WO2023184688A1 - VARIANT FONCTIONNEL DE β-GALACTOSIDASE, VECTEUR D'EXPRESSION DE β-GALACTOSIDASE HUMAINE MÉDIÉ PAR VAA ET UTILISATION ASSOCIÉE - Google Patents

VARIANT FONCTIONNEL DE β-GALACTOSIDASE, VECTEUR D'EXPRESSION DE β-GALACTOSIDASE HUMAINE MÉDIÉ PAR VAA ET UTILISATION ASSOCIÉE 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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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

L'invention concerne une β-galactosidase fonctionnelle, telle qu'un variant fonctionnel de la β-galactosidase humaine, et son utilisation pharmaceutique. L'invention concerne également une cassette d'expression de gène de β-galactosidase humaine optimisée, un vecteur de virus adéno-associé recombinant qui porte la cassette d'expression, un procédé de préparation correspondant et une utilisation pharmaceutique associée. La cassette d'expression peut exprimer efficacement la β-galactosidase humaine fonctionnelle in vivo. Après l'administration d'un médicament comprenant la cassette d'expression ou le vecteur viral par injection intraventriculaire et injection intraveineuse, la β-galactosidase est exprimée en continu dans l'ensemble du corps, ce qui permet de dégrader de manière efficace le substrat de ganglioside GM1 avec une accumulation aberrante et d'atténuer et d'éliminer les symptômes de maladie.
PCT/CN2022/095638 2022-03-31 2022-05-27 VARIANT FONCTIONNEL DE β-GALACTOSIDASE, VECTEUR D'EXPRESSION DE β-GALACTOSIDASE HUMAINE MÉDIÉ PAR VAA ET UTILISATION ASSOCIÉE WO2023184688A1 (fr)

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