基础上还具有点突变I353L、G245D、R299(L/A/F/Q)中的一种。
在一个优选的实施方案中，表达框中所包含的GLB1基因序列所编码的人β-半乳糖苷酶的氨基酸序列是SEQ ID NO:11-16任一项所示的序列。
在一个优选的实施方案中，表达框中所包含的GLB1基因序列所编码的人β-半乳糖苷酶的氨基酸序列是SEQ ID NO:10所示的序列。
在本发明的一些实施方案中，表达框中所包含的编码β-半乳糖苷酶的核苷酸序列(即GLB1基因序列)与天然存在的野生型GLB1基因相同或基本相同，即与后者具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高、或100％的同一性。其中所述天然存在的野生型GLB1基因的序列例如是SEQ ID NO:9所示的序列。
在本发明的一些实施方案中，表达框中所包含的GLB1基因序列是经过密码子优化的，在表达相同氨基酸序列的同时比天然存在的野生型GLB1基因含有更多人体内最常见或更常见的密码子形式，从而提高表达速度和表达量。在一些优选的实施例中，所述经过密码子优化的序列是SEQ ID NO:2所示的序列。
在一些实施方案中，表达框中所包含的GLB1基因序列与SEQ ID NO:2所示的序列具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高、或100％的同一性。
在本发明的另一些实施方案中，为了在最终表达的β-半乳糖苷酶中引入R299(L/A/F/Q)点突变，将GLB1基因编码序列中第895-897位置上编码精氨酸的密码子(例如，AGA)改变为CTG/GCT/TTT/CAA(以起始密码子ATG为第1-3位置)。
在本发明的另一些实施方案中，为了在最终表达的β-半乳糖苷酶中引入G245D点突变，将GLB1基因编码序列中第733-735位置上编码甘氨酸的密码子(例如，GGC)改变为GAC。
在本发明的另一些实施方案中，为了在最终表达的β-半乳糖苷酶中引入I353L点突变，将GLB1基因编码序列中第1057-1059位置上编码异亮氨酸的密码子(例如，ATC)改变为CTG。
在一些实施方案中，表达框中所包含的GLB1基因序列是SEQ ID NO:17、19、21、23、25或27所示的序列。
在一些实施方案中，表达框中所包含的GLB1基因序列是SEQ ID NO:2或9所示的序列。
在第一方面，本发明提供了一种优化的人GLB1基因表达框。
在一个实施方案中，所述表达框包含：
(1)高活性启动子序列，所述启动子在中枢神经系统和外周组织都能够高效启动目的基因转录，避免被沉默；
(2)编码功能性β-半乳糖苷酶的核苷酸序列，例如人GLB1基因序列，或其经过密码子优化后的编码序列；以及任选地
(3)可选的调控序列，例如polyA信号序列，等等，
其中功能性β-半乳糖苷酶具有不显著低于天然存在的人野生型β-半乳糖苷酶的体内活性，从而能够有效降解GM1神经节苷脂底物以避免和消除其贮积，并缓解或完全消除疾病症状。
在一个更具体的实施方案中，所述表达框包含：
(1)如SEQ ID NO:1所示的启动子序列或者与其具有至少约90％同一性的启动子序列；
(2)编码功能性β-半乳糖苷酶的GLB1基因序列，所述酶具有与天然存在的人野生型β-半乳糖苷酶相同或基本相同的氨基酸序列，即与后者具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高、或100％的序列同一性，并且具有不显著低于后者的体内活性，从而能够有效降解GM1神经节苷脂底物以避免和消除其贮积，并缓解或完全消除疾病症状，
例如，
(i)编码如SEQ ID NO:10-16任一项所示的氨基酸序列的核苷酸序列；
(ii)如SEQ ID NO:2或9所示的核苷酸序列；
(iii)编码β-半乳糖苷酶变体酶(例如，包含氨基酸序列第299位或第245位或第353位的突变)的核苷酸序列，例如SEQ ID NO:17、19、21、23、25或27所示的核苷酸序列；
(iv)与(i)-(iii)的核苷酸序列互补的核苷酸序列；
(v)与(i)-(iii)的核苷酸序列编码相同的β-半乳糖苷酶，但因遗传密码的简并性而与(i)-(iii)的核苷酸序列不同的核苷酸序列；或
(vi)与(i)至(v)任一项所述的核苷酸序列具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高(例如99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％、99.9％)同一性的序列。
在一个更优选的实施方案中，所述表达框包含(3)，且(3)是如SEQ ID NO:4所示的或者与其具有至少约90％同一性的BGH polyA序列。
在一个更优选的实施方案中，所述表达框进一步包含：
(4)至少一个(例如，1－5个)串联的SEQ ID NO:3所示的人miR-142-3p靶序列，例如，具有1个，或2或3个串联的人142-3P靶序列，即1x、2x或3x142-3P，优选地例如具有2个串联的人142-3P靶序列，即2x 142-3P。
在一个具体实施方案中，本发明的优化的人GLB1基因表达框具有如SEQ ID NO:5、7、18、20、22、24、26或28所示的核苷酸序列或与SEQ ID NO:5、7、18、20、22、24、26或28具有至少约90％同一性的序列。
在一个具体实施方案中，本发明的优化的人GLB1基因表达框两端与AAV病毒载体的ITR序列连接。
在一个具体实施方案中，本发明的优化的人GLB1基因表达框的表达产物为功能性β-半乳糖苷酶，能够降解GM1神经节苷脂；此外，可选地，所述表达框还能够至少部分地抑制生物体的免疫反应。
在第二方面，本发明提供了一种病毒载体，其包含本发明的第一方面的人GLB1基因表达框。
在一个具体的实施方案中，所述病毒载体包含如SEQ ID NO:2、9、17、19、21、23、25或 27所示的序列。
在一个具体的实施方案中，所述病毒载体包含如SEQ ID NO:5、7、18、20、22、24、26或28所示的序列。
在一个具体的实施方案中，所述病毒载体包含本发明的人GLB1基因表达框，并且所述人GLB1基因表达框两端与AAV病毒载体的ITR序列连接。
在某些具体的实施方案中，所述病毒载体包含例如如SEQ ID NO:6或8所示的序列。
在一些实施方案中，本发明的病毒载体是重组腺相关病毒载体AAV，包括但不限于选自AAV1、AAV2、AAV3B、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAVrh.10或它们的组合的血清型的重组腺相关病毒载体，优选地为重组AAV5、AAV3B、AAV8、AAV9载体。
在一些优选的实施方案中，本发明的病毒载体是AAV9病毒载体。在一些优选的实施方案中，所述AAV9为重组的AAV9.
在一些实施方案中，本发明的病毒载体的基因组可以自我互补形成双链DNA分子。
在第三方面，本发明提供了功能性β-半乳糖苷酶变体酶，其具有与天然存在的人野生型β-半乳糖苷酶相同或基本相同的氨基酸序列，即与后者具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高、或100％的序列同一性，并且具有不显著低于后者的体内活性，从而能够有效降解GM1神经节苷脂底物以避免和消除其贮积，并缓解或完全消除疾病症状。
在一些具体的实施方案中，所述功能性β-半乳糖苷酶变体酶相对于天然存在的人野生型β-半乳糖苷酶包含氨基酸序列第299位或第245位或第353位的突变。在一个优选的实施方案中，所述功能性β-半乳糖苷酶变体酶相对于天然存在的人野生型β-半乳糖苷酶包含第299位的突变。在一些更具体的实施方案中，所述功能性β-半乳糖苷酶变体酶相对于天然存在的人野生型β-半乳糖苷酶包含以下(i)-(iii)的氨基酸序列突变中的任一项：(i)在氨基酸序列第299位，由精氨酸突变为亮氨酸、丙氨酸、苯丙氨酸或谷氨酰胺；(ii)在氨基酸序列第245位，由甘氨酸突变为天冬氨酸；(iii)在氨基酸序列第353位的突变，由异亮氨酸突变为亮氨酸。
在一些实施方案中，本发明还提供了编码上述变体酶的核酸分子。
在第四方面，本发明提供了一种用于预防和/或治疗GM1神经节苷脂沉积病的药物，例如基因药物等，其中所述药物是包含本发明的功能性β-半乳糖苷酶变体酶、优化的人GLB1基因表达框的病毒载体。通过静脉注射本发明的所述药物，例如基因药物，能够在所需要的组织、器官、部位(例如，中枢神经系统)中特异性表达功能性人β-半乳糖苷酶和/或弥补β-半乳糖苷酶活性，发挥预防和/或治疗GM1神经节苷脂沉积病或其引起的疾病或与其相关联的疾病。在一些实施方案中，所述GM1神经节苷脂沉积病是GLB1基因缺陷导致的。
在第五方面，本发明提供了本发明的功能性β-半乳糖苷酶变体酶、优化的人GLB1基因表达框和本发明的病毒载体的用途，用于制备预防和/或治疗GM1神经节苷脂沉积病的药物，例如，用于制备预防和/或治疗GLB1基因缺陷的药物。
在一些实施方案中，本发明的预防和/或治疗药物的给药方式为单次或多次脑室内注射和/或单次或多次静脉注射。
在一些实施方案中，本发明的病毒载体通过脑室内注射或静脉注射方式给药，优选地，通过一次性地脑室内注射或静脉注射方式给药，减少了反复施用药物所带给患者的痛苦。在一些更优选的实施方案中，本发明的病毒载体通过脑室内注射和静脉注射双重方式给药，例如，一次性给药。
在一些实施方案中，本发明的病毒载体在进入体内后，能够在细胞内稳定而持续地表达β-半乳糖苷酶。
在一些实施方案中，本发明的病毒载体在通过静脉注射方式给药后，在全身尤其是心脏、肝脏、脾脏、肺脏、肾脏、肌肉、外周血等组织、器官和部位中持续表达β-半乳糖苷酶，表现出持续存在的高水平酶活性，并有效降解GM1神经节苷脂底物以避免和消除其贮积。
在一些实施方案中，本发明的病毒载体在通过脑室内注射方式给药后，在全身尤其是中枢神经系统等组织、器官和部位中持续表达β-半乳糖苷酶，表现出持续存在的高水平酶活性，并有效降解GM1神经节苷脂底物以避免和消除其贮积。
在一些实施方案中，本发明的病毒载体在通过脑室内注射和静脉注射方式给药后，在全身尤其是中枢神经系统、心脏、肝脏、脾脏、肺脏、肾脏、肌肉、血清等组织、器官和部位中持续表达β-半乳糖苷酶，表现出持续存在的高水平酶活性，并有效降解GM1神经节苷脂底物以避免和消除其贮积。
在一些实施方案中，本发明的病毒载体在通过脑室内注射方式和/或静脉注射方式给药后，在中枢神经系统的组织、器官和部位中持续表达β-半乳糖苷酶，表现出持续存在的高水平酶活性，该酶活性达到或超过正常个体中相同组织、器官和部位的酶活性，并有效降解GM1神经节苷脂底物以避免和消除其贮积，并缓解或完全消除疾病症状。
在一些实施方案中，本发明的病毒载体在通过脑室内注射方式和/或静脉注射方式给药后，在中枢神经系统的组织、器官和部位中持续表达β-半乳糖苷酶，表现出持续存在的高水平酶活性，该酶活性低于正常个体中相同组织、器官和部位的酶活性，但其仍然能够有效降解GM1神经节苷脂底物以避免和消除其贮积，并足以缓解或完全消除疾病症状。
在一些实施方案中，本发明的病毒载体在通过脑室内注射方式和/或静脉注射方式给药后，在外周组织、器官和部位中持续表达β-半乳糖苷酶，表现出持续存在的高水平酶活性，该酶活性达到或超过正常个体中相同组织、器官和部位的酶活性，并有效降解GM1神经节苷脂底物以避免和消除其贮积，并缓解或完全消除疾病症状。
在一些实施方案中，本发明的病毒载体在通过脑室内注射方式和/或静脉注射方式给药后，在外周组织、器官和部位中持续表达β-半乳糖苷酶，表现出持续存在的高水平酶活性，该酶活性低于正常个体中相同组织、器官和部位的酶活性，但其仍然能够有效降解GM1神经节苷脂底物以避免和消除其贮积，并足以缓解或完全消除疾病症状。
在一些实施方案中，本发明的构建体中含有一个或多个miR-142-3P靶序列片段，使得整个病毒构建体在保留β-半乳糖苷酶的表达水平的同时，大幅度降低在巨噬细胞中的转基因表达，从而降低免疫原性，提升治疗效果。
在本发明的大部分实施方案中，通过对病毒载体结构的多层次优化，实现了功能性β-半乳糖苷酶(例如，野生型酶或变体酶)在全身大部分组织、器官和部位中的高效、特异性表达，同时降低了有可能引起的免疫反应，提高了病毒载体作为基因药物的有效性和安全性。
附图简述
结合以下附图一起阅读时，将更好地理解以下详细描述的本发明的优选实施方案。出于说明本发明的目的，图中显示了目前优选的实施方案。然而，应当理解本发明不限于图中所示实施方案的精确安排和手段。
图1显示了rAAV9-CAR-coGLB1质粒载体的一个实例的结构图。
图2显示了质粒rAAV9-CAR-coGLB1体外表达的验证实验，通过X-gal细胞染色。A.X-gal染色的HEK-293细胞(100×)；B.X-gal染色的转染了携带GLB1基因的质粒的HEK-293细胞(100×)；C.X-gal染色的HEK-293细胞(200×)；D.X-gal染色的转染了携带GLB1基因的质粒的HEK-293细胞(200×)。
图3显示了rAAV9-CAR-coGLB1超滤浓缩SDS-PAGE结果图。可见明显、清晰的特异性AAV病毒的衣壳蛋白VP1、VP2和VP3电泳条带。
图4显示了体外转染细胞β-半乳糖苷酶蛋白的活性测定，转染了pRDAAV-CAR-coGLB1的细胞酶活性测定为430nmol/mg·h，而对照组为100。
图5显示了体外感染HEK-293、U-87MG、RAW264.7细胞β-半乳糖苷酶的活性测定。
图6显示了新生鼠IV治疗20周龄纯合突变模型鼠及对照组小鼠的大脑核团β-Gal酶活性检测结果。
图7显示了新生鼠ICV治疗8周龄纯合突变模型鼠及对照组小鼠的大脑核团β-Gal酶活性检测结果。
图8显示了新生鼠ICV+IV双途径治疗8周龄纯合突变模型鼠及对照组小鼠的大脑核团β-Gal酶活性检测结果。
图9显示了新生鼠IV治疗20周龄纯合突变模型鼠及对照组小鼠的外周组织β-Gal酶活性检测结果。
图10显示了新生鼠ICV治疗8周龄纯合突变模型鼠及对照组小鼠的外周组织β-Gal酶活性检测结果。
图11显示了新生鼠ICV治疗16周龄纯合突变模型鼠及对照组小鼠的组织β-Gal酶活性检测结果。
图12显示了新生鼠ICV+IV双途径治疗8周龄纯合突变模型鼠及对照组小鼠的外周组织β-Gal酶活性检测结果。
图13显示了新生鼠IV治疗20周龄纯合突变模型鼠及对照组小鼠的血清β-Gal酶活性检测结果。
图14显示了新生鼠ICV治疗8周龄纯合突变模型鼠及对照组小鼠的血清β-Gal酶活性检测结果。
图15显示了新生鼠ICV治疗16周龄纯合突变模型鼠及对照组小鼠的血清β-Gal酶活性检测结果。
图16显示了新生鼠ICV+IV双途径治疗8周龄纯合突变模型鼠及对照组小鼠的血清β-Gal酶活性检测结果。
图17显示了尾静脉注射治疗模型鼠行为学测试结果。左图为上肢悬吊实验结果，可见rAAV9-CAR-coGLB1病毒对于悬吊时间有剂量依赖性的改善。右图为转棒疲劳实验，可见rAAV9-CAR-coGLB1病毒对于小鼠在棒上的停留时间有剂量依赖性的改善。上述改善在第8周最为明显。
图18显示了尾静脉注射治疗模型鼠脑组织Luxol Fast Blue(LFB)染色。第一排图像来自大脑皮层组织的镜下可见，第二、三排分别来自丘脑和海马组织。最左一列为GM1小鼠给予高剂量rAAV9-CAR-coGLB1病毒，第二至四列分别为GM1小鼠给予低剂量rAAV9-CAR-coGLB1病毒，GM1小鼠给予生理盐水对照，和野生型小鼠对照。第一、四列基本无蓝染，第二列可见微量蓝染(箭头所指)，第三列可见大量蓝染蓄积(箭头所指)。
图19显示了尾静脉注射治疗模型鼠大脑皮层免疫荧光(绿色：GM1神经节苷脂；蓝色：DAPI)。可见未注射模型小鼠的免疫荧光染色显示出明显的绿色信号积累，相比之下HD组和LD组小鼠GM1信号强度较弱。
图20(a)显示了用包含不同的点突变的β-半乳糖苷酶蛋白的编码核酸体外转染细胞，然后进行活性测定的结果。HEK-293细胞未转染对照组为64±11nmol/mg·h，转染了rAAV9-CAR-coGLB1的细胞酶活性测定为509±129nmol/mg·h，转染了rAAV9-CAR-coGLB1 R299L的细胞酶活性测定为718±113nmol/mg·h，转染了rAAV9-CAR-coGLB1 G245D的细胞酶活性测定为588±167nmol/mg·h，转染了rAAV9-CAR-coGLB1 I353L的细胞酶活性测定为280±83nmol/mg·h。图20(b)显示了质粒浓度梯度细胞转染酶活测定，两组所转染的质粒分别为rAAV9-CAR-coGLB1 R299L和rAAV9-CAR-coGLB1。
图21(a)显示了X-Gal细胞染色原位检测β-gal酶活性的结果，比例尺100μm，细胞分别用编码携带R299L、R299A、R299Q、R299F突变的β-gal酶的质粒载体、编码野生型β-gal酶的质粒载体转染(设置一组未转染对照)。图21(b)显示了平行的上述6组细胞进行Western  blot检测β-半乳糖苷酶蛋白表达量的结果。图21(c)显示了平行的上述6组细胞进行体外酶活测定的结果。
图22(a)显示了rAAV9-coGLB1及rAAV9-GLB1 R299L病毒点杂交滴度检测.图22(b)显示了rAAV9-coGLB1及rAAV9-GLB1 R299L以不同MOI感染HEK-293细胞后全蛋白酶活结果。
发明详述
本发明公开了一种预防和/或治疗GM1神经节苷脂贮积症的药物，例如，基因药物，涉及该药物的设计、小量制备及功能验证。
除非下文中另外定义，否则本说明书中所用的全部技术与科学术语具有如本发明所属领域的普通技术人员通常理解的相同含义。本文所提及的全部出版物、专利申请、专利和其他参考文献通过引用的方式完整地并入。此外，本文中所述的材料、方法和例子仅是说明性的并且不意在是限制性的。本发明的其他特征、目的和优点将从本说明书及附图并且从后附的权利要求书中显而易见。
I.定义
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。
术语“编码”指核酸中的特定的核苷酸序列的内在特性，其作为用于在生物学过程中合成具有确定核苷酸序列(例如rRNA、tRNA和mRNA)或确定氨基酸序列和源自其的生物学特性的其他聚合物和大分子的模板。因此，如果对应于该基因的mRNA的转录和翻译在细胞或其他生物系统中产生蛋白质，则该基因、cDNA或RNA编码蛋白质。编码链(其核苷酸序列与mRNA序列相同，且通常在序列表中提供)和非编码链(用作基因或cDNA的转录模板)都可以称为编码该基因或cDNA的蛋白质或其他产物。
术语“蛋白质”和“多肽”在本文中可互换使用，指包含氨基酸残基的聚合物序列。如无特别说明，本文中均使用遵照IUPAC-IUB生物化学命名联合委员会(Joint Commission on Biochemical Nomenclature(JCBN))定义的氨基酸的单字母和三字母代码。单字母X是指二十种氨基酸中的任一种。还应理解，由于遗传密码的简并性，多肽可由一种以上核苷酸序列编码。氨基酸序列中的突变可如下命名：亲本氨基酸的单字母代码，接着是位置编号，之后是变体氨基酸的单字母代码。例如，将第299位的精氨酸(R)突变为亮氨酸(L)表示为“R299L”。有时，在序列中，使用斜线(/)来限定可替换的多种选项，例如“R299(L/A/F/Q)”指299位的精氨酸(R)可突变为亮氨酸(L)或丙氨酸(A)或苯丙氨酸(F)或谷氨酰胺(Q)。
术语“表达”指启动子驱动的具体核苷酸序列的转录和/或翻译。
术语“β-半乳糖苷酶(β-galactosidase lipoprotein lipase,β-gal)”是一种糖苷水解酶(EC3.2.1.23)，属于糖苷水解酶蛋白35家族，它能水解半乳糖与其有机结构间形成的β-糖苷键，催化β-半乳糖苷水解成单糖。它也可以切裂岩藻糖苷和阿拉伯糖苷，但效率很低。β-半乳糖苷酶的几种不同底物包括神经节苷脂GM1、乳糖神经酰胺、乳糖和各种糖蛋白。它由人类GLB1基因编码，该基因位于第3号染色体p22上，包含16个外显子，全长62.5kb。野生型GLB1基因CDS序列如SEQ ID NO:9所示。
如本文中所用，术语“功能性β-半乳糖苷酶”(或者，等同于，功能性GLB1)是指具有全长野生型(天然)人β-半乳糖苷酶(例如NCBI Reference Sequence:NP_000395.3所示)的氨基酸序列的酶、其变体(例如，具有保守氨基酸置换的变体，或具有相当的或改进的功能的变体)、其片段，所述变体或片段提供至少约10％、至少约20％、至少约30％、至少约40％、 至少约50％、至少约60％、至少约70％、至少约75％、至少约80％、至少约90％、或大约相同、或大于100％的全长野生型(天然)β-半乳糖苷酶的生物活性水平。在一个实施方案中，本发明提供了一种功能性β-半乳糖苷酶变体酶，所述β-半乳糖苷酶变体酶为相对于天然存在的人野生型β-半乳糖苷酶(β-gal)包含氨基酸序列第299位或第245位或第353位的突变，优选地具有的为第299位的突变。在一个更具体的实施方案中，所述β-半乳糖苷酶变体酶相对于天然存在的人野生型β-半乳糖苷酶(GLB1)包含以下(i)-(iii)的氨基酸序列突变中的任一项：(i)在氨基酸序列第299位，由精氨酸突变为亮氨酸、丙氨酸、苯丙氨酸或谷氨酰胺；(ii)在氨基酸序列第245位，由甘氨酸突变为天冬氨酸；(iii)在氨基酸序列第353位的突变，由异亮氨酸突变为亮氨酸。在另一个实施方案中，本发明还提供了编码上述功能性β-半乳糖苷酶变体酶的核酸分子。
如本文中所用，术语“保守氨基酸置换”或“保守氨基酸取代”是指将氨基酸改变、置换或取代成具有相似生物化学性质(例如电荷、疏水性和大小)的不同氨基酸，这是本领域技术人员公知的。
如本文中所描述的，如果在野生型蛋白质的原始序列的基础上发生一个或多个氨基酸位点的突变(例如取代、缺失或添加)，那么具有这样的序列的蛋白相对于野生型蛋白质而言即被称为“变体”或“突变体”。所述突变对蛋白质的功能、活性产生的影响可能是提升的(即，例如，活性增加)、相当的(即，例如，活性基本类似)或减退或消除的(即，例如，功能异常或丧失)。在某些情况下，出于某些特定的目的，可以用变体蛋白代替天然的野生型蛋白进行基因治疗，只要所述变体蛋白的活性足以达到治疗效果，例如，用β-半乳糖苷酶变体代替野生型β-半乳糖苷酶对GM1神经节苷脂沉积病进行基因治疗。
在本发明的一个或多个实施方案中，通过使用带有核酸序列点突变的突变构建引物对进行PCR扩增，向所得到的GLB1基因序列编码区中引入设计的核苷酸替换，从而通过翻译突变的密码子而表达得到带有氨基酸序列点突变的β-半乳糖苷酶变体。相比于野生型β-半乳糖苷酶，酶变体可能具有减弱的、相当的、或提升的活性。
“密码子优化(codon-optimization)”是一种改进目的蛋白表达水平的技术，最初的指导思想是，密码子具有简并性，但多个同义密码子在细胞内所对应的tRNA的丰度是不同的，因此通过替换同义密码子，以更好地避免稀有密码子，利用偏爱密码子(Preferred codons)，即可以达到提升翻译效率从而提高表达水平的效果。在优化过程中，除了寻求利用更高丰度的tRNA，同时还应当尽可能地考虑简化mRNA的二级结构、优化重复序列、消除酶切位点、调整GC含量等因素，全面重新设计基因编码序列。尽管在将来自其他物种的编码序列在宿主细胞中表达的情形下会更倾向于使用密码子优化技术，但这一策略事实上对于绝大多数基因工程都是有益的。
“信号序列”是连接至蛋白质的N-端部分的氨基酸的序列，其促进蛋白质分泌至细胞外。细胞外蛋白质的成熟形式没有信号序列，其在分泌过程期间被切除。
术语“N端”指N端的最末氨基酸，术语“C端”指C端的最末氨基酸。
术语“有效连接”意指指定的各组分处于一种允许它们以预期的方式起作用的关系。
如下进行序列之间序列同一性的计算：为确定两个氨基酸序列或两个核酸序列的同一性百分数，将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方 案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在http://www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。在又一个优选的实施方案中，使用GCG软件包中的GAP程序(在http://www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum 62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4)，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
额外地或备选地，可以进一步使用本文所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。
术语“调控序列”或“表达控制序列”是指这样的核酸序列，其诱导、抑制或以其它方式控制与之有效连接的编码核酸序列的蛋白质转录。调控序列可以是例如起始序列、增强子序列、内含子序列和启动子序列等。
术语“CAR启动子”是指这样的元件，其由发明人将人CMV病毒的增强子序列进行结构优化而获得，避免了传统CMV启动子在肝脏沉默的特性，能够在包括肝脏在内的全身各部位和组织广泛表达。优选地，CAR启动子的核酸序列如序列SEQ ID No:1所示。
存在多种通过常规手段来测量β-半乳糖苷酶及其变体的表达和活性水平的测定法。参见，例如，本说明书实施例中所用的。
描述核酸或蛋白质时所用的术语“外源的”是指核酸或蛋白质不是天然存在于其存在的染色体或宿主细胞的位置。外源核酸序列也指衍生自并插入相同宿主细胞或受试者但以非天然状态存在的序列，例如，不同的拷贝数，或受不同调控元件的控制。
如本文中所用，术语“腺相关病毒(Adeno-associated virus，AAV)”因在腺病毒制品中发现而得名。AAV是微小病毒科(Parvovirus)成员，包含多种血清型，其基因组为单链DNA。
AAV是依赖性病毒，需要其它病毒如腺病毒、单纯疱疹病毒、人乳头瘤病毒、或辅助因素提供辅助功能蛋白才能复制。
最早分离到的AAV病毒是血清型2型AAV(AAV2)。AAV2基因组长约4.7kb，基因组两端为长度145bp的“反向末端重复序列”(inverted terminal repeat,ITR)，呈回文-发卡结构。基因组中有两个大开放阅读框(ORF)，分别编码Rep和Cap基因。已将AAV2的全长基因组克隆至大肠杆菌质粒中(Samulski RJ等人，Proc Natl Acad Sci USA.1982；79:2077-2081.Laughlin CA等人，Gene.1983；23:65-73)。
ITR是AAV载体基因组的顺式作用元件，在AAV病毒的整合、拯救、复制和基因组包装中发挥重要作用。ITR序列中包含Rep蛋白结合位点(Rep binding site，RBS)和末端解链位点trs(terminal resolution site)，能够被Rep蛋白结合识别并在trs处产生切口。ITR序列还可形成独特的“T”字母型二级结构，在AAV病毒的生活周期中发挥重要作用。
AAV2基因组其余部分可分为2个功能区，Rep基因区和Cap基因区。
Rep基因区编码Rep78、Rep68、Rep52和Rep40四种Rep蛋白。Rep蛋白对于AAV病毒的复制、整合、拯救和包装都具有重要作用。其中Rep78和Rep68与ITR中的末端解链位点trs和GAGY重复基序特异性结合，启动AAV基因组由单链向双链的复制过程。ITR中trs和GAGC重复基序和/或GAGY重复基序是AAV基因组复制的中心，因此虽然在各种血清型的AAV病毒中ITR序列都不尽相同，但是都能形成发卡结构和存在Rep结合位点。在AAV2基因组图谱位置19处有p19启动子，启动分别表达Rep52和Rep40。Rep52和Rep40具有ATP 依赖的DNA解旋酶活性，但没有结合DNA的功能。
Cap基因编码AAV病毒的衣壳蛋白VP1、VP2和VP3。其中，VP3分子量最小，但数量最多，在成熟的AAV颗粒中VP1、VP2、VP3的比例大致为1:1:10。VP1是形成有感染性的AAV所必需的；VP2协助VP3进入细胞核；VP3是组成AAV颗粒的主要蛋白。
如本文中所用，术语“AAV载体”是人们随着对AAV病毒生活周期及其相关分子生物学机制的了解，将野生型AAV病毒改造成的一种高效的外源基因转移工具，即AAV载体。改造后的AAV载体基因组中只包含AAV病毒的ITR序列和携带待转运的外源基因表达框。AAV病毒包装需要的Rep和Cap蛋白通过其他外源质粒反式提供，由此降低了Rep和Cap基因包装入AAV载体可能带来的危害。进一步地，AAV病毒本身不具有致病性，这使得AAV载体成为公认的最安全的病毒载体之一。删除AAV病毒的一侧ITR序列中的D序列和trs序列还能够使包装得到的重组AAV病毒载体所携带基因组自我互补，形成双链，显著提高AAV载体的体内外转导效率(Wang Z等人，Gene Ther.2003；10(26):2105-2111；McCarty DM等人，Gene Ther.2003；10(26):2112-2118)。包装得到的病毒成为scAAV(self-complementary AAV)病毒，即所谓的双链AAV病毒。它不同于双侧ITR均未突变的ssAAV(single-stranded AAV)，即传统的AAV病毒。
scAAV病毒载体的包装容量更小，仅为ssAAV病毒载体包装容量的一半，约为2.2kb-2.5kb，但感染细胞后转导效率更高。
AAV病毒血清型众多，不同的血清型具有不同的组织感染嗜性，因此应用AAV载体能够将外源基因转运至特定的器官和组织(Wu Z等人，Mol Ther.2006；14(3):316-327)。某些血清型AAV载体还可穿越血脑屏障，将外源基因导入至大脑神经元中，为靶向大脑的基因转导提供了可能(Samaranch L等人，Hum Gene Ther.2012；23(4):382-389)。
此外，AAV载体的理化性质稳定，对酸、碱和高温体现出较强的耐受性(Gruntman AM等人，Hum Gene Ther Methods.2015；26(2):71-76)，容易开发出稳定性较高的生物制品。
现有技术中对AAV载体具有相对成熟的包装系统，这便于规模化生产AAV载体。
目前常用的AAV载体包装系统主要包括三质粒共转染系统、腺病毒作为辅助病毒的系统、单纯疱疹病毒(Herpes simplex virus type 1，HSV1)作为辅助病毒的包装系统、以及基于杆状病毒的包装系统。每种包装系统都各具特点，本领域技术人员可以根据需要做出合适的选择。
三质粒转染包装系统因无需辅助病毒，安全性高，是应用最为广泛的AAV载体包装系统，也是目前国际上主流的生产系统。略显不足的是，高效大规模转染方法的缺失限制了三质粒转染系统在AAV载体大规模制备中的应用。
Yuan等建立以腺病毒为辅助病毒的AAV大规模包装系统(Yuan Z等人，Hum Gene Ther.2011；22(5):613-624)，该系统生产效率高，但包装系统中腺病毒在最后AAV成品中的痕量存在，影响了AAV成品的安全性。
HSV1作为辅助病毒的包装系统是另一类应用较为广泛的AAV载体包装系统。伍志坚和Conway等几乎同时在国际上提出了以HSV1为辅助病毒的AAV2载体包装策略(伍志坚，吴小兵等，科学通报，1999，44(5)：506-509；Conway JE等人，Gene Ther.1999，6：986-993)。随后Wustner等提出了以HSV1为辅助病毒的AAV5载体包装策略(Wustner JT等人，Mol Ther.2002，6(4)：510-518)。在此基础上，Booth等利用两个HSV1分别携带AAV的Rep/Cap基因和AAV的反向末端序列(Inverted terminal repeat，ITR)/外源基因表达框，然后用这两个重组HSV1病毒共同感染生产细胞，包装产生AAV病毒(Booth MJ等人，Gene Ther.2004；11:829-837)。Thomas等进一步建立双HSV1病毒AAV生产的悬浮细胞系统(Thomas DL等人，Gene Ther.2009；20:861-870)，使更大规模的AAV病毒生产成为可能。
Urabe等利用三个杆状病毒分别携带AAV的结构基因、非结构基因和ITR/外源基因表达框，构建了AAV载体的杆状病毒包装系统。考虑到杆状病毒携带外源基因的不稳定性，随后减 少了生产系统中所需杆状病毒的个数，逐渐从最开始的需要三个杆状病毒到需要两个或一个杆状病毒(Chen H.，Mol Ther.2008，16(5)：924-930；Galibert L.等人，J Invertebr Pathol.2011；107 Suppl:S80-93)以及一个杆状病毒组合一株诱导细胞株策略(Mietzsch M等人，Hum Gene Ther.2014；25:212-222，Mietzsch M等人，Hum Gene Ther.2015；26(10):688-697)。
由于上述特点，AAV载体逐渐成为一种广泛应用于基因治疗，特别是遗传病的基因治疗的外源基因转运工具。截至2016年8月，世界上批准的基于AAV载体的基因治疗临床试验方案有173项(http://www.abedia.com/wiley/vectors.php)。更为重要的是，基于AAV载体的脂蛋白脂酶基因治疗药物Glybera已于2012年被欧洲药监局批准上市，成为西方世界批准的第一个基因治疗药物(
Figure PCTCN2022095638-appb-000001
S.，Mol Ther.2012；20(10):1831-1832)；血友病B(Kay MA等人，Nat Genet.2000；24(3):257-261)和先天性黑蒙症(RPE65基因突变引起)(Jacobson SG等人，Arch Ophthalmol.2012；130(1):9-24)的AAV载体基因治疗药物均取得不错的临床试验效果，预期在不久的将来会上市销售，造福广大患者。
术语“载体基因组(vg)”是指包装在rAAV衣壳内形成rAAV载体的核酸序列。在一个实施方案中，载体基因组至少包含5’至3’的AAV2 5’ITR、编码功能性GLB1的核酸序列和AAV2 3’ITR。也可以选择来自除AAV2以外的不同来源AAV的ITR。此外，载体基因组可以包含指导功能性GLB1表达的调控序列。
如本文中所用，术语“miRNA(microRNA)”是指广泛存在于人类和动物体内的长度为18至25个核苷酸(nucleotide，nt)的单链非编码RNA。
1993年miRNA首先在秀丽隐杆线虫(C.elegans)中发现。秀丽隐杆线虫中lin-4基因能够下调lin-14基因的表达，但lin-4基因的编码产物不是蛋白质，而是一种小RNA分子，这表明自身编码的小RNA分子能够调节基因的表达。随后，多种类似的小RNA分子在不同的物种和细胞中相继发现，miRNA开始成为该类小RNA的统称。
miRNA调节人类大约60％基因的表达(Friedman RC等人，Genome Res.2009；19:92-105)，在多种生理和病理过程中发挥重要作用。
miRNA基因通常位于基因组的外显子、内含子和基因间区中(Olena AF等人，J Cell Physiol.2010；222:540-545；Kim VN等人，Trends Genet.2006；22:165-173)。在细胞内，miRNA的产生过程如下所述。首先，在细胞核中，miRNA基因由RNA聚合酶II或III启动转录产生初始产物pri-microRNA；pri-microRNA自我折叠部分序列形成茎环结构。随后，由核糖核酸酶III Drosha和DGCR8分子组成的加工复合体作用于pri-microRNA，切去多余序列，留下60nt左右的茎环结构，即前体miRNA分子pre-microRNA。然后，在转运蛋白Exportin-5的协助下，pre-microRNA从细胞核进入细胞质中，经Dicer酶加工去掉其茎环结构中的环形部分，变为双链RNA分子。最后，双链RNA分子被AGO2等蛋白因子结合，其中一条链发生降解，另一条链和蛋白因子形成RNA诱导的沉默复合物(RNA induced silencing complex，RISC)。RISC识别mRNA中的靶序列，通过降解mRNA分子、促进mRNA分子3’端去腺苷化和抑制翻译来降低mRNA的表达水平，在转录后水平调节基因的表达(Fabian MR等人，Annu Rev Biochem.2010；79:351-379)。因此，利用细胞内高表达的miRNA，在外源基因的3’UTR(untranslated region)插入该miRNA的靶序列，能够有效地抑制外源基因在导入细胞中的表达。
miR-142-3p是一种miRNA，其在造血干细胞系来源细胞中高表达。
免疫细胞均分化来源于造血干细胞系，因此利用miRNA抑制基因表达的原理(Kim VN.Nat Rev Mol Cell Biol.2005；6(5):376-385)，携带miR-142-3p靶序列的基因表达会在免疫细胞中受到明显抑制，从而降低机体产生针对基因表达产物免疫反应的概率(Dismuke DJ等人，Curr Gene Ther.2013；13(6):434-452)。
β-半乳糖苷酶功能障碍通常由GLB1基因变异，导致无法表达的到正常序列的β-半乳糖苷酶前体蛋白，进而使得细胞内β-半乳糖苷酶活性显著下降甚至缺失，会导致其底物无法被降 解而异常沉积，例如，GM1神经节苷脂沉积，进而使得机体发生严重代谢和功能障碍，体现为严重的疾病，例如，GM1神经节苷脂沉积病。
术语“治疗”指意欲改变正在接受治疗的个体中疾病之天然过程的临床介入。想要的治疗效果包括但不限于防止疾病出现或复发、减轻症状、减小疾病的任何直接或间接病理学后果、降低病情进展速率、改善或缓和疾病状态，以及缓解或改善预后。对于GM1神经节苷脂贮积症，病情的严重程度与异常沉积的GM1神经节苷脂的总量正相关，与体内功能异常的β-gal的残留活性呈反相关。因此，包含本发明的β-半乳糖苷酶变体酶，和/或本发明的表达框的载体(例如但不限于，本发明的AAV载体)转入体内表达的外源性β-半乳糖苷酶将发挥代偿作用，降解异常沉积的GM1神经节苷脂，从而从一个或多个方面降低病情的严重程度，即发挥治疗作用。在某些实施方案中，异常沉积的GM1神经节苷脂得到有效降解，可表现为以下情形中的一项或多项：脑脊液(CSF)中的GLB1药效学和生物学活性增加；血清中的GLB1药效学和生物学活性增加；患者的平均寿命(存活期)延长；GM1神经节苷脂病的疾病进展延迟(通过达成时的年龄、丧失时的年龄以及维持或获取适合年龄的发育和运动里程碑的患者所占的百分比中的一个或多个进行评估)以及基于贝利婴幼儿发展量表的年龄当量认知、粗大运动、精细运动、接受性和表达性交流评分的变化、文兰适应性行为量表的每个域的标准评分的变化中的一个或多个的神经认知发展改善；吞咽困难、步态功能、运动技能、语言和/或呼吸功能得到改善；癫痫频率降低和/或癫痫发作年龄提高；24个月大时饲管独立的可能性提高。世界卫生组织提供了适合年龄的发育和运动里程碑的实例。参见例如Wijnhoven T.M.等人，(2004).WHO多中心生长参考研究中对粗大运动发展的评估(Assessment of gross motor development in the WHO Multicentre Growth Reference Study)《食物营养公报(Food Nutr Bull)》25(增刊1)：S37-45。在某些实施方案中，为了确定异常沉积的GM1神经节苷脂得到有效降解，可检测以下中的一项或多项：CSF和血清β-半乳糖苷酶活性、CSF GM1浓度以及血清和尿液硫酸角质素；脑MRI改变；肝和脾体积；EEG和视觉诱发电位(VEP)。在某些实施方案中，异常沉积的GM1神经节苷脂得到有效降解，可表现为以下情形中的一项或多项：寿命(存活期)延长；对饲管的需求减少；癫痫发生率、频率和时长降低以及癫痫发作延迟；生活质量改善，例如，如通过PedsQL进行测量；神经认知衰退的进展减慢和/或神经认知发展改善，例如，适应性行为、认知、语言(接受性和表达性交流)和运动功能(粗大运动、精细运动)发展改善或改善；达到运动里程碑的年龄较早并且丧失运动里程碑的年龄较晚；脑组织体积(大脑皮质和其它较小结构)和心室体积增大延迟、包含胼胝体、尾状核和壳状核以及小脑皮质的脑子结构的大小减小延迟以及脑萎缩和容量变化稳定；丘脑和基底神经节中异常T1/T2信号强度进展延迟；CSF和血清中的β-gal酶活性增加；CSF GM1浓度降低；血清和/或尿液硫酸角质素水平降低、己糖胺酶活性降低；脑中的炎性应答减少；异常肝和脾体积延迟；异常EEG和视觉诱发电位(VEP)延迟；和/或吞咽困难、步态功能、运动技能、语言和/或呼吸功能改善。在某些实施方案中，通过包含本发明的表达框的载体(例如但不限于，本发明的AAV载体)转入患者体内并表达的外源性β-半乳糖苷酶的活性可以高于、等于或低于正常个体体内的β-半乳糖苷酶的活性，并发挥代偿作用，降解异常沉积的GM1神经节苷脂。在进一步的实施方案中，通过包含本发明的表达框的载体(例如但不限于，本发明的AAV载体)转入患者体内并表达的外源性β-半乳糖苷酶的活性可以高于、等于或低于正常个体体内的β-半乳糖苷酶的活性，并发挥代偿作用，降解异常沉积的GM1神经节苷脂、但未使得患者体内(例如，中枢，外周)的GM1神经节苷脂的残留量低至正常个体体内的GM1神经节苷脂的含量或与其相当，但仍然足以消除或部分消除未经治疗时的临床症状，并使得病情得以缓解。
用于本文时，“预防”包括对疾病或特定疾病的症状的发生或发展的阻止或抑制。在一些实施方式中，具有GLB1基因异常的受试者，或处于高水平的此类风险之中的受试者，或处于高水平的GM1神经节苷脂异常沉积的风险之中的受试者，或已经有相对较高水平的GM1神 经节苷脂异常沉积、但尚未表现出一定程度的临床症状的受试者，是预防性方案实施的候选对象。通常，术语“预防”是指在GM1神经节苷脂沉积病发生前，特别是在具有GLB1基因缺陷的受试者中于GM1神经节苷脂沉积病发生前的药物施用。
II.表达框
在一个方面，本发明提供了一种功能性人β-半乳糖苷酶的表达框，其包含编码功能性β-半乳糖苷酶的核苷酸序列，以及指导其表达的调控序列，例如上游调控序列和下游调控序列。
在一个实施方案中，表达框包含如本文所述的编码功能性β-半乳糖苷酶的核苷酸序列和指导其表达的调控序列。在一些实施方案中，所述功能性β-半乳糖苷酶的氨基酸序列是如SEQ ID NO:10-16任一项所示的序列。在又一个实施方案中，编码功能性β-半乳糖苷酶的核苷酸序列与SEQ ID NO:2或9具有至少70％、80％、90％的同一性，例如，具有至少95％、96％、97％、98％、99％或更高、或100％的同一性。在另一些实施方案中，编码功能性β-半乳糖苷酶的核苷酸序列与SEQ ID NO:17、19、21、23、25或27具有至少70％、80％、90％的同一性，例如，具有至少95％、96％、97％、98％、99％或更高、或100％的同一性。
在另一个实施方案中，编码功能性GLB1的核苷酸序列在其5’端连接了SEQ ID NO:1所示的启动子序列，或者在其5’端连接了具有与SEQ ID NO:1具有至少约90％同一性(例如，具有至少95％、96％、97％、98％、99％或更高的同一性)的启动子序列的编码功能性β-半乳糖苷酶的核苷酸序列。
在一些实施方案中，编码功能性β-半乳糖苷酶的核苷酸序列选自
(i)编码如SEQ ID NO:10-16任一项所示的氨基酸序列的核苷酸序列；
(ii)如SEQ ID NO:2或9所示的核苷酸序列；
(iii)编码β-半乳糖苷酶变体酶(例如，包含氨基酸序列第299位或第245位或第353位的突变)的核苷酸序列，例如如SEQ ID NO:17、19、21、23、25、27或29所示的核苷酸序列；
(iv)与(i)-(iii)的核苷酸序列互补的核苷酸序列；
(v)与(i)-(iii)的核苷酸序列编码相同的β-半乳糖苷酶，但因遗传密码的简并性而与(i)-(iii)的核苷酸序列不同的核苷酸序列；或
(vi)与(i)-(iii)任一项所述的核苷酸序列具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高(例如99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％、99.9％)同一性的序列。
在一个实施方案中，指导功能性β-半乳糖苷酶表达的调控序列包含启动子，例如，鸡β-actin启动子、CMV启动子等。优选地，调控序列包含如SEQ ID NO:1所示的CAR启动子序列或者与其具有至少约90％同一性(例如，具有至少95％、96％、97％、98％、99％或更高同一性)的启动子序列。与使用CMV启动子相比较，使用所述CAR启动子能够避免CMV启动子进入体内(尤其是肝脏)表达易被甲基化沉默的缺点；相比于其他常用启动子，例如鸡β-actin启动子等，CAR启动子序列较短，利于病毒包装，因此表达量有显著提高。
在一个实施方案中，调控序列进一步包含可降低功能性β-半乳糖苷酶在免疫相关细胞(如抗原呈递细胞、巨噬细胞)中的表达，减弱功能性β-半乳糖苷酶表达带来的免疫反应的序列，由此显著降低针对外源性β-半乳糖苷酶蛋白发生免疫反应的概率，所述序列为例如一个或多个(例如，1－8个、2－7个、3个、4个、5个或6个)串联的与miR-142-3p互补的靶序列，由此，本发明的表达框转录得到编码功能性β-半乳糖苷酶的mRNA的非翻译区(例如，5’端非翻译区和/或3’端非翻译区)中含有人miR-142-3p靶序列，从而能够有效地抑制功能性GLB1在免疫相关细胞(如抗原呈递细胞)中的表达，抑制免疫反应。
在一个实施方案中，调控序列包含一个或多个表达增强子，例如，TBG增强子、CMV增强子等。
在一个实施方案中，调控序列包含聚腺苷酸化信号(polyA)，例如，人生长激素(hGH)聚腺苷酸化序列、SV40polyA、BGH polyA。在一个具体实施方案中，调控序列包含如SEQ ID NO:4所示的或者与其具有至少约90％同一性(例如，与其具有至少95％、96％、97％、98％、99％或更高同一性)的BGH polyA序列。在一个或多个具体的实施方案中，在聚腺苷酸化信号(polyA)的引导下，正确转录的mRNA尾部(3’端)上被加入数十-数百个腺苷酸。在一个具体的实施方案中，上述元件中的至少两个依次连接，构成本发明的表达框。
在一些优选的实施方案中，在本发明的表达框中，上述元件中的至少两个依次连接时，在元件的序列之间还具有间隔序列，也可称为连接序列、接头、连接子等。这些序列是为了方便进行克隆构建和后续鉴定、提高基因转录和翻译效率等目的而添加的，例如Kozak序列、内切酶作用位点、通用引物序列、批次标签等，只要其不干扰或抑制表达框的表达效果，不影响各个元件之间的协同作用，则这样的间隔序列被视为在本发明的技术方案的保护范围之内，并且无需刻意去除。本领域技术人员根据常规技术和公知常识能够知晓并使用这样的间隔序列。
在一个更具体的实施方案中，所述间隔序列的长度为0-20个核苷酸。在一个优选的实施方案中，所述间隔序列包含KpnI酶切位点GGTACC；在一个优选的实施方案中，所述间隔序列包含Kozak序列GCCACC；在一个优选的实施方案中，所述间隔序列包含EcoRI酶切位点GAATTC；在一个优选的实施方案中，所述间隔序列包含SalI酶切位点GTCGA；在一个优选的实施方案中，所述间隔序列包含KpnI酶切位点和Kozak序列的组合；在一个优选的实施方案中，所述间隔序列包含EcoRI酶切位点和SalI酶切位点的组合。
III.病毒载体
又在一个方面，本发明提供了一种病毒载体，其为人造的重组病毒颗粒，其中，包含编码功能性GLB1的表达框的复制缺陷型病毒基因组序列被包装在病毒衣壳或包膜中，从而所述重组病毒颗粒不能产生子代病毒体，但保留了感染靶细胞的能力。
在一个实施方案中，病毒载体的基因组序列不包含编码病毒复制所需的酶的基因，因此，认为在基因疗法中使用病毒载体是安全的，因为在不存在病毒复制所需的酶的情况下，子代病毒体的复制和感染是不会发生的。
本发明的重组病毒载体优选地可以是重组的腺相关病毒(AAV)，然而也可以是腺病毒、博卡病毒(Bocavirus)、AAV/博卡病毒杂交体、单纯疱疹病毒或慢病毒等递送基因同样有效的常用载体。
生产重组病毒载体(例如，重组AAV载体)的包装细胞系可以是原核细胞或真核细胞(例如，人类细胞、昆虫细胞或酵母细胞)，其含有通过任何方式(例如电穿孔、磷酸钙沉淀、显微注射、转化、病毒感染、转染和原生质体融合)引入细胞中的外源DNA。包装细胞系细胞包括但不限于大肠杆菌细胞、酵母细胞、人类细胞、非人类细胞、哺乳动物细胞、非哺乳动物细胞、昆虫细胞、HEK293细胞、肝细胞、肾细胞、神经胶质细胞、肿瘤细胞或干细胞。
如本文所用，术语“靶细胞”是指其中期望表达功能性β-半乳糖苷酶的靶细胞。靶细胞的示例包括但不限于中枢神经系统、心脏、肝脏、脾脏、肺脏、肾脏、肌肉中的功能细胞、支持性细胞、基质细胞等等，例如神经细胞、胶质细胞、心肌细胞、肝细胞。在一些实施方案中，将载体体内递送至靶细胞。
在一个优选的实施方案中，病毒载体是重组腺相关病毒(rAAV)载体，其包含AAV衣壳和包装在其中的载体基因组。rAAV载体用于治疗GLB1基因缺陷引起的疾病如GM1神经节苷脂沉积病。载体基因组包含AAV 5’反向末端重复序列(ITR)或AAV 5’ΔITR、编码功能性GLB1的核酸序列、指导GLB1在靶细胞中表达的调控序列、以及AAV 3’ITR或 AAV 3’ΔITR。所述ΔITR是删除了D序列和末端解链位点trs的ITR。ITR是在载体生产期间负责基因组的复制和包装的遗传元件，并且是产生rAAV所需的唯一病毒顺式元件。可以选择来自不同来源AAV的ITR。在一个实施方案中，ITR来自与病毒颗粒的衣壳不同的AAV。
在一个优选的实施方案中，当病毒载体包含本发明的人GLB1基因表达框时，所述表达框的两端各自与一段ITR序列连接。
除非另有说明，否则本文描述的AAV衣壳、ITR和其它AAV组分可以容易地从任何AAV中选择，包括但不限于通常被鉴定为AAV1、AAV2、AAV3B、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAVrh.10或它们的组合的血清型的AAV。在一个实施方案中，AAV衣壳是AAV5衣壳或其变体。在一个实施方案中，AAV衣壳是AAV3B衣壳或其变体。在一个实施方案中，AAV衣壳是AAV8衣壳或其变体。在一个实施方案中，AAV衣壳是AAV9衣壳或其变体。在某些实施方案中，衣壳蛋白由rAAV载体名称中术语“AAV”之后的数字或数字和字母的组合来指定。
在本发明的一个或多个实施方案中，本发明的重组病毒载体包含本发明的人GLB1基因表达框。
在一个实施方案中，提供一种这样的rAAV，其包含选自AAV血清型5(AAV5)、血清型3B(AAV3B)、血清型8(AAV8)、血清型9(AAV9)的衣壳；并且在其基因组中包含SEQ ID NO:2、5-9和17-28中任一项的或与SEQ ID NO:2、5-9和17-28中任一项具有至少约90％同一性的序列。
在本发明的一个或多个实施方案中，本发明的重组病毒载体在感染宿主细胞后表达β-半乳糖苷酶。
如本文中所用的，AAV“变体”是指衍生自已知的AAV序列的任何AAV序列，包括具有保守氨基酸置换的那些AAV序列，以及与AAV的氨基酸或核酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少97％、至少99％或更大的序列同一性的序列。在另一个实施方案中，AAV衣壳包括可以包含与任何描述或已知的AAV衣壳序列相比高达约10％的变异的变体。即，AAV衣壳与本文提供的和/或本领域已知的AAV衣壳具有约90％的同一性至约99.9％的同一性，约95％至约99％的同一性或约97％至约98％的同一性。在一个实施方案中，AAV衣壳与AAV衣壳变体具有至少95％、96％、97％、98％、99％或更高的同一性。当确定AAV衣壳的百分同一性时，可以对任何变体蛋白(例如vp1、vp2或vp3)进行比较。
IV.病毒载体的制备方法
本发明的重组腺相关病毒(AAV)载体可以使用已知的技术产生。此类方法涉及培养包装细胞，其包含编码AAV衣壳的核酸序列；功能性Rep基因；如本文描述的表达框，其侧接有AAV反向末端重复序列(ITR)或ΔITR；和足够的辅助功能，以允许将表达框包装到AAV衣壳蛋白中。
本文还提供了包装细胞，其包含编码AAV衣壳的核酸序列；功能性Rep基因；如本文描述的表达框，其侧接有AAV反向末端重复序列(ITR)或ΔITR；和足够的辅助功能，以允许将表达框包装到AAV衣壳蛋白中。在一个实施方案中，宿主细胞是HEK 293细胞。
可以利用本领域已知的产生rAAV的其它方法。合适的方法可以包括但不限于杆状病毒表达系统或通过酵母生产。
V.病毒载体的用途
本发明的病毒载体作为一种基因药物，能够通过静脉注射和/或脑室内注射，在全身尤其是中枢神经系统、心脏、肝脏、脾脏、肺脏、肾脏、肌肉、血清等组织、器官和部位高效地、持续地、特异性表达预防和/或治疗严重GM1神经节苷脂沉积病的功能性GLB1。
通过静脉注射和/或脑室内注射本发明的病毒载体，在患者的不能正常表达GLB1基因的细胞中，表达外源性功能性β-半乳糖苷酶，且所表达的功能性β-半乳糖苷酶可持续、高效地实现对GM1神经节苷脂的降解，使其沉积消除，从而全部或部分地缓解或消除疾病的症状。并且一次给药即可持续降解体内的GM1神经节苷脂。
在一些实施方案中，本发明的病毒载体可以用于预防和/或治疗GM1神经节苷脂沉积病或其引起的疾病或与其相关联的疾病。在另一些实施方案中，本发明的病毒载体可以用于预防和/或治疗GLB1基因缺陷导致的GM1神经节苷脂沉积病或其引起的疾病或与其相关联的疾病。
VI.制备药物的用途
在一些实施方案中，本发明的表达框可以用于制备药物，所述药物用来预防和/或治疗GM1神经节苷脂沉积病或其引起的疾病或与其相关联的疾病，或者用来预防和/或治疗GLB1基因缺陷导致的GM1神经节苷脂沉积病或其引起的疾病或与其相关联的疾病。在一些优选的实施方案中，所述药物制备成通过口服、腹腔内、静脉和/或脑室给药等形式，更优选地是静脉和/或脑室给药的形式。
在另一些实施方案中，本发明的病毒载体可以用于制备药物，所述药物用来预防和/或治疗GM1神经节苷脂沉积病或其引起的疾病或与其相关联的疾病，或者用来预防和/或治疗GLB1基因缺陷导致的GM1神经节苷脂沉积病或其引起的疾病或与其相关联的疾病。在一些优选的实施方案中，所述药物制备成通过口服、腹腔内、静脉和/或脑室给药等形式，更优选地是静脉和/或脑室给药的形式。
在另一些实施方案中，本发明的功能性β-半乳糖苷酶及其变体酶、编码功能性β-半乳糖苷酶及其变体酶的核酸分子，可以用于制备药物，所述药物用来预防和/或治疗GM1神经节苷脂沉积病或其引起的疾病或与其相关联的疾病，或者用来预防和/或治疗GLB1基因缺陷导致的GM1神经节苷脂沉积病或其引起的疾病或与其相关联的疾病。
在一些实施方案中，本发明还提供了所得的药物组合物，其包含本发明的β-半乳糖苷酶及其功能性变体酶、核酸分子、优化的人GLB1基因表达框、病毒载体、基因药物、或上述的任意组合。
实施例
下面对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。如无特殊说明，实施例中涉及的各种反应试剂均可以通过商业渠道购买得到。
实施例1.β-半乳糖苷酶进行外源性替代的方案设计和克隆构建
我们初步设计了这样的GM1神经节苷脂沉积病的基因治疗方案：利用携带β-半乳糖苷酶编码基因的AAV载体在患者体内表达外源性β-半乳糖苷酶，替代/弥补患者体内功能缺失的自身β-半乳糖苷酶，发挥降解GM1神经节苷脂的作用。
为了验证该方案的可行性，我们首先在HEK-293细胞内表达外源性β-半乳糖苷酶，确认其功能活性相比于正常HEK-293细胞中β-半乳糖苷酶活性具有大幅提升的水平。
此外，我们还设计了并克隆了一些β-半乳糖苷酶变体，并同时验证了部分β-半乳糖苷酶变体的活性。
携带Rep和Cap蛋白表达基因的辅助质粒pRep2Cap9、辅助质粒pADHelper和HEK-293细胞来自北京瑞希罕见病基因治疗技术研究所；大肠杆菌DH5α感受态细胞购自北京擎科生物 科技有限公司。Lipofectamine2000为Invitrogen公司产品；4-甲基伞形酮-β-D-吡喃半乳糖苷(4-Methylumbelliferyl-β-D-galactopyranoside，4-MU-β-D-Gal)底物购自卡博森斯公司；4-甲基伞形酮(4-Methylumbelliferone，4-MU)标准品购自Sigma-Aldrich；β-半乳糖苷酶抗体购自Novus公司；β-Tublin抗体和辣根酶标记山羊抗小鼠IgG(H+L)购自中杉金桥公司；5-溴-4-氯-3-吲哚-β-D-半乳糖苷(X-gal)底物、铁氰化钾溶液和亚铁氰化钾溶液为北京索莱宝公司产品。
首先，以pUC57为基础质粒，引入了新霉素抗性neo基因和ITR片段，构建得到pRDAAV质粒载体。随后通过在pRDAAV质粒载体中插入本发明表达框或组成表达框的目的片段，得到携带表达天然型GLB1基因表达框的重组AAV9病毒质粒构建体，命名为rAAV9-CAR-coGLB1，其中包含的主要元件如下：
(1)ITR，AAV载体中唯一的来源于AAV基因组的顺式作用元件，是制备AAV载体的必需元件。
(2)CAR promoter，由人CMV病毒的增强子序列经结构优化组成，核酸序列如序列SEQ ID No:1所示。
(3)coGLB1，密码子优化的人源β-半乳糖苷酶CDS编码基因，核酸序列如序列SEQ ID No:2所示
(4)BGH polyA，牛生长激素的多聚核苷酸加尾信号，核酸序列如序列SEQ ID No:4所示。
包含上述(2)、(3)和(4)元件的GLB1表达框如SEQ ID No:5所示，其中在元件(2)、(3)之间包含了连接序列GGTACCGCCACC(KpnI酶切位点和Kozak序列)，在元件(3)、(4)之间包含了连接序列GAATTCGTCGA(EcoRI及SalI酶切位点)。
进一步地，以rAAV9-CAR-coGLB1构建体为基础，利用EcoRI及SalI酶切位点，以酶切连接的方式插入2x142-3P，将此构建体命名为rAAV9-CAR-coGLB1-2x142-3P，插入mir-142-3P靶序列的序列如SEQ ID No:3所示。
该构建体中的表达框序列如SEQ ID No:7所示。
表达变体β-gal酶的载体构建
比较生物学研究发现，为了适应不同的环境压力，不同物种对特定酶的活性有不同的需要。因此推测其他物种尤其是脊椎动物中可能存在更高催化活性的β-gal。为此，发明人比对了人和若干种脊椎动物的β-gal蛋白质氨基酸序列，寻找差异位点用于基因改造，以期得到更高催化活性的β-gal。根据从NCBI数据库下载获取的人(NP_000395.3)、斑马鱼(NP_001017547.1)、鼠(NP_033882.1)、猫(NP_001009860.1)、狗(NP_001032730.1)、猪(XP_020927212.1)、恒河猴(XP_028699438.1)的β-gal蛋白序列，使用Clustal X软件对人源β-gal 149位至522位和其他物种进行了多序列比对分析。排除文献中已报道的致病性突变位点，最终选择了位于β-gal TIM桶状结构域(残基1-359)的193、194、197、208、245、246、284、299和353位点及位于β结构域Ⅰ(残基397-514)的459和488位点进行定点突变，构建12个GLB1基因突变体。
方法为采用表1中的突变引物进行PCR扩增，利用基因同源重组的方法将rAAV9-CAR-coGLB1质粒中的coGLB1基因序列替换为携带突变位点的基因片段(mtGLB1)。通过基因测序鉴定重组质粒，测序结果显示已获得预期的GLB1突变体(结果略)。
表1突变体构建引物
Figure PCTCN2022095638-appb-000002
实施例2.构建体所表达的β-半乳糖苷酶及其功能性变体的活性验证
对于表达野生型β-半乳糖苷酶的构建体rAAV9-CAR-coGLB1，采用X-Gal染色法原位检测转染后细胞中的β-gal酶活性。
利用HEK-293为实验细胞，将rAAV9-CAR-coGLB1质粒进行体外转染。将HEK-293细胞均匀铺于六孔细胞培养板中的6个孔，待每孔的细胞密度达为60-70％时，取其中3孔利用Lipofectamine 2000(Invitrogen,美国)转染质粒，余下3孔细胞不转染，作为阴性对照孔。待转染48h后，对细胞进行X-Gal底物染色，验证构建的携带优化GLB1基因的质粒能够在体外转染中表达β-半乳糖苷酶蛋白。染色液工作液配制：1mg/mL X-gal，5mmol/L铁氰化钾，5mmol/L亚铁氰化钾，2mmol/LMgCl2，以PBS溶液稀释(pH＝4.5)，现用现配。
吸除细胞培养液，用1mL PBS溶液洗涤1次，加入1mL 4％PFA溶液，覆盖整个生长表面。室温固定15min。吸除4％PFA溶液，用1mL PBS(pH＝4.5)溶液洗涤细胞3次，每次3min。吸除PBS溶液，每孔加入1mL染色工作液，覆盖整个生长表面。37℃孵育6h～过夜。孵育完成后吸弃染色液，用1mL PBS溶液漂洗1次，光学显微镜下观察细胞蓝染情况。
X-Gal在β-半乳糖苷酶的催化下水解后呈蓝色。如图2所示，在显微镜明场下观察细胞，与阴性对照相比，转染细胞中的蓝色斑点显著增加(最明显处例如图中箭头所指位置)，证明rAAV9-CAR-coGLB1质粒能够有效的在体外细胞表达具有活性的β-半乳糖苷酶蛋白。
这一结果初步表明，本发明所设想的利用所设计的构建体进行基因治疗的方案是可行的。
进一步，利用X-Gal法测定实施例1中所获得的克隆构建体在细胞中的表达产物的酶活性。
方法如下：如之前所述的方法转染细胞。待转染48h后，收集细胞，采用反复冻融后离心的方式提取细胞总蛋白。利用Pierce BCA Protein Aaasy Kit(ThermoFisher,美国)分别测定转染rAAV9-CAR-coGLB1质粒、rAAV9-CAR-coGLB1-2x142-3P质粒及空白细胞的总蛋白浓度。根据结果，取细胞提取的全蛋白样本，按每孔30μL(约含5-10μg蛋白)，加入30μL 0.75mM 4-甲基伞形酮-β-D-吡喃半乳糖苷(4-MU-β-D-Gal)混匀。设置阴性对照，将30μL待测样品替换为30μL PBS缓冲液；37℃温育反应30min；吸取120μL在冰箱预冷的Na 2CO 3终止液加入反应体系终止反应，充分混匀后在激发光为360nm，发射光为460nm处，使用酶标仪测定游离4-甲基伞形酮荧光强度，将已知浓度的4-甲基伞形酮(Sigma-Millipore)标品使用Na 2CO 3终止液梯度稀释为标准曲线。以4-甲基伞形酮为标准计算酶活性，实验重复3次。β-gal酶活性以每毫克蛋白每小时生成的4-甲基伞形酮的量表示，即单位为nmol/mg·h。
结果如图4和图20(a)所示。
在图4中的柱形清楚表明体外转染rAAV9-CAR-coGLB1质粒能够大幅提高细胞内β-半乳糖苷酶的活性(近4倍)。
根据图20(a)，与未转染对照组相比，转染编码携带F193Y、A194T、R208H、S246N、I353L、N459L、Y488F突变的β-gal酶突变体的质粒虽然提高了该细胞中的β-半乳糖苷酶活性，但提升量显著低于转染编码野生型β-gal酶的细胞(均有P＜0.05)。F197Y、I284N和Y488S突变体过表达后，细胞酶活与未转染对照组相近(P>0.05)。过表达G245D突变体的细胞较转染pRDAAV-CAR-coGLB1的细胞活性无明显变化(P>0.05)，提示G245D突变对酶活性影响不大。过表达I353L突变体的细胞所展现出的β-半乳糖苷酶活性约为转染pRDAAV-CAR-coGLB1的细胞酶活性的50％-60％，但仍然远高于未转染对照组。而作为已知会降低酶活性的对照突变体N488Q，其过表达导致细胞酶活低于未转染细胞，和预期相符。突变体中活性最突出的为R299L，由其相应质粒转染的细胞中β-半乳糖苷酶活性相对于转染野生型GLB1的细胞提升了30％～40％，差异具有统计学显著性(p<0.00005)。
为了进一步验证突变体R299L具有更高的催化活性，HEK-293细胞分别转染了系列浓度梯度(每孔100、200、400、600、800ng)的pRDAAV-CAR-coGLB1和pRDAAV-CAR-coGLB1R299L质粒。转染48小时后按前述方法进行检测，结果显示(参见图20(b))，转染野生型和变体GLB1细胞中的β-gal酶活性分别与野生型(线性关系R2＝0.944)或突变体GLB1质粒(线性关系R2＝0.973)的转染量存在很好的线性关系，并且在浓度梯度中任一点值浓度下，过表达突变体β-gal酶R299L的细胞中的酶活性都高于野生型。这一结果进一步证实β-gal酶的突变体R299L具有更高的酶活性。
按照前述的元件设计和克隆步骤所构建的带有突变R299L、G245D和I353L的构建体分别是pRDAAV-CAR-coGLB1 R299L、pRDAAV-CAR-coGLB1 G245D和pRDAAV-CAR-coGLB1 I353L。三者的基因序列分别如SEQ ID No:17、25和27所示；相应的表达框序列分别如SEQ ID No:18、26和28所示。
实施例3 β-gal酶299位氨基酸取代变体的进一步验证
发明人根据β-gal酶变体R299L的活性测定结果进一步设想这一位点上的其他突变方式同样有希望获得高活性的β-gal酶变体。为此，按照实施例1所述方法构建了R299A、R299Q、R299F突变体质粒载体，引物序列如表2所示。将相同量编码野生型和变体GLB1质粒转染HEK-293细胞，48小时后比较β-gal酶活性和蛋白表达量。
表2突变体构建引物
Figure PCTCN2022095638-appb-000003
采用X-Gal染色法原位检测细胞中β-gal酶活性(方法步骤同实施例2)，结果参见图21(a)所示：转染R299L质粒组X-Gal阳性细胞率最高、染色最深，R299A组染色高于coGLB1组而略低于R299L组，R299Q、R299F组染色低于coGLB1组。另外，所有质粒转染组细胞X-gal染色都强于未转染的HEK-293阴性对照细胞。所有结果表明，我们改造所获得的突变体中，在299位的点突变往往有益于β-Gal酶活性的提升，其中R299L具有最高的β-Gal酶活性。
还实施了荧光底物法比较酶活性。方法如下：收获转染后48h的HEK-293细胞，提取总蛋白后定量样本蛋白浓度。取30μL 4-MU-β-D-Gal底物溶液(0.75mM，pH＝4.5)与30μL样本于37℃温育0.5h，加入120μL碳酸钠终止液(pH＝10.7)终止反应。将Perkin Elmer酶标仪设置为激发波长365nm，发射波长445nm，测定游离4-MU荧光强度。将已知浓度的4-MU标准品用碳酸钠终止液进行梯度稀释，以4-MU含量为横坐标，荧光值为纵坐标，绘制标准曲线。根据标准曲线计算样本生成的4-MU产物量。β-gal酶活性以每毫克蛋白每小时生成
Figure PCTCN2022095638-appb-000004
积，gL；T：反应时间，h；反应孔终止后4-MU产物浓度y(nmol/mL)。
结果如图21(c)所示，过表达变体酶R299A和R299L的细胞中酶活相近，都较coGLB1提升30％～40％左右，而过表达变体酶R299F与R299Q的细胞酶活则较coGLB1下降约20％，但显著高于未转染的HEK-293细胞酶活。荧光底物法的结果与X-Gal染色法的结果相符。
从酶活结果推断，299位点突变为直径更小的亮氨酸和丙氨酸时，可使β-gal酶活水平提升。而当299位氨基酸突变为具有极性侧链的谷氨酰胺时，其酶活水平与天然氨基酸序列的人β-半乳糖苷酶无显著差异。
为了验证蛋白表达量，我们采用了Western blot进行检测。
方法如下：HEK-293细胞转染48h后，提取细胞总蛋白，经12.5％SDS-聚丙烯酰胺凝胶电泳分离样品，每条泳道上样量10μg蛋白。电泳结束后将蛋白转移到PVDF膜上，与β-半乳糖苷酶抗体和β-Tublin内参抗体共同孵育。使用辣根过氧化物酶标记的羊抗鼠免疫球蛋白G(IgG)抗体作为二抗，在5％脱脂牛奶中进行杂交。与生物发光试剂反应，检测目的蛋白的表达。
结果如图21(b)所示，各转染组细胞中，β-半乳糖苷酶表达水平相近，且显著高于未转染的对照HEK-293细胞。携带coGLB1及其变体编码序列的质粒载体都能在细胞中正确表达β-半乳糖苷酶蛋白，85kDa大小的前体与64kDa大小的成熟体β-半乳糖苷酶条带存在于所有转染细胞样本中。结果提示外源基因成功过表达。
因此，过表达R299L与R299A变体的细胞中酶活升高不是蛋白表达量更高带来的，而是突变体催化活性提高的结果。
按照前述的元件设计和克隆步骤所构建的带有突变R299A、R299F和R299Q的构建体分别是pRDAAV-CAR-coGLB1 R299A、pRDAAV-CAR-coGLB1 R299F和pRDAAV-CAR-coGLB1 R299Q。三者的基因序列分别如SEQ ID No:19、21和23所示；相应的表达框序列分别如SEQ ID No:20、22和24所示。
实施例4 rAAV9-CAR-coGLB1病毒的制备及检定
重组AAV病毒的包装：携带Rep和Cap蛋白表达基因的辅助质粒pRep2Cap9、辅助质粒pADHelper和HEK-293细胞来自北京瑞希罕见病基因治疗技术研究所。HEK-293细胞扩大培养，在细胞生长至密度约为80％时，使用以构建的携带优化GLB1基因的AAV9载体质粒rAAV9-CAR-coGLB1以及辅助质粒pRep2Cap9和pADHelper共转染。转染48h后，收获细胞和培养上清，添加10U/mL浓度的DRase消化病毒外核酸。通过超滤浓缩的方式提升病毒浓度。纯化得到rAAV9-CAR-coGLB1浓缩病毒液。进行SDS-PAGE检测，结果参见图3所示，可见明显、清晰的特异性AAV病毒的衣壳蛋白VP1、VP2和VP3电泳条带。
实施例5 体外感染有效性
5.1病毒构建体活性测定
铺设HEK-293(ATCC CRL-1573)、U-87MG(ATCC HTB-14)、RAW264.7(ATCC TIB-71)三种不同种属、类型的细胞作为实验细胞于24孔板，置于CO 2生化培养箱。37℃培养直至细胞密度达到约60％-70％，将rAAV9-CAR-coGLB1病毒和rAAV9-CAR-coGLB1-2x142-3P病毒进行体外感染。每种细胞取rAAV9-CAR-coGLB1病毒感染3孔，取rAAV9-CAR-coGLB1-2x142-3P病毒感染另3孔，并余下3孔不感染作为阴性对照孔。病毒感染后6h向培养基中加入0.1％培养基体积的丁酸钠加强表达。
感染后48小时收取细胞，采用反复冻融后离心的方式提取细胞总蛋白。利用Pierce BCA Protein Aaasy Kit(ThermoFisher,美国)分别测定感染细胞、空白细胞的总蛋白溶液浓度，用于β-半乳糖苷酶的酶活检测实验，评估病毒的β-半乳糖苷酶体外表达效果。细胞蛋白提取方法和酶活检测实验方法同实施例2。
在提取细胞总蛋白后，将上述提取的蛋白分别取10μg用于测定β-半乳糖苷酶蛋白酶活。结果如图5所示，构建的病毒通过体外感染，能够在细胞内有效的表达β-半乳糖苷酶蛋白。比较两种病毒的感染结果，其中rAAV9-CAR-coGLB1病毒感染HEK-293、U-87MG、RAW264.7后都能极大提升细胞内β-半乳糖苷酶的酶活水平，符合预期。而rAAV9-CAR-coGLB1-2x142-3P病毒感染HEK-293、U-87MG细胞同样能够提升β-半乳糖苷酶酶活水平，且结果与rAAV9-CAR-coGLB1病毒相当，但在RAW264.7小鼠巨噬细胞中β-半乳糖苷酶酶活水平大幅降低，说明在治疗过程中加入miRNA 142-3P靶序列片段的病毒能够在广泛的体细胞和神经细胞中保留β-半乳糖苷酶的表达水平，而能大幅度降低在巨噬细胞中的转基因表达，降低免疫原性，提升治疗效果。
表3不同细胞系病毒载体感染实验酶活结果(单位：nmol/(mg·h)
Figure PCTCN2022095638-appb-000005
5.2 AAV介导的GLB1突变体与野生型GLB1体外表达活性比较
使用点杂交法得到病毒物理滴度(参见图22(a))rAAV9-coGLB1(2.75×10 13vg/mL)和rAAV9-coGLB1-R299L(1.83×10 13vg/mL)。设置三个MOI值(2×10 4、5×10 4和1×10 5)的病毒感染HEK-293细胞。
结果如图22(b)显示，相对于无病毒感染对照组，感染病毒后细胞β-gal酶活性升高。低感染复数(MOI)时rAAV9-coGLB1和rAAV9-coGLB1-R299L组酶活性均有少量提高，但无显著差异,据我们推测这一结果可能是由于MOI较小导致感染效率不足。相比之下，以中MOI感染的病毒显著提高了细胞酶活性，但随之再提高病毒感染复数则没有进一步提高酶活性。中高MOI感染时，rAAV9-coGLB1-R299L组酶活性比rAAV9-coGLB1组提升约30％，且两种病毒在高浓度组的活性达到了统计学显著性差异。这一结果表明，通过AAV9病毒载体表达的β-半乳糖苷酶R299L变体较野生型具有更高的酶活性。
实施例6 在模型小鼠体内有效性评价实验
6.1 GM1神经节苷脂贮积症模型鼠
应用CRISPR/cas9介导的Cyagen基因组工程技术，对小鼠Glb1基因座上与人coGLB1G453R相对应的p.G455R(NM_009752.2:c.1363g>a)进行了编辑。通过同源性定向修复将寡核苷酸G455R(GGA→AGA)突变位点导入外显子14，获得携带G455R点突变的GM1神经节苷脂贮积症模型鼠(该模型参见Liu S,Feng Y,Huang Y,et al.A GM1 gangliosidosis mutant mouse model exhibits activated microglia and disturbed autophagy[J].Experimental biology and medicine(Maywood,N.J.),2021,246(11):1330-1341.)。
6.2方法：小鼠处死取材及酶活检测具体步骤
(1)处死及取材：实验完毕后，将全部小鼠处死，摘眼球取血获得每只小鼠的血液，处理获得血清。解剖小鼠并提取关键组织心、肝、脾、肺、肾、肌肉、脑(可细分为皮质、丘脑、脑干、小脑)的蛋白样品。
(2)组织蛋白提取：加入PBS 2+和蛋白酶抑制剂匀浆组织，悬液放入-70℃冻结10min，取出后37℃水浴2min融化，冻融重复3次。4℃离心(12000rpm/10min)取上清即为组织蛋白溶液。
(3)血液样品处理：获得的血液放在非抗凝EP管内，室温静置3h，离心(3000rpm，离心10min)，得到的上清液即为血清，将上清移至另一清洁EP管保存。
(4)提取小鼠组织全蛋白，并采用Pierce BCA Protein Aaasy Kit(ThermoFisher,美国)分别测定各组织蛋白提取液浓度。将小鼠的不同组织提取液用PBS溶液稀释至浓度相近后测定酶活。
(5)酶活检测：取各组织样本30μL(约含5-10μg蛋白)，加30μL 0.75mM 4-甲基伞形酮-β-D-吡喃半乳糖苷(4-MU-β-D-Gal)混匀。设置阴性对照将30μL待测样品替换为30μL PBS 2+缓冲液；37℃温育反应30min；吸取120μL在冰箱预冷的NaCO 3终止液加入反应体系终止反应，充分混匀后在激发光为360nm，发射光为460nm处，使用酶标仪测定游离4-甲基伞形酮荧光强度，将已知浓度的4-甲基伞形酮(Sigma-Millipore)标品使用Na 2CO 3终止液梯度稀释为标准曲线。以4-甲基伞形酮为标准计算酶活性，实验重复3次。β-gal酶活性以每毫克蛋白每小时生成的4-甲基伞形酮的量表示，即单位为nmol/mg·h。
6.3颞浅静脉注射rAAV9-CAR-coGLB1病毒提高模型小鼠不同组织器官的β-半乳糖苷酶
本实验中GLB1基因点突变纯合突变的新生模型小鼠(P2)共12只，随机平均分为2组。1组作为实验组通过颞浅静脉注射rAAV9-CAR-coGLB1病毒，注射剂量为每只3.40×10 10vg 病毒载体。另1组作为阴性对照组，每只注射50μL PBS；并另取1组6只C57BL/6N野生型小鼠作为正常鼠对照组3。
注射病毒20周后，将全部小鼠处死，摘眼球取血获得每只小鼠的血液，处理获得血清。解剖小鼠并提取关键组织心、肝、脾、肺、肾、脑(可细分为皮质、丘脑、脑干、小脑)的蛋白样品。提取小鼠组织全蛋白，并采用Pierce BCA Protein Aaasy Kit(ThermoFisher,美国)分别测定各组织蛋白提取液浓度。
结果如图6、图9、图13、表4所示，表明我们设计的基因治疗药物经颞浅静脉注射治疗新生模型小鼠后，能够在多组织广泛有效地感染细胞并表达产生具有活性的β-半乳糖苷酶蛋白。其中非脑部组织β-半乳糖苷酶的提升程度大于脑部组织。
表4新生鼠IV治疗20周龄及野生型小鼠的外周组织及脑核团β-Gal酶活性检测结果(单位：nmol/(mg·h))
Figure PCTCN2022095638-appb-000006
6.4侧脑室注射rAAV9-CAR-coGLB1病毒提高模型小鼠不同组织器官的β-半乳糖苷酶
本实验中取GLB1基因点突变纯合突变的新生模型小鼠(P2)共18只，随机分为3组。组1共10只鼠，作为低剂量组通过侧脑室注射rAAV9-CAR-coGLB1病毒进行治疗，注射剂量为每只8.50×10 9vg病毒载体。组2共6只鼠，作为高剂量组通过侧脑室注射rAAV9-CAR-coGLB1病毒进行治疗，注射剂量为每只3.40×10 10vg病毒载体。组3共9只鼠作为阴性对照组，每只注射5μL PBS；并另取1组9只C57BL/6N野生型小鼠作为正常鼠对照组4。
注射病毒8周后，组1中4只鼠、组3中3只鼠、组4中3只鼠处死，摘眼球取血获得每只小鼠的血液，处理获得血清。解剖小鼠并提取关键组织心、肝、脾、肺、肾、脑(可细分为皮质、丘脑、脑干、小脑)的蛋白样品。具体方法同上文。
注射病毒16周后，将余下全部小鼠处死，组织取材和处理同上。
结果如图7、图10、图11、图14、图15和表5所示
表5新生鼠ICV治疗8周龄纯合突变模型及野生型小鼠的脑核团组织β-Gal酶活性检测结果(单位：nmol/(mg·h))
Figure PCTCN2022095638-appb-000007
Figure PCTCN2022095638-appb-000008
6.5侧脑室、静脉双途径注射rAAV9-CAR-coGLB1提高模型小鼠不同组织器官的β-半乳 糖苷酶
本实验中取GLB1基因点突变纯合突变的新生模型小鼠(P2)共12只，随机平均分为2组。组1作为实验组通过侧脑室加颞浅静脉双途径注射rAAV9-CAR-coGLB1病毒进行治疗，注射剂量为每只侧脑室注射8.50×10 9vg和颞浅静脉注射2.55×10 10vg病毒载体，共计3.40×10 10vg/只。组2作为阴性对照组，每只与实验鼠注射相同体积的PBS溶液；并另取1组6只C57BL/6N野生型小鼠作为正常鼠对照组3。
注射病毒8周后，将全部小鼠处死，摘眼球取血获得每只小鼠的血液，处理获得血清。解剖小鼠并提取关键组织心、肝、脾、肺、肾、脑(可细分为皮质、丘脑、脑干、小脑)的蛋白样品。具体方法同上文。
结果如图8、图12、图16和表6所示。
表6新生鼠ICV+IV双途径治疗8周龄纯合突变模型及野生型小鼠的脑核团组织β-Gal酶活性检测结果(单位：nmol/(mg·h))
Figure PCTCN2022095638-appb-000009
结果分析
野生型鼠在心、肝、脾、肺、肾、脑组织及血清中都能检测到远高于空白值的β-Gal酶活，但在肌肉中酶活数值较低。将实验鼠脑组织细分为小脑、脑干、皮质、丘脑核团分别检测时，发现不同核团间的酶活分布较为均匀。
作为未注射病毒的2月龄对照模型小鼠血清中的β-半乳糖苷酶的活性极低，几乎完全丧失。 而在外周组织器官中，肝、脾、肺、肾内的残余酶活同样显著低于同龄野生型小鼠，其中肺、肾酶活约只有野生型鼠的1％、脾酶活约为野生型鼠4％左右，肝酶活约为野生型鼠17％左右，但模型鼠心脏酶活只比野生型略低。脑核团中模型鼠的脑干、皮质、小脑和丘脑核团部分酶活都显著低于野生型对照鼠。
分析以上三种注射方式并比较其效果。如图7、图10、图11、图14、图15和表5所示，新生鼠侧脑室注射治疗药物后的模型小鼠，于2月龄处死解剖，其心、肝、脾、肺、肾、脑组织的β-半乳糖苷酶均有显著上升。在外周组织中与未注射病毒的模型小鼠相比，没有突出的酶活提高，仅在肝脏中表现出酶活的改善。比较血清酶活，未注射模型鼠血清酶活极低，而ICV治疗鼠的血清酶活提高了约6倍。脑组织核团中，ICV治疗鼠酶活有明显改善，提升最多的为小脑和丘脑部分，且超过了野生型鼠的脑组织酶活水平，但治疗鼠脑核团中酶活分布不如野生型鼠均匀，相较之下皮质部分酶活水平最高。
如图6、图9、图13、表4所示，新生鼠IV治疗的5月龄小鼠，在脑组织中，完全没有酶活改善，符合注射途径差异和随治疗时间推移酶活下降的客观规律。心、肝、脾、肺中治疗鼠的酶活有所改善。其中心脏酶活最高，为野生型鼠的3.5倍高，应为AAV9的转导特性所致。
两种注射方式治疗侧重点不一，但能够明确ICV治疗对脑部酶活改善的有效性，且对于血清酶活也有一定提高。IV注射主要对外周部分组织和血清酶活有所改善。
如图8、图12、图16和表6所示，侧脑室、静脉双途径注射是对于单途径注射方法的优化。新生鼠ICV注射AAV载体后，可以观察到目的基因在脑组织的高转导效率和高强度表达。比较之下IV注射对于中枢神经系统的转导效率和表达强度明显低于ICV注射，但在血清和心脏、肝脏、肾脏、脾脏这些重要组织器官中酶活提升效果非常显著，在对新生模型鼠侧脑室、静脉双途径注射治疗后2个月，小鼠外周组织酶活能够提升到接近野生型鼠的水平，其中心脏组织和脑组织酶活甚至高于野生型对照鼠。虽然以神经系统症状为主，但GM1神经节苷脂贮积症患者在神经系统及外周组织脏器中均有一定病理表现，而使用双途径注射的方法可兼顾对神经系统和外周重要脏器的保护，避免GM1神经节苷脂底物的贮积，比单一途径注射具有更广泛的治疗效果。
由以上结果同样可知，在本发明的基因替代治疗中，即使外源表达的酶活性低于野生型酶，同样能够通过持续降解累积底物而对患病的受试者起到治疗作用。
实施例7  尾静脉注射rAAV9-CAR-coGLB1病毒治疗4周龄模型小鼠
本实施例中取GLB1基因点突变纯合突变的4周龄模型小鼠共24只，随机平均分为3组。组1作为高剂量实验组通过尾静脉注射rAAV9-CAR-coGLB1病毒进行治疗，注射剂量为每只3×10 13vg/kg。组2作为低剂量实验组通过尾静脉注射rAAV9-CAR-coGLB1病毒进行治疗，注射剂量为每只8×10 13vg/kg。组3作为未注射病毒治疗的阴性对照组；并另取1组8只C57BL/6N野生型小鼠作为正常鼠对照组4。
7.1行为学测试
上肢悬吊实验(Forelimb suspension test)：将小鼠轻置于高架的金属丝上，确保小鼠上肢握住金属丝后开始计时，直到它们从杆子上掉下来或者达到最大试验时间10分钟。每只小鼠每次实验测量3次，每次测试间隔至少10分钟。取重复实验的最长时间记为上肢悬吊时间，作为每只鼠的最终测试结果。结果如图17左图所示。
转棒疲劳实验(Rotarod test)：使用加速转棒器测试每组鼠的运动能力。每次实验中，小鼠都要接受三次旋转实验。转棒转速会逐渐从5rpm加速到40rpm。当老鼠从转棒上掉落下或者在棒上的时间超过3分钟，则停止本次测试。每次测试间隔时间为15分钟。记录三次重复测试中的最长时间，作为每只鼠的最终结果。结果如图17右图所示。
7.2 rAAV9-CAR-coGLB1病毒对GM1小鼠神经节苷脂积累的影响
按如下步骤制备小鼠大脑切片
脑组织在4％多聚甲醛中固定24h以上，然后置于磷酸盐缓冲盐水(PBS)中。组织样品在脱水机上进行处理，使用JB-P5包埋机进行包埋，切成4微米切片，安装在玻片上，在烤箱中脱水。
按如下步骤进行Luxol Fast Blue(LFB)染色
切片脱蜡和复水处理:
切片放入二甲苯I 20min，二甲苯II 20min，无水乙醇I 5min，无水乙醇II 5min，75％乙醇5min，然后用自来水冲洗。将LFB染色溶液a在60℃的烤箱中预热30分钟。将载玻片浸入LFB染色溶液a中，用膜覆盖，温育1小时，并用自来水洗涤。将载玻片置于LFB染色液b中2s，直接浸入LFB染色液c中15s进行背景分化，然后洗涤终止反应。重复分化步骤，直到髓鞘染成蓝色，而在显微镜下观察到的背景几乎保持无色。将载玻片放于无水乙醇I溶液中5min，无水乙醇II溶液中5min，无水乙醇III溶液中5min，二甲苯I溶液中5min，二甲苯II溶液中5min。
TUNEL染色
脱蜡时，切片分别在2种不同浓度的二甲苯溶液中孵育20min，2种不同浓度的纯乙醇溶液中孵育10min，然后分别在95％、90％、80％和70％乙醇溶液中孵育5min。加入蛋白酶k工作液使组织完全覆盖，37°c孵育20min，用PBS(ph7.4)洗涤3次(每次5min)，室温下孵育20min，再用PBS(ph7.4)洗涤3次(每次5min)。稍微干燥后，将缓冲液加入到组织中，然后在室温下孵育10分钟。将含有TDT酶dUTP和缓冲液(TUNEL染色试剂盒)的溶液按1:5:50的比例加入到组织样品中，然后在37℃的湿箱中孵育1h。然后加入1:200的链霉亲和素-辣根过氧化物酶(HRP)和TBST混合物，使组织完全覆盖，切片放入37℃培养箱中30min。随后，将载玻片置于PBS(ph7.4)中，在脱色振荡器上洗涤三次，每次洗涤5分钟。稍微干燥。然后将DAB显色剂加入到组织中，并将细胞核用苏木精染色溶液复染1分钟。最后，将切片在纯水中洗涤，脱水，并置于二甲苯中以使组织透明。
如图18所示，我们观察到神经节苷脂1+NS小鼠脑髓鞘贮存蓝染。在高剂量(HD)和低剂量(LD)组小鼠中，大部分脑区的LFB染色基本正常，表明AAV基因治疗后的GM1小鼠神经节苷脂底物贮存清除效果良好。
免疫荧光
如上操作获得大脑切片，进行抗原修复3％牛血清白蛋白(BSA)50μL室温下封闭45min，加含0.3％Tween 20的3％BSA稀释的一抗GM1后置于湿盒，4℃过夜，洗涤后加人3％BSA稀释的荧光二抗抗体50μL，孵育60min，洗涤后使用0.1μg/ml DAPI 50μL染核，室温下5min。封片剂封片后激光共聚焦显微镜下拍照。
如图19所示，与LFB染色结果一致的是，未注射模型小鼠的免疫荧光染色显示出明显的GM1神经节苷脂积累。HD组和LD组小鼠GM1信号强度较弱。与未注射模型小鼠相比，HD和LD小鼠GM1的平均荧光密度显著降低(图2C)。治疗组小鼠脑组织神经节苷脂GM1水平不完全正常，但与治疗组小鼠相比有明显改善。
总的来说，rAAV9-CAR-coGLB1病毒能明显、持久地减少GM1神经节苷脂在模型小鼠脑内的贮积。

Claims (23)

  1. 一种功能性β-半乳糖苷酶变体酶，所述β-半乳糖苷酶变体酶相对于天然存在的人野生型β-半乳糖苷酶(β-gal)包含氨基酸序列第299位或第245位或第353位的突变，优选地包含第299位的突变。
  2. 如权利要求1所述的功能性β-半乳糖苷酶变体酶，其相对于天然存在的人野生型β-半乳糖苷酶包含以下(i)-(iii)的氨基酸序列突变中的任一项：
    (i)在氨基酸序列第299位，由精氨酸突变为亮氨酸、丙氨酸、苯丙氨酸或谷氨酰胺；
    (ii)在氨基酸序列第245位，由甘氨酸突变为天冬氨酸；
    (iii)在氨基酸序列第353位，由异亮氨酸突变为亮氨酸。
  3. 核酸分子，其编码如权利要求1或2所述的功能性β-半乳糖苷酶变体酶。
  4. 如权利要求3所述的核酸分子，其特征在于具有如SEQ ID NO:17、19、21、23、25或27所示的核苷酸序列，或因遗传密码的简并性而与之不同的核苷酸序列。
  5. 一种优化的人GLB1基因表达框，其包含：
    (1)如SEQ ID NO:1所示的启动子序列或者与其具有至少约90％同一性的启动子序列；和
    (2)编码功能性β-半乳糖苷酶的GLB1基因序列，所述功能性β-半乳糖苷酶为天然存在的人野生型β-半乳糖苷酶或如权利要求1所述的β-半乳糖苷酶变体酶。
  6. 如权利要求5所述的人GLB1基因表达框，其中(2)所述的编码功能性β-半乳糖苷酶的GLB1基因序列选自以下(i)-(vi)中任一项：
    (i)编码如SEQ ID NO:10-16任一项所示的氨基酸序列的核苷酸序列；
    (ii)如SEQ ID NO:2或9所示的核苷酸序列；
    (iii)与权利要求3或4的核酸分子相同的核苷酸序列；
    (iv)与如(i)-(iii)的核苷酸序列互补的核苷酸序列；
    (v)与(i)-(iii)的核苷酸序列编码相同的β-半乳糖苷酶，但因遗传密码的简并性而与(i)-(iii)的核苷酸序列不同的核苷酸序列；
    (vi)与(i)至(v)任一项所述的核苷酸序列具有至少70％、80％、90％、95％、96％、97％、98％、99％或更高同一性的序列。
  7. 根据权利要求5-6任一项所述的基因表达框，其中在相邻的元件(1)和(2)之间具有间隔序列，所述间隔序列的长度不超过20个核苷酸。
  8. 根据权利要求5-6任一项所述的优化的人GLB1基因表达框，其进一步包含(3)如SEQ ID NO:4所示的或者与其具有至少约90％同一性的BGH polyA序列。
  9. 根据权利要求8所述的基因表达框，其中在相邻的元件(1)和(2)之间和/或(2)和(3)之间具有间隔序列，所述间隔序列的长度不超过20个核苷酸。
  10. 根据权利要求5-6任一项或权利要求8所述的优化的人GLB1基因表达框，其进一步包含(4)至少一个(例如，1－5个)串联的SEQ ID NO:3所示的人miR-142-3p靶序列，例如，具有1个，或2或3个串联的人142-3P靶序列，即1x、2x或3x142-3P。
  11. 根据权利要求10所述的基因表达框，其中在相邻的元件(1)和(2)之间、(2)和(3)之间、和/或(3)和(4)之间，具有间隔序列，所述间隔序列的长度不超过20个核苷酸。
  12. 根据权利要求5-9任一项所述的基因表达框，其特征在于，其核酸序列如SEQ ID NO:5、18、20、22、24、26或28所示或与SEQ ID NO:5、18、20、22、24、26或28具有至少约90％同一性。
  13. 根据权利要求10-11任一项所述的基因表达框，其特征在于，其核酸序列如SEQ ID NO:7所示或与SEQ ID NO:7具有至少约90％同一性。
  14. 一种病毒载体，其特征在于，包含权利要求5-13中任一项所述的优化的人GLB1基因表达框。
  15. 根据权利要求14所述的病毒载体，其特征在于，其包含SEQ ID NO:2、5-9和17-28中任一条序列，或包含与SEQ ID NO:2、5-9和17-28中任一条具有至少约90％同一性的序列并且能够在感染宿主细胞后表达功能性β-半乳糖苷酶。
  16. 一种用于预防和/或治疗GM1神经节苷脂沉积病的基因药物，其包含权利要求5-13中任一项所述的优化的人GLB1基因表达框或权利要求14或15所述的病毒载体，并且所述基因药物是通过静脉注射和/或脑室内注射到体内以特异性表达人β-半乳糖苷酶来达到预防和/或治疗GM1神经节苷脂沉积病的目的。
  17. 根据权利要求14或15所述的病毒载体或根据权利要求16所述的基因药物，其特征在于，
    (1)所述病毒载体是重组腺相关病毒载体；和/或
    (2)所述病毒载体的基因组可自我互补形成双链DNA分子。
  18. 根据权利要求17所述的病毒载体或基因药物，其特征在于，所述重组腺相关病毒载体是选自AAV1、AAV2、AAV3B、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAVrh.10或它们的组合的血清型的重组腺相关病毒载体，优选的血清型为AAV5、AAV3B、AAV8、AAV9。
  19. 根据权利要求1或2所述的功能性β-半乳糖苷酶变体酶、根据权利要求3或4所述的核酸分子、根据权利要求5-13中任一项所述的优化的人GLB1基因表达框或根据权利要求14、15、17和18中任一项所述的病毒载体的用途，用于制备预防和/或治疗GM1神经节苷脂沉积病和/或其他疾病的药物。
  20. 根据权利要求19所述的用途，其特征在于，所述其他疾病是由GM1神经节苷脂沉积病引起的疾病或与GM1神经节苷脂沉积病相关联的疾病。
  21. 根据权利要求19所述的用途，其特征在于，所述药物的给药方式为静脉注射和/或脑室内注射。
  22. 根据权利要求19-21任一项所述的用途，其特征在于，所述药物的一次给药可持续降解体内的GM1神经节苷脂。
  23. 一种药物组合物，其包含权利要求1或2的功能性β-半乳糖苷酶变体酶、权利要求3或4的核酸分子、权利要求5-13中任一项的优化的人GLB1基因表达框、权利要求14、15、17和18中任一项的病毒载体、权利要求16-18中任一项的基因药物或其任意组合。
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