WO2023035687A1 - 用于治疗庞贝氏病的基因治疗构建体、药物组合物和方法 - Google Patents

用于治疗庞贝氏病的基因治疗构建体、药物组合物和方法 Download PDF

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WO2023035687A1
WO2023035687A1 PCT/CN2022/095637 CN2022095637W WO2023035687A1 WO 2023035687 A1 WO2023035687 A1 WO 2023035687A1 CN 2022095637 W CN2022095637 W CN 2022095637W WO 2023035687 A1 WO2023035687 A1 WO 2023035687A1
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sequence
vector
gaa
promoter
recombinant aav
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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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Definitions

  • the present invention relates to gene therapy, more specifically to a construct for gene therapy of Pompe disease, a pharmaceutical composition comprising the construct and a method for treating Pompe disease.
  • the key to gene therapy drugs is to use appropriate carrier materials to transfer foreign genes into recipient cells, and achieve the purpose of disease treatment through transcription and expression.
  • the commonly used gene therapy vectors mainly include viral and non-viral vectors.
  • Viral vectors are widely used due to their natural characteristics, which can efficiently introduce foreign genes into recipient cells.
  • adeno-associated virus (AAV) vectors have good safety and high-efficiency transfection of various target tissues. Guide, become one of the most active carriers of gene therapy in vivo.
  • the promoter can determine the expression efficiency and tissue expression profile of the cloned gene to a large extent. Therefore, in the field of gene therapy, in order to meet the needs of gene therapy, it is often necessary to construct new promoters that meet the needs based on specific therapeutic purposes. At the same time, there is also an objective need to provide a variety of promoter options in this field.
  • the gene capacity of viral vectors as transgene delivery tools is limited.
  • the total packaging capacity is approximately 4.8 kb.
  • the total packaging capacity is approximately half that, approximately 2.5 kb. Therefore, in consideration of the size of the gene construct, it is particularly important to select an appropriate combination of vector gene elements to ensure that the target gene is expressed at an appropriate level in the desired tissue (or multiple tissues).
  • constitutive expression of the transgene in all or most cell types is desired, for example, when the disease or condition being treated affects multiple tissues.
  • Some constitutive promoters have been proposed in the art, such as human elongation factor 1, cytomegalovirus promoter CMV, chicken actin promoter CBA, and synthetic CAG promoter containing CMV enhancer, etc.
  • human elongation factor 1 cytomegalovirus promoter CMV
  • CBA cytomegalovirus promoter
  • synthetic CAG promoter containing CMV enhancer etc.
  • the effect of using constitutive promoters often varies due to factors such as specific applied diseases or diseased tissues, administration methods, etc., and in some cases may bring about higher drug immunogenicity and/or animal toxicity , thereby limiting the application of gene therapy drug constructs.
  • gene therapy there is a continuing need to provide safer gene therapy constructs suitable for more efficient transduction of disease-associated tissues.
  • Pompe disease also known as acid ⁇ -glucosidase deficiency or glycogen storage disease type II (GSD II), is a systemic lysosomal storage disease that mainly affects the muscles and also affects the central nervous system.
  • GAA functional acid ⁇ -glucosidase
  • lysosomes resulting in the inability of glycogen to be converted into glucose for utilization, resulting in glycogen in the lysosomes of cells in the patient's body, especially in the It is caused by intracellular accumulation in peripheral organ tissues such as skeletal muscle and cardiac muscle and central nervous system (including brain and spinal cord).
  • Pompe disease can be diagnosed by enzymatic activity detection, which detects the activity of ⁇ -glucosidase.
  • Pompe disease can be divided into: infantile; and late-onset.
  • Individuals with infantile-onset Ponzi's disease have extremely low residual GAA enzyme activity, and show more severe symptoms such as dyspnea, generalized muscle weakness, and cardiopulmonary failure, and are often fatal.
  • Individuals with childhood-to-adult-onset Pompe disease have slower disease progression due to higher residual GAA enzyme activity.
  • This milder form of Pompe disease is also called late-onset Pompe disease (LOPD).
  • LOPD late-onset Pompe disease
  • Heart muscle defects are often absent in individuals with LOPD, but muscle weakness can lead to severe breathing problems and respiratory failure.
  • ERT enzyme replacement therapy
  • ERT has the advantage of continuously improving cardiac dysfunction and preventing heart failure.
  • ERT has shown limitations with regard to the affected skeletal muscle and CNS system. Individual patients receiving ERT can have widely variable skeletal muscle responses. One of the factors for this variability in response is thought to be possibly related to the formation of high titers of anti-drug antibodies.
  • Studies in animals and humans have suggested that antibodies formed against the GAA enzyme can reduce the efficacy of ERT.
  • ERT drugs cannot cross the blood-brain barrier and cannot treat CNS lesions and affected respiratory motor neurons. Severe progressive neurodegeneration has been reported in individual infants receiving ERT. Brain MRI studies in long-term survivors of ERT also revealed slowly progressive white matter damage.
  • a further limitation of ERT is the complete lack or only insufficient clearance of glycogen in certain tissue types, such as smooth muscle of the blood vessels, eye, gastrointestinal tract and respiratory system.
  • Pompe disease as an autosomal recessive monogenic disorder, is caused by pathological mutations in the acid ⁇ -glucosidase (GAA) gene (including various nonsense mutations that lead to loss or reduction of GAA enzyme activity mutations and missense mutations). Therefore, as an alternative or supplement to ERT, gene therapy approaches have been proposed to overcome GAA gene defects in individual patients.
  • GAA acid ⁇ -glucosidase
  • Darin J Falk et al. 2013, Intrapleural Administration of AAV9 Improves Neural and Cardiorespiratory Function in Pompe Disease, doi:10.1038/mt.2013.96
  • AAV9 to carry the recombinant GAA gene under the control of the constitutive promoter CMV and the tissue-specific promoter DES , to treat Pompe disease mice by intrathoracic injection.
  • the results showed that GAA enzyme activity was increased in the heart, but almost no GAA enzyme activity was detected in the liver.
  • Enyu Deng et al. (MOLECULAR THERAPY Vol.5, No.4, 2002; doi:10.1006/mthe.2002.0563) used an AAV vector (Ad CMV-GAA) carrying a constitutive promoter CMV and a recombinant GAA gene, by intravenous injection Mice to treat Pompe disease.
  • Ad CMV-GAA Ad CMV-GAA carrying a constitutive promoter CMV and a recombinant GAA gene
  • both vectors efficiently transduced the heart, resulting in glycogen clearance, and transduction of the diaphragm and central nervous system observed on tissue sections. guide.
  • AAVB1-treated mice showed stable weight gain and recovery of limb strength.
  • the liver GAA levels of AAV-treated animals were significantly lower than those of wild-type, and GAA levels in the trachea, medulla, neck, thoracic and GAA activity in the lumbar spinal cord were below the detection limit of the enzyme assay.
  • the viral capsid PHP.B was used to construct AAV viral vector, glycogen content was reduced to wild-type levels in brain and heart and significantly reduced in skeletal muscle after a single intravenous injection of AAV-PHP.B-CB-GAA in 2-week-old GAA KO mice .
  • the transduction efficiency of PHP.B-CB-hGAA was sufficient to prevent the accumulation of glycogen in the brain of GAAKO mice and rescue the associated neural phenotypes.
  • this unusually high CNS targeting of the PHP.B capsid was limited to a specific transgenic mouse model.
  • the inventors After in-depth research, the inventors have proposed a new artificially synthesized constitutive promoter that can be used to reduce the central nervous system burden of Pompe disease and correct peripheral organ involvement after intravenous injection, and has low drug immunogenicity. Novel AAV viral vectors for said promoters, and uses thereof.
  • the invention provides a mutant promoter comprising SEQ ID NO: 4 or a polynucleotide having at least 95% identity or one or several nucleotide changes to SEQ ID NO: 4, And the polynucleotide has a mutation from T to C or G or A, especially T to C, in position 562-572 of SEQ ID NO:4, preferably position 568.
  • the mutated promoters of the present invention increase the expression of a gene of interest to which it is functionally linked relative to an unmutated reference promoter, especially in mammalian cells or tissues.
  • the strong promoter activity of the mutant promoters of the present invention makes them particularly suitable for therapeutic use in Pompe disease.
  • the present invention provides expression constructs, vectors, host cells, and pharmaceutical compositions thereof comprising the mutant promoters of the present invention.
  • the present invention provides a recombinant AAV viral vector comprising a mutant promoter of the present invention and a polynucleotide encoding acid alpha glucosidase GAA.
  • the viral vectors of the present invention may be ssAAV or scAAV viral vectors.
  • the viral vector of the present invention comprises an AAV capsid protein with muscle and/or nervous system targeting, such as an AAV9 serotype capsid protein.
  • the present invention provides the application method of the recombinant virus vector of the present invention in the treatment or prevention of said disease or defect in Pompe disease or a subject with acid glucosidase deficiency, and also provides a method for preparing Use in a medicament for the prophylaxis or treatment of said disease or deficiency.
  • the methods of the invention result in increased levels of GAA enzyme activity and decreased glycogen storage in peripheral and central nervous system tissues of the subject.
  • the central nervous system burden of Pompe disease and the peripheral organ involvement can be corrected advantageously after intravenous injection, and at the same time, it has the advantage of low drug immunogenicity.
  • Figures 1A-1D show schematic diagrams of the pscAAV-CAR-Gluc vector, pscAAV-CAR-MutC-Gluc vector, pscAAV-CAR-MutA-Gluc vector, and pscAAV-CAR-MutG-Gluc vector, respectively.
  • Figure 2 shows that, in the cell test in vitro, compared with BHK-21 cells without transfection plasmid (i.e., blank control), the pscAAV-CAR-Gluc vector and the pscAAV-CAR-MutC-Gluc vector were transfected , pscAAV-CAR-MutA-Gluc vector, and pscAAV-CAR-MutG-Gluc vector BHK-21 cells, the measured Gluc level changes.
  • ** means p ⁇ 0.01.
  • Figures 3A-3C show, respectively, after IV injection of mice with recombinant AAV vector carrying CAR and CAR-Mut promoter (SEQ ID NO: 1), dissected mouse brain tissue (Fig. 3A), heart tissue (Fig. 3B) and Gluc levels detected in liver tissue (Fig. 3C).
  • ** means p ⁇ 0.01.
  • Figures 4A-4D show schematic diagrams of the structures of pRDAAV-CMV-EGFP vector, pRDAAV-CAR-Mut-EGFP vector, pRDAAV-CAR-Mut-coGAA vector, and pRDAAV-CAR-Mut-coGAA-2 ⁇ 142-3P vector, respectively.
  • Figure 5 shows the level of GAA enzyme activity measured in virus-infected and non-infected cells in an in vitro cultured cell assay.
  • BHK cells BHK-21 blank cells not infected with virus;
  • rAAV9-CAR-Mut-coGAA-142-3p BHK-21 cells infected with recombinant AAV9 virus rAAV9-CAR-Mut-coGAA-2 ⁇ 142-3P;
  • ** indicates p ⁇ 0.01.
  • Figure 6 shows that in the in vivo evaluation experiment of GAA -/- model mice, after a single IV injection of recombinant AAV9 virus rAAV9-CAR-Mut-coGAA-2 ⁇ 142-3P, the dissected mouse heart, liver, GAA enzyme activities detected in muscle, kidney, lung, and spleen tissues.
  • Model control group model mice injected with PBS, as a negative control; low dose group: model mice injected with 5E12vg/kg recombinant AAV9 virus; medium dose group: model mice injected with 1.1E13vg/kg recombinant AAV9 virus; high dose Group: model mice injected with 3E13vg/kg recombinant AAV9 virus; wild-type control group: 129 wild-type mice.
  • FIG. 7A-7D show histopathological staining analysis after a single IV injection of recombinant AAV9 virus rAAV9-CAR-Mut-coGAA-2 ⁇ 142-3P administered in GAA ⁇ / ⁇ model mice in vivo evaluation experiment 1.
  • Figure 7A shows the results of H&E staining of liver tissue (upper row: magnification of 100 times; lower row: magnification of 400 times);
  • Figure 7B shows the results of H&E staining of cardiomyocytes;
  • Figure 7C shows the results of H&E staining of skeletal muscle cells;
  • Figure 7D shows the results of skeletal muscle and cardiac muscle PAS staining of cells.
  • Gaa -/- tissue section of model mice in PBS administration group
  • LD tissue section of model mice given low dose of recombinant AAV9 virus
  • MD tissue section of model mice given medium dose of recombinant AAV9 virus
  • HD Tissue sections of model mice given high doses of recombinant AAV9 virus
  • AAV treated tissue sections of model mice treated with recombinant AAV9 virus.
  • FIG. 8A-8C show histopathological staining analysis after a single IV injection of recombinant AAV9 virus rAAV9-CAR-Mut-coGAA-2 ⁇ 142-3P administered in GAA ⁇ / ⁇ model mice in vivo evaluation experiment 2.
  • Fig. 8A shows the result of PAS staining in brain tissue
  • Fig. 8B shows the result of PAS staining in spinal cord tissue
  • Fig. 8C shows the result of PAS staining in cerebellum tissue.
  • Gaa -/- the tissue section of the model mouse of the PBS administration group
  • WT the tissue section of the 129 wild-type mouse
  • 3E13vg/kg the tissue section of the model mouse injected with 3E13vg/kg recombinant AAV9 virus
  • 6.8E13vg/ kg tissue section of model mice injected with 6.8E13vg/kg recombinant AAV9 virus.
  • Figure 9 shows GAA enzymes measured in brain tissue after a single IV injection of recombinant AAV9 virus rAAV9-CAR-Mut-coGAA-2 ⁇ 142-3P administered in GAA ⁇ / ⁇ model mice in vivo evaluation experiment 2 vitality level.
  • model control model mice of PBS administration group; 6.8E13vg/kg: model mice injected with 6.8E13vg/kg recombinant AAV9 virus.
  • Figure 10 shows the survival curves of mice recorded after a single IV injection of PBS or recombinant AAV9 virus rAAV9-CAR-Mut-coGAA-2 ⁇ 142-3P in GAA -/- model mice in vivo evaluation experiment 4.
  • Gaa -/- model mice in PBS administration group;
  • AAV treatment 1.1E13vg/kg model mice injected with 1.1E13vg/kg recombinant AAV9 virus.
  • the invention discloses gene therapy constructs, pharmaceutical compositions and methods for treating Pompe disease or acid glucosidase deficient subjects, especially the construction, preparation and application of recombinant AAV vectors for delivering GAA.
  • recombinant adeno-associated virus can be represented by the AAV virus serotype from which the capsid is derived alone, or by the AAV virus serotype from which the capsid and genomic ITR sequences are derived.
  • the identifier "/" is used for separation, followed by the serotype of origin of the capsid and before the identifier "/" by the serotype of origin of the ITR.
  • the number 9 in the expression recombinant AAV9 indicates that the recombinant adeno-associated virus has a capsid from the AAV9 serotype; while the number before the identifier "/" in the expression recombinant AAV2/9 indicates that the recombinant adeno-associated virus has The wild-type or variant ITR sequence from AAV2, while the number after the identifier "/" indicates that the recombinant adeno-associated virus has a capsid protein from AAV9.
  • Acid ⁇ -glucosidase or “acid glucosidase” or GAA are used interchangeably herein to refer to the ability to hydrolyze ⁇ -1-4 bonds in maltose and other linear oligosaccharides to degrade lysosomal Lysosomal enzymes of excess glycogen.
  • the GAA-encoding gene When the GAA-encoding gene is expressed in the cell, the GAA polypeptide will be synthesized in the cytoplasm, glycosylated in the ER, and linked to a high-mannose sugar chain at the N-terminus.
  • the high mannose sugar chains on GAA can be further modified to add mannose-6-phosphate (M6P). Through the interaction of M6P with the M6P receptor, GAA is delivered into the lysosome, where it functions in the degradation of glycogen.
  • M6P mannose-6-phosphate
  • GAA examples include, but are not limited to, enzyme proteins having the amino acid sequence of full-length wild-type (native) human GAA (as shown in Unipro database accession number UniProtKB-P10253), mature forms thereof, variants thereof (e.g., with conserved amino acid substituted variants), and fragments thereof.
  • Human GGA has a conserved hexapeptide WIDMNE at amino acid residues 516-521, which is required for GAA protein activity.
  • variants and fragments of GAA can also be used, as long as the variant or fragment retains the activity of hydrolyzing glycogen, and for example provides 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% of the enzymatic activity level of full-length wild-type (native) human GAA.
  • the GAA polypeptide comprises the amino acid sequence of SEQ ID NO:13, or the amino acid sequence of residues 70-952 of SEQ ID N:13; the amino acid sequence of residues 123-952 of SEQ ID NO:13 Sequence, the amino acid sequence of residues 204-952 of SEQ ID NO: 13, or at least 90%, or at least 95%, 96%, 97%, 98%, 99% or more identity to any of the preceding sequences amino acid sequence.
  • the first 27 amino acids of the human GAA polypeptide are typical signal peptides for lysosomal and secreted proteins. GAA can be targeted to lysosomes through this signal peptide.
  • a GAA polypeptide of the invention comprises a lysosome-targeting signal peptide, such as the native signal peptide sequence from a human GGA polypeptide.
  • a GAA polypeptide of the invention comprises a signal peptide from a heterologous lysosomal targeting protein.
  • the polynucleotide sequence encoding a GAA polypeptide comprises a wild-type GAA nucleic acid sequence.
  • the polynucleotide sequence encoding the GAA polypeptide is human codon-optimized (i.e., codon-optimized for expression in human cells) for, e.g., enhancing the expression of said polynucleotide Expression and/or stability in vivo.
  • the polynucleotide sequence encoding GAA comprises the polynucleotide sequence of SEQ ID NO:10.
  • ETR or "enzyme replacement therapy” refers herein to a therapeutic procedure for the treatment of Pompe disease or acid glucosidase deficiency in which recombinant GAA protein is administered to a subject in need thereof.
  • Recombinant GAA proteins for ETR can be produced in engineered mammalian cell lines such as CHO cells, or in the milk of transgenic animals such as transgenic rabbits.
  • conservative amino acid substitution is the substitution or substitution of an amino acid for a different amino acid with a side chain having similar biochemical properties (eg, charge, hydrophobicity, and size).
  • Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologues and alleles.
  • the following 8 groups contain mutually conservative substitutions of amino acids: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N) , glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine acid (C), methionine (M) (see eg, Creighton, Proteins (1984)).
  • Those skilled in the art can easily detect the conservation of amino acid or nucleotide changes in a specific polypeptide sequence or nucleotide sequence by conventional technical means, such as functional assays.
  • sequence “identity” is used to describe the similarity in sequence structure between two amino acid sequences or polynucleotide sequences.
  • sequences can be aligned for optimal comparison purposes (e.g., the first and second amino acid sequences or nucleic acid sequences can be compared for optimal alignment) gaps may be introduced in one or both or non-homologous sequences may be discarded for comparison purposes).
  • the length of the reference sequence aligned for comparison purposes 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.
  • amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the comparison of sequences and the calculation of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available at http://www.gcg.com available), use the Blossum 62 matrix or the PAM250 matrix with gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6 to determine the distance between two amino acid sequences. percent identity.
  • using the GAP program in the GCG software package (available at http://www.gcg.com), using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70 or 80 and Length weights of 1, 2, 3, 4, 5 or 6 determine 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.
  • the term "host cell” refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells.
  • the host cell is any type of cell system that can be used to produce a recombinant AAV vector of the invention, for example, mammalian cells (such as HEK 293 cells suitable for production of recombinant AAV by a three-plasmid packaging system) and insect cells (eg sf9 cells suitable for production of recombinant AAV by the baculovirus packaging system).
  • regulatory sequence refers to a nucleic acid sequence that induces, represses, or otherwise controls the transcription of a protein of an encoding nucleic acid sequence to which it is operably linked. Regulatory sequences can be, for example, initiation sequences, enhancer sequences, intron sequences, and promoter sequences, among others.
  • exogenous or heterologous are used interchangeably when describing a nucleic acid or protein to mean that the nucleic acid or protein is not naturally present at the chromosomal or host cell location in which it is present.
  • An exogenous nucleic acid sequence also refers to a sequence that is derived from and inserted into the same host cell or subject but exists in a non-native state, eg, the sequence is present in a different copy number, or is under the control of a different regulatory element.
  • an "isolated" polynucleotide eg, isolated DNA or isolated RNA
  • an "isolated" nucleic acid is enriched at least about 10-fold, 100-fold, 1000-fold, 10,000-fold or more relative to the starting material.
  • an "isolated" polypeptide refers to a polypeptide that is at least partially separated from at least some other components of the native organism or virus in which it is contained. In some embodiments, an “isolated” polypeptide is enriched at least about 10-fold, 100-fold, 1000-fold, 10,000-fold or more relative to the starting material.
  • isolated or purified a viral vector means that the viral vector is partially separated from at least some components of the starting material comprising it. In some embodiments, an "isolated” viral vector is enriched at least about 10-fold, 100-fold, 1000-fold, 10,000-fold or more relative to the starting material.
  • viral vector refers to a viral particle (eg, an AAV viral particle) capable of serving as a delivery vehicle for a nucleic acid of interest.
  • a viral vector comprises a capsid and a viral genome (for example, viral DNA) packaged therein, and the target nucleic acid to be delivered is inserted into the viral genome.
  • recombinant AAV viral vectors in order to generate recombinant virus particles that can deliver the nucleic acid of interest to tissues or cells, it is usually only necessary to retain the inverted terminal repeat (ITR) cis element in the genome, while the rest required for viral packaging Sequences can be provided in trans.
  • ITR inverted terminal repeat
  • the recombinant AAV viral vectors of the present invention comprise a capsid and a recombinant viral genome packaged therein, wherein the recombinant viral genome comprises or consists of one or more exogenous genes located between two AAV ITR sequences.
  • Source nucleotide sequence composition The two ITR sequences located at the 5' and 3' ends of the recombinant viral genome (i.e., 5'ITR and 3'ITR) may be the same or different.
  • AAV "inverted terminal repeat” refers herein to a cis-acting element from the AAV viral genome that plays an important role in the integration, rescue, replication, and genome packaging of the AAV virus.
  • the ITR sequence of the natural AAV virus contains a Rep protein binding site (Rep binding site, RBS) and a terminal unzipping site trs (terminal resolution site), which can be recognized by the Rep protein and generate a nick at the 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.
  • AAV2 The earliest isolated AAV virus, AAV2, has "inverted terminal repeats" (ITRs) with a palindrome-hairpin structure of 145 bp located at both ends of the genome. Later, different ITR sequences were found in various serotypes of AAV viruses, but they all formed hairpin structures and had Rep binding sites.
  • ITRs inverted terminal repeats
  • Traditional recombinant AAV viral vectors based on these wild-type ITR sequences are generally single-stranded AAV vectors (ssAAV), and the viral genome is packaged in the AAV capsid in a single-stranded form.
  • the genome carried by the recombinant AAV virus vector obtained by packaging can be self-complementary to form a double chain (Wang Z et al., Gene Ther. 2003; 10(26):2105-2111; McCarty DM et al., Gene Ther. 2003; 10(26):2112-2118).
  • the virus thus packaged is a double-stranded AAV virus, that is, scAAV (self-complementary AAV) virus.
  • the packaging capacity of the scAAV viral vector is smaller, only half of the packaging capacity of the ssAAV viral vector, about 2.2kb-2.5kb, but the transduction efficiency after infection of cells is higher.
  • ITR in relation to AAV encompasses wild-type ITRs and variant IRTs.
  • Wild-type ITRs can be from any native AAV virus, such as an AAV2 virus.
  • the wild-type ITR contains a Rep protein binding site (Rep binding site, RBS) and a terminal unzipping site trs (terminal resolution site), which can be recognized by the Rep protein and generate a nick at the trs.
  • the wild-type ITR sequence can form a unique "T" letter-shaped secondary structure, which plays an important role in the life cycle of AAV virus.
  • a variant ITR is a non-native ITR sequence which may, for example, be derived from any wild-type AAV ITR sequence and which comprises a deletion, substitution, and/or addition of one or more nucleotides relative to the wild-type ITR, and/ Or truncated, but still functional, ie, can be used to generate ssAAV viral vectors or scAAV viral vectors.
  • a variant ITR is an AAV ITR sequence (also referred to herein as a ⁇ ITR) that has been deleted for a functional trs site and optionally a D region sequence.
  • wild-type ITRs are combined with ⁇ ITRs to generate self-complementary recombinant AAV viral vectors (scAAV).
  • scAAV self-complementary recombinant AAV viral vectors
  • two wild-type ITRs are used in combination to generate single-chain recombinant AAV viral vectors (ssAAV).
  • the AAV proteins VP1, VP2 and VP3 are capsid proteins that interact to form the AAV capsid.
  • Different serotypes of AAV viruses have different tissue infection tropisms, and foreign genes can be transferred to specific organs and tissues by selecting the source serotype of the recombinant AAV virus vector capsid (Wu Z et al., Mol Ther.2006; 14(3):316-327).
  • the recombinant AAV virus vector can have different targeting properties by selecting the source serotype of the capsid.
  • the capsid of the recombinant AAV virus is from an AAV serotype that targets neuronal cells.
  • the recombinant AAV viral vector comprises a capsid from AAV9.
  • the recombinant AAV viral vector comprises a capsid from AAV9 and an ITR from AAV2.
  • immune-associated miRNA is a miRNA that is preferentially expressed in cells of the immune system, such as antigen-presenting cells.
  • the expression level of an immune-related miRNA in an immune cell is high, especially at least 2-fold, 3-fold, 4-fold high, relative to its expression level in a non-immune cell (e.g., a reference cell, such as a HEK293 cell). times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times.
  • the immune system cells expressing immune-related miRNAs are B cells, T cells, T killer cells, T helper cells, dendritic cells, macrophages, monocytes, vascular endothelial cells, or other immune cells.
  • the immune-related miRNA is miR-142-3P.
  • 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. Therefore, using the principle of miRNA inhibition of gene expression, the expression of genes carrying miR-142-3p target sequences will be significantly inhibited in immune cells, thereby reducing the body's ability to produce immune responses against gene expression products. probability.
  • treatment refers to clinical intervention intended to alter the natural course of disease in the individual being treated. Desirable therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, reduction of the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • the recombinant AAV virus of the present invention after administration to a Pompe disease or GAA-deficient subject, preferably after systemic administration, reduces the number of affected tissues (especially, skeletal muscle, cardiac muscle, Lysosomal glycogen storage in the diaphragm and central nervous system).
  • the recombinant AAV virus of the invention after administration to a Pompe disease or GAA-deficient subject, preferably after systemic administration, improves central nervous system damage in the subject.
  • the recombinant AAV virus of the present invention improves skeletal muscle, myocardial damage in the subject after administration to Pompe disease or GAA-deficient subjects, preferably after systemic administration.
  • the recombinant AAV virus of the present invention improves the subject's nervous system (including brain, spinal cord and/or cerebellum tissue) after administration to a subject with Pompe disease or GAA deficiency, preferably after systemic administration.
  • Pathological changes caused by diseases In some embodiments, glycogen accumulation in glial cells in brain tissue is improved.
  • the recombinant AAV virus of the invention prolongs the survival of a subject after administration to a Pompe disease or GAA-deficient subject, preferably after systemic administration.
  • prevention includes the inhibition of the occurrence or development of a disease or symptoms of a particular disease.
  • subjects predisposed to developing Pompe disease are candidates for prophylactic regimens.
  • prevention refers to hospital intervention before at least one symptom of a disease occurs. Therefore, in one embodiment, prophylaxis includes the administration of the gene therapy drug of the present invention before the onset of symptoms of Pompe disease in a subject with a GAA gene defect, so as to delay the development of the disease or prevent the appearance of the disease.
  • Promoter is a specific DNA sequence that RNA polymerase recognizes, binds, and initiates transcription.
  • Eukaryotic class II (class II) promoters are involved in the transcriptional control of protein-coding genes, usually located upstream of the gene coding region, and regulate the timing and location of gene transcription through the interaction with transcription factors (transcription factors, TFs).
  • This type of promoter includes 5 types of action elements: basic promoter, initiator, upstream element, downstream element and response element. Different combinations and sequence changes of these elements endow multiple effects on the functional activity of the promoter (Tang Fang, Tu Huizhen. Research progress on eukaryotic promoters [J]. Forestry Science and Technology Development. 2015,29(2):7-12 .).
  • a synthetic mutant constitutive promoter CAR-Mut is provided.
  • the constitutive promoter of the present invention can effectively promote the expression of exogenous genes in various tissues, so it is especially suitable for use in the treatment method of the present invention to correct the peripheral organ involvement of Pompe disease and reduce the burden on the central nervous system.
  • the present invention provides a mutant promoter comprising a polynucleotide selected from:
  • the polynucleotide has a mutation at nucleotide 562-572 of SEQ ID NO:4 or a position corresponding thereto, preferably the mutation is a T mutation at nucleotide 568 or the corresponding position It is C or G or A, more preferably T is mutated to C.
  • the mutant promoter of the present invention increases the expression of a gene of interest functionally linked thereto, e.g., makes said gene of interest Gene expression is increased by 1%-70%, eg, at least 5%, 10%, 20%, 30%, 40%, or at least 50%, 60%.
  • the mutant promoter of the present invention increases the expression of the gene of interest functionally linked thereto in mammalian cells or tissues, for example, increases the expression of the gene of interest in mammals relative to the reference promoter.
  • the mammal is a human or a non-human mammal, eg, a mouse, a rat and a non-human primate.
  • the promoter comprises a nucleotide sequence selected from any one of SEQ ID NOs: 1 to 3, or differs therefrom by one or several nucleotide substitutions, deletions and/or additions and has Nucleotide sequences with equivalent promoter activity.
  • the promoter comprises or consists of the nucleotide sequence of SEQ ID NO:1.
  • any promoter functional assay known in the art such as the luciferase reporter gene expression assay in Example 1
  • the reference promoter such as SEQ ID NO: 1-3
  • the promoter to be tested under the same test conditions, compared with the reference promoter (such as SEQ ID NO: 1-3), if the promoter to be tested has the same or substantially the same activity, such as the reference promoter activity ⁇ 10%, preferably ⁇ 5%, or more preferably ⁇ 1%, the promoter to be tested can be considered to have equivalent promoter activity.
  • the present invention also encompasses expression cassettes, recombinant vectors and host cells comprising the promoter and the coding nucleotide sequences functionally linked thereto, and the use of the expression cassettes, vectors or host cells to develop mammalian cells Compositions and methods for delivering an encoding polynucleotide to or to an individual.
  • the invention provides expression constructs.
  • the expression construct of the present invention comprises the promoter of the present invention and can be advantageously used for the expression of the GAA-encoding nucleic acid sequence in desired tissues or cells of patients with Pompe disease or acid glucosidase deficiency.
  • the following elements of the expression construct of the invention are functionally linked to each other in transcriptional direction:
  • any CAR-Mut promoter of the invention especially the promoter of SEQ ID NO: 1,
  • the polynucleotide of coding target gene for example, the polynucleotide sequence of coding ⁇ -acid glucosidase (GAA), preferably people's codon-optimized people's GAA polypeptide coding sequence, more preferably the sequence of SEQ ID NO:10,
  • GAA ⁇ -acid glucosidase
  • At least one (e.g. 2-4) immune-related miRNA binding site especially miR-142 binding site, e.g. a miR comprising at least one (e.g. one or two) sequence of SEQ ID NO: 11 -142 binding site,
  • a transcription terminator such as a polyA signal sequence, preferably selected from the group consisting of SV40 late polyA sequence, rabbit ⁇ -globin polyA sequence, bovine growth hormone polyA sequence, or any variant thereof, more preferably comprising SEQ ID NO: 13 or a bovine growth hormone polyA sequence having at least 95% identity thereto.
  • the expression construct also includes two ITR sequences.
  • the expression construct may comprise elements arranged as follows: 5'ITR-promoter-GAA coding sequence-miRNA binding site-polyA-3'ITR.
  • the 5'ITR and 3'ITR are the same.
  • the 5'ITR and the 3'ITR are different and one (preferably the 3'ITR) is a ⁇ ITR lacking a functional trs site.
  • the 5'ITR and 3'ITR in the expression construct are the same, both comprising or consisting of the sequence of SEQ ID NO:5.
  • the 5'ITR and 3'ITR in the expression construct are different, wherein the 5'ITR comprises or consists of the sequence of SEQ ID NO:5 and the 3'ITR comprises or consists of the sequence of SEQ ID NO:6 .
  • the promoter used in the expression construct of the present invention may be the CAR-Mut promoter described in any of the above embodiments of the present invention.
  • the promoter comprises or consists of the nucleotide sequence of SEQ ID No:1.
  • the promoter comprises or consists of the nucleotide sequence of SEQ ID NO:2.
  • the promoter comprises or consists of the nucleotide sequence of SEQ ID NO:3.
  • the expression construct of the present invention may comprise a Kozak sequence located upstream of the initiation codon of the GAA-encoding nucleic acid sequence to facilitate translation of GAA.
  • the Kozak sequence used in the present invention may be a consensus sequence defined as GCCRCC, wherein R is a purine (ie A or G), and wherein said sequence is located upstream of the start codon.
  • the Kozak sequence in the nucleic acid sequence of the expression construct of the present invention, has a 5'-GCCACC-3' sequence. Other different Kozak sequences can also be used. Kozak sequences can be screened against sequence libraries, and enhancement of translation efficiency can be assessed using routine means known in the art.
  • recombinant nucleic acids comprising reporter genes or recombinant GAA genes with different Kozak sequences
  • the expression constructs of the present invention further comprise one or more immune-related miRNA binding sites, i.e., miRNA target sequences, located in the 3'UTR of the GAA-encoding nucleic acid sequence of interest.
  • miRNA binding sites i.e., miRNA target sequences
  • the inclusion of miRNA binding sites in expression constructs allows for modulation (eg, repression) of expression of a gene of interest in cells and tissues producing the corresponding miRNA.
  • the expression constructs of the invention comprise one or more miRNA binding sites so that expression of GAA can be downregulated in a cell type specific manner.
  • the expression construct of the invention comprises one or more miRNA binding sites, wherein said miRNA is expressed in an antigen presenting cell, thereby reducing the expression of GAA by the expression construct of the invention in said antigen presenting cell s efficiency.
  • one or more miRNA binding sites are located in the 3' untranslated region (3'UTR) of the GAA-encoding gene, e.g., between the last codon of the GAA-encoding nucleotide sequence and the polyA sequence .
  • the expression construct comprises one or more (e.g., 1, 2, 3, 4, 5 or more) miRNA binding sites that down-regulate the GAA gene from immune cells (e.g., , the expression of antigen-presenting cells APC, such as macrophages and dendritic cells, etc.).
  • immune cells e.g., the expression of antigen-presenting cells APC, such as macrophages and dendritic cells, etc.
  • the incorporation of such immune-relevant miRNA binding sites in expression constructs can result in reduced expression of the GAA gene of interest in antigen presenting cells bearing the miRNA, and thereby reduce or inhibit the subject's production of Anti-GAA immune response.
  • the expression construct comprises one or more miR-142 binding sites (also referred to herein as miR-142 target sequence), such as the miR-142-3P target sequence of SEQ ID NO: 11 , or its tandem repeats, such as 2, 3, 4, 5, 6 tandem repeats, preferably 2 tandem recombinations, such as the miR-142-3P target sequence of SEQ ID NO: 12.
  • the miRNA binding site can reduce expression of the recombinant AAV vector in antigen presenting cells.
  • the miRNA binding site can reduce the immunogenicity of the recombinant AAV vector.
  • the recombinant AAV vector comprising the miRNA binding site elicits a low immune response in the subject.
  • the recombinant AAV vector comprising the miRNA binding site elicits low anti-GAA serum titers in the subject following administration relative to a recombinant AAV vector control without the miRNA binding site.
  • said administration is intravenous administration.
  • the anti-GAA serum titer is determined after 1-6 weeks of administration, such as 5 weeks, preferably, the serum titer is reduced by about 1 to 10 times, for example, about 2 times, relative to the control. 3x, 4x, 5x, 6x, 7x or 8x.
  • the expression constructs of the invention comprise at least one polyA tail located downstream of the polynucleotides encoding the GAA and miRNA binding sites.
  • Any suitable polyA sequence may be used, including but not limited to hGHpolyA, BGHpolyA, SV40 late polyA sequence, rabbit ⁇ -globin polyA sequence, or any variant thereof.
  • polyA is BGHpolyA, such as the polyA shown in SEQ ID NO:7, or has at least 80%, 85%, 90%, 95%, 96%, 97% with SEQ ID NO:7, A polyA polynucleotide sequence having 98% or 99% nucleotide sequence identity.
  • the GAA-encoding nucleic acid contained in the expression constructs of the present invention can be any polynucleotide encoding functional GAA enzymatic activity.
  • the nucleic acid encodes a human full-length GAA sequence, e.g., the sequence of SEQ ID NO: 13, or a fragment thereof, e.g., starting between residues 1-204 of SEQ ID NO: 14 and ending at residues 952, or the GAA enzyme fragment at the corresponding position.
  • the GAA comprises a native lysosome-targeting signal peptide (i.e., in the case of SEQ ID NO: 13, the signal peptide of amino acids 1-27).
  • the GAA may comprise a signal peptide from a heterologous source, such as a signal peptide from a human lysosomal targeting or secreted protein.
  • a heterologous source such as a signal peptide from a human lysosomal targeting or secreted protein.
  • heterologous signal peptides include, but are not limited to: signal peptides from immunoglobulins (eg IgG), cytokines (eg IL-2), insulin. See eg WO2018046774.
  • the expression construct of the present invention comprises a GAA-encoding nucleic acid sequence, wherein said nucleic acid sequence encodes a polypeptide having GAA enzymatic activity, wherein said polypeptide comprises: a sequence with SEQ ID NO: 13, or a sequence with SEQ ID
  • the sequence of amino acids 70-952 of NO:13, the sequence with amino acids 123-952 of SEQ ID NO:13, or the sequence with amino acids 204-952 of SEQ ID NO:13 has at least 95%, at least 97%, at least Amino acid sequences of 98%, or at least 99% or greater sequence identity.
  • the polypeptide has about the same glycogenolytic activity as compared to the reference GAA protein of SEQ ID NO: 13, e.g., the GAA enzymatic activity of the polypeptide is at least about 95%, about 96%, of the reference GAA protease activity, Around 97%, 98%, 99% or higher.
  • Assays for determining GAA enzymatic activity are known in the art. Any such assay can be used by one skilled in the art to determine suitable GAA polypeptides for use in the expression constructs, recombinant AAV viral vectors, and methods and uses of the invention.
  • the codons used to encode the GAA polypeptide are preferably codon-optimized.
  • the GAA-encoding nucleic acid used in the expression construct of the present invention comprises the polynucleotide sequence of SEQ ID NO: 13, or at least about 95%, about 96%, about 97%, 98%, Polynucleotide sequences with 99% or greater nucleotide sequence identity.
  • the invention also provides vectors comprising expression constructs of the invention.
  • the vector is a plasmid (eg, a plasmid used for recombinant viral particle production).
  • the vector is a viral vector, such as a recombinant AAV vector or a baculovirus vector.
  • the genome of the recombinant AAV vector is single-stranded (eg, single-stranded DNA).
  • the genome of the recombinant AAV vector is self-complementary.
  • the vector is a baculovirus vector (eg, an Autographa californica nuclear polyhedrosis virus (AcNPV) vector).
  • the present invention also provides host cells, such as mammalian cells or insect cells, comprising the expression construct or vector of the present invention.
  • the cells can be used to produce recombinant AAV viruses.
  • the invention provides recombinant AAV vectors.
  • the recombinant AAV vectors of the invention are particularly useful in the treatment of Pompe disease or acid glucosidase deficiency.
  • the recombinant AAV vector comprises a capsid and nucleic acid located within the capsid, also referred to herein as the "genome of the recombinant AAV vector.”
  • the genome of the recombinant AAV vector contains multiple elements, including but not limited to two inverted terminal repeats (ITRs, i.e., 5'-ITR and 3'-ITR), and other elements located between the two ITRs, including the promoter , a heterologous gene, and a polyA tail.
  • ITRs inverted terminal repeats
  • at least one immune-related miRNA binding site may be included between the two ITRs.
  • adeno-associated virus includes, but is not limited to, AAV of any serotype, such as AAV types 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and AAV with artificial changes The capsid protein of AAV.
  • AAV adeno-associated virus
  • the genome sequences of various serotypes and artificial AAVs and their native inverted terminal repeat (ITR) sequences, Rep proteins and capsid cap proteins are known in the art. These sequences can be found in public databases such as GenBank or in the literature.
  • the present invention provides a recombinant AAV viral vector comprising a capsid, wherein the capsid is composed of a capsid protein capable of crossing the blood-brain barrier, such as AAV9, AAVPHP.B, AAVPHP.eB capsid protein.
  • the recombinant AAV vectors of the invention transduce neuronal cells of the central nervous system (CNS), as well as peripheral non-neuronal cells.
  • recombinant AAV vectors are capable of targeting and transducing muscle and neuronal cells following systemic administration.
  • the recombinant AAV vector is capable of targeting and transducing the peripheral organs and central nervous system of a subject following systemic administration.
  • recombinant AAV vectors are capable of targeting and transducing multiple tissues (e.g., brain, spinal cord, skeletal muscle, heart, and liver) in a subject following systemic administration, and preferably, in the form of The recombinant AAV vector resulted in higher expression and/or enzymatic activity of the foreign gene of interest (GAA in this application) in the targeted and transduced tissue compared to control subjects administered the recombinant AAV vector.
  • the recombinant AAV vectors of the invention have a capsid from an AAV9 serotype (also referred to herein as an AAV9 vector); Bulk ITR sequences (also referred to herein as AAV2/9 vectors).
  • the two ITR sequences of the recombinant AAV vector of the present invention are full-length ITRs (for example, about 125-145 bp in length, and contain a functional Rep binding site (RBS) and a terminal melting site ( trs)).
  • full-length functional ITRs are used to produce single-chain recombinant AAV vectors (ssAAV).
  • one of the ITRs of the recombinant AAV vector is truncated.
  • truncated ITRs lack functional terminal melting sites trs and are used to produce self-complementary recombinant AAV vectors (scAAV vectors).
  • the recombinant AAV vector of the present invention comprises a wild-type AAV ITR, such as a wild-type AAV2 ITR, such as the ITR sequence shown in SEQ ID NO:5.
  • the recombinant AAV vectors of the invention comprise variant ITRs with one or more modifications, such as nucleotide additions, deletions and/or substitutions, relative to the wild-type AAV ITR, e.g., relative to the wild-type AAV2 ITR A ⁇ ITR that is truncated and lacks a functional trs site, such as the ⁇ ITR sequence shown in SEQ ID NO:6.
  • the present invention provides a recombinant adeno-associated virus (AAV) vector, wherein said recombinant AAV vector comprises in its genome:
  • ITR inverted terminal repeat
  • any CAR-Mut promoter according to the invention, especially the promoter of SEQ ID NO: 1,
  • GAA human alpha-acid glucosidase
  • a transcription terminator such as a polyA signal sequence, preferably selected from the group consisting of the SV40 late polyA sequence, the rabbit ⁇ -globin polyA sequence, the bovine growth hormone polyA sequence, or any variant thereof.
  • the polynucleotide encoding GAA is human codon optimized, preferably the codon optimization is used to enhance the expression efficiency and/or stability of the polynucleotide in vivo, More preferably, said polynucleotide comprises the sequence of SEQ ID NO:10.
  • both ITRs of the recombinant AAV viral vector are wild-type AAV2 ITR sequences, or one of the ITRs is an AA2 ⁇ ITR sequence lacking a functional terminal melting site (trs).
  • the recombinant AAV vector is a ssAAV vector. In other embodiments, the recombinant AAV vector is a scAAV vector.
  • the recombinant AAV vector comprises a capsid protein from an AAV9 serotype, preferably, the recombinant AAV vector is an AAV2/9 vector.
  • AAV vector packaging systems mainly include three-plasmid co-transfection system, adenovirus as helper virus system, Herpes simplex virus type 1 (HSV1) as helper virus packaging system, and baculovirus-based packaging system. system.
  • HSV1 Herpes simplex virus type 1
  • baculovirus-based packaging system baculovirus-based packaging system.
  • Each packaging system has its own characteristics, and those skilled in the art can make appropriate choices according to needs.
  • the three-plasmid transfection packaging system is the most widely used AAV vector packaging system because it does not require helper virus and is highly safe. It is also the mainstream production system in the world.
  • the slight disadvantage is that the absence of an efficient large-scale transfection method limits the application of the three-plasmid transfection system in the large-scale preparation of AAV vectors.
  • Yuan et al. established a large-scale packaging system for AAV using adenovirus as a helper virus (Yuan Z et al., Hum Gene Ther. 2011; 22(5):613-624).
  • the system has high production efficiency, but the adenovirus in the packaging system is in the Finally, there are traces in AAV finished products, which affects the safety of AAV finished products.
  • the packaging system of HSV1 as a helper virus is another kind of AAV vector packaging system that is widely used.
  • Wu Zhijian and Conway et al. proposed the AAV2 vector packaging strategy (Wu Zhijian, Wu Xiaobing, etc., Science Bulletin, 1999, 44 (5): 506-509 with HSV1 as the helper virus in the world; 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 used two HSV1 to carry the rep/cap gene of AAV and the inverted terminal repeat (ITR)/exogenous gene expression cassette of AAV respectively, and then co-infected with these two recombinant HSV1 viruses Producer cells, packaged to produce AAV virus (Booth MJ, et al. Gene Ther. 2004; 11:829-837). Thomas et al. further established a suspension cell system for AAV production of dual HSV1 viruses (Thomas DL et al., Gene Ther. 2009; 20:861-870), making larger-scale AAV virus production possible.
  • Urabe et al. constructed a baculovirus packaging system for AAV vectors using three baculoviruses carrying AAV structural genes, nonstructural genes, and ITR/exogenous gene expression cassettes. Considering the instability of baculoviruses 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.2011; 107Suppl:S80-93) and a baculovirus combined with an induced cell line strategy ( (2012)zsch M et al., Hum Gene Ther. 2014;25:212-222, Ricozsch M et al., Hum Gene Ther. 2015;26(10):688-697).
  • the recombinant AAV viral vectors of the invention can be produced using any suitable method known in the art.
  • the recombinant AAV virus of the present invention is produced using a three-plasmid packaging system.
  • the recombinant AAV virus of the present invention is produced using a baculovirus packaging system.
  • the invention provides a cell comprising: (i) a first vector encoding one or more adeno-associated virus rep proteins and/or one or more adeno-associated virus cap proteins; and (ii) a second vector comprising any of the expression constructs of the invention described herein.
  • the cells of the invention can be used for the production of recombinant AAV viral vectors of the invention.
  • the present invention also provides a method for producing a recombinant AAV viral vector, wherein the method comprises the steps of:
  • cell comprising: (i) a first vector encoding one or more adeno-associated virus rep proteins and/or one or more adeno-associated virus cap proteins; and (ii) comprising A second vector for any expression construct of the invention;
  • the first vector is a plasmid and the second vector is a plasmid; said cells are mammalian cells, optionally wherein said mammalian cells are HEK293 cells.
  • the cells may provide other functions, or partial functions, required for the production of infectious recombinant AAV virions.
  • the cell further comprises a third helper plasmid vector. Cells of the invention can be readily prepared by transiently co-transfecting the first plasmid vector, the second plasmid vector, and/or the third helper plasmid.
  • the functions required for the production of infectious AAV particles are provided by adenoviral genes, wherein a third helper plasmid provides the adenoviral genes VA, E2A, and E4; the remaining adenoviral gene products required for production are provided by stably expressing adenoviral
  • the host cell provides the E1 gene. See, eg, T Matsushita et al., Adeno-associated virus vectors can be efficiently produced without helper virus. Gene Therapy (1998) 5, 938-945.
  • the first vector is a baculovirus vector and the second vector is a baculovirus vector; said cells are insect cells, optionally wherein said insect cells are sf9 cells.
  • the Rep and Cap proteins of AAV are respectively provided by two separate first baculovirus vectors; in other embodiments, the Rep and Cap proteins of AAV are provided simultaneously by one first baculovirus vector.
  • two baculoviruses encoding the GAA gene of interest and the Rep and Cap proteins of AAV, respectively can be produced by, for example, the Bac-to-AAV system, and the two baculoviruses can be used to co-infect Spodoptera frugiperda (Sf9 ) insect cells to produce the cells of the invention. See, eg, Galibert L. et al., J Invertebr Pathol. 2011; 107 Suppl:S80-93.
  • the present invention provides a pharmaceutical composition comprising a recombinant AAV viral vector of the present invention.
  • the pharmaceutical composition of the present invention preferably comprises a pharmaceutically acceptable excipient, diluent or carrier.
  • the pharmaceutical compositions of the present invention may be formulated in any suitable preparation form.
  • Suitable pharmaceutically acceptable excipients, diluents or carriers for formulation are well known in the art and include, for example, phosphate buffered saline, water, emulsions, such as oil/water emulsions, various types of Wet agent, sterile solution, etc.
  • Preparations can be formulated by conventional methods, and administered to subjects in appropriate doses.
  • Administration of a suitably formulated composition can be achieved in different ways, eg. Administration is by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal. The particular route of administration depends, inter alia, on the type of carrier included in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and other clinical factors.
  • the dosage for any one patient will depend on many factors, including the patient's size, body surface area, age, sex, the particular active agent to be administered, the timing and route used, and the type and phase of the drug used. Infections or diseases, general health conditions, and combinations of other medications.
  • the pharmaceutical compositions of the present invention may include a second active agent.
  • the second active agent is a recombinant GAA protein for ERT, such as recombinant GAA protein from the milk of a transgenic animal or a productive mammalian cell line.
  • the second active agent is a bronchodilator.
  • the pharmaceutical composition of the present invention may contain components capable of reducing side effects (eg anti-drug immune response) upon drug administration.
  • the component may be an immunosuppressant.
  • compositions of the present invention may be administered by any suitable route, including systemic administration and topical administration.
  • the pharmaceutical composition of the present invention is used for systemic administration, especially intravenous injection.
  • the present invention provides a pharmaceutical composition comprising a recombinant AAV vector of the present invention, wherein said pharmaceutical composition is an intravenous formulation, or a lyophilized stable formulation suitable for formulation as an intravenous formulation.
  • the pharmaceutical composition of the present invention is suitable for local administration, for example, directly in or near the organ or tissue to be treated in a subject.
  • the present invention relates to methods of treating diseases using the recombinant AAV vectors of the present invention or pharmaceutical compositions comprising the same.
  • the disease is Pompe disease.
  • the disease is acid alpha glucosidase deficiency.
  • the method comprises: administering any of the recombinant AAV vectors or pharmaceutical compositions of the invention to a subject in need thereof.
  • the recombinant AAV vector or pharmaceutical composition can be administered by any suitable route, including but not limited to, intramuscular, subcutaneous, intraspinal, intracerebroventricular, intrathecal, intravenous, intradiaphragmatic, intrathoracic, intraperitoneal.
  • the recombinant AAV vector or pharmaceutical composition of the present invention is delivered to a subject by systemic administration, especially intravenous administration.
  • the treatment is therapeutic.
  • the treatment is prophylactic.
  • the subject is a mammal, wherein the mammal is especially a human, primate, dog, horse, cow, especially a human subject.
  • treatment includes any one or more of: (1) preventing or delaying the onset of Pompe disease; (2) alleviating Pompe disease (3) reduce or prevent the onset and/or worsening of at least one symptom of Pompe disease; (4) improve neurodegeneration and/or subject behavior associated with Pompe disease; and (5 ) to prolong the survival of the subjects.
  • Pompe disease subjects who may be treated include IOPD and LOPD patients.
  • the subject is an IOPD patient.
  • the subject is a patient with LOPD.
  • the present invention provides the use of the recombinant AAV viral vector of the present invention for driving the expression of a polynucleotide encoding ⁇ -acid glucosidase (GAA) in mammalian cells (especially human cells), or in the preparation of Use in medicines for driving polynucleotides encoding ⁇ -acid glucosidase (GAA) expressed in mammalian cells or in one or more tissues or organs in mammalian (especially human) bodies,
  • GAA ⁇ -acid glucosidase
  • the medicament is for expressing GAA in the heart, liver, muscle, central nervous system (including brain and spinal cord) of a mammal,
  • the medicament is administered systemically, eg by intraperitoneal (i.p.), intramuscular (i.m.), intraarterial or intravenous (i.v.) injection, preferably intravenous injection.
  • intraperitoneal i.p.
  • intramuscular i.m.
  • intraarterial i.v.
  • intravenous i.v.
  • the present invention provides a method for treating Pompe disease subjects or subjects with acid glucosidase deficiency, and the recombinant AAV vector of the present invention is used for the treatment of Pompe disease.
  • the treatment comprises administering to the subject any one or more recombinant AAV vectors of the invention, preferably, by systemic administration, such as intraperitoneal (i.p.), intramuscular (i.m.), intraarterial or intravenous ( i.v.)
  • the recombinant AAV vector is administered by injection, preferably intravenous injection.
  • the GAA polypeptide is expressed in the subject's heart, liver, muscle, central nervous system (including brain and spinal cord) after administration of the recombinant AAV vector of the present invention.
  • recombinant AAV vector administration results in a reduction in lysosomal glycogen storage in the subject's skeletal muscle, myocardium, diaphragm, and central nervous system, and preferably induces no or low immunogenicity .
  • administration of a recombinant AAV vector of the invention improves cardiac, respiratory, and/or skeletal muscle function in a subject.
  • the administration of the recombinant AAV vector of the present invention can prevent or improve the central nervous system of the subject, such as brain, spinal cord and/or neurons, from pathological changes caused by glycogen storage, such as progressive neurodegeneration. In some embodiments, administration of the recombinant AAV vectors of the invention prolongs the survival of the subject.
  • the present invention also provides the following methods and the use of the recombinant AAV vector of the present invention in the preparation of medicaments for the following methods:
  • the recombinant AAV viral vectors of the invention are administered in combination with another therapeutic agent or procedure.
  • Therapeutic drugs or therapeutic procedures that may be administered in combination with the recombinant AAV vectors of the present invention may be selected from immunomodulators, bronchodilators, acetylcholinesterase inhibitors, respiratory muscle strength training (RMST), enzyme replacement therapy (ERT), and/or Diaphragmatic pacing therapy.
  • Example 1 CAR-Mut promoter construction and characterization
  • CA promoter composed of the enhancer sequence of human CMV virus and the basal promoter of chicken ⁇ -actin protein
  • the 3' end of the sequence was introduced into the human TATA box binding protein-related factor 1 gene (GenBank: NG_012771.2)
  • the intron sequence from position 62804 to position 62890 was named CAR promoter.
  • CAR-MutC with the mutation T568C, the sequence is shown in SEQ ID NO: 1
  • CAR-MutA with mutation T568A, sequence shown in SEQ ID NO: 2
  • CAR-MutG with mutation T568G, sequence shown in SEQ ID NO: 3
  • the pscAAV-CAR-Gluc plasmid vector shown in Figure 1A was constructed, including:
  • ITR from the 3' end of the AAV2 genome (GenBank No.AF043303), the sequence of which is shown in SEQ ID NO:5;
  • Gluc the nucleotide sequence encoding the luciferase reporter gene
  • BGH polyA bovine growth hormone
  • the CAR promoter in the pscAAV vector was replaced with the CAR-Mut promoter (SEQ ID No.1, 2, or 3) to obtain pscAAV-CAR-Mut-Gluc ( Figures 1B-1D) Vectors. Briefly, the CAR-Mut promoter sequence was synthesized and XhoI and KpnI restriction sites were added at both ends, respectively. The synthesized sequence was cloned into pUC57simple vector (GenScript Biotechnology, Nanjing) to obtain pUC57-CAR-Mut.
  • the well-grown BHK-21 cells were passaged to 24-well plates, and when the density reached 60%, Lipofectamine2000 (Invitrogen, USA) was used to transfect pscAAV-CAR-Gluc, pscAAV-CAR-MutC-Gluc, pscAAV according to the manufacturer's instructions - 3 wells each for CAR-MutA-Gluc and pscAAV-CAR-MutG-Gluc. 48 hours after transfection, 100 ⁇ L of the supernatant was taken from each well, the Gluc level was detected with a Glomax96 microplate luminometer (Promega), and data analysis was performed using the detector software.
  • Lipofectamine2000 Invitrogen, USA
  • the three-plasmid packaging system was used to package and purify the recombinant AAV virus to obtain rscAAV9-CAR-Mut-Gluc and rscAAV9-CAR-Gluc recombinant virus.
  • the AAV Rep and Cap protein expression plasmid pAAV-R2C9 construct the AAV Rep and Cap protein expression plasmid pAAV-R2C9.
  • the capsid protein coding sequence also known as Cap9
  • the sequence between the HindIII and PmeI restriction sites in the pAAV-RC plasmid was obtained to obtain the pAAV-R2C9 plasmid.
  • the pAAV-R2C9 plasmid comprises the cap gene of complete AAV9 and the rep gene of AAV2, and provides four kinds of Rep proteins (Rep78, Rep68, Rep52 and Rep40) necessary for packaging when three plasmids are co-transfected and packaged to prepare recombinant AAV9 virus AAV9 capsid protein.
  • AAV vector plasmid pscAAV-CAR-Gluc and pscAAV-CAR-Mut-Gluc
  • helper plasmid pHelper, from AAV Helper Free System, Agilent Technologies
  • AAV Rep and Cap protein expression plasmid pAAV-R2C9 After mixing according to the molar ratio of 1:1:1, HEK293 cells were transfected by the calcium phosphate method.
  • the cells and culture supernatant were harvested, and the recombinant AAV virus was isolated and purified by cesium chloride density gradient centrifugation to obtain rscAAV9-CAR-Gluc and rscAAV9-CAR-Mut-Gluc.
  • the genomic titer of the prepared recombinant AAV virus (rAAV) was measured by dot hybridization.
  • the specific process is as follows:
  • CAR-Mut-F 5'-GTTCCCATAGTAACGCCAATAGGG-3' (SEQ ID NO:8)
  • PCR method was used to specifically amplify the CAR-Mut promoter to obtain a DNA probe fragment with a length of 175bp.
  • the pscAAV-CAR-Mut-Gluc plasmid and its 2 times The ratio gradient dilution solution was used as the standard, and the 2-fold ratio gradient dilution of the rAAV sample was used as the detection sample.
  • the standard and detection samples are spotted on the hybridization membrane, and the membrane is hybridized with the probe. Please refer to Molecular Cloning Experiment Guide (Fourth Edition) for details of the operation process.
  • Use ImigeJ software to scan in grayscale, compare the hybridization signals of the sample point and the series of standard points, and analyze and calculate the rAAV sample titer.
  • mice Gluc levels were detected after the recombinant AAV vectors carrying CAR and CAR-Mut promoters were injected into mice to characterize the functional activity of the promoters. Specifically, a total of 18 6-week-old C57BL/6J wild mice were randomly divided into 3 groups. Group 1 mice were injected with rscAAV9-CAR-Gluc through the tail vein at a dose of 1 ⁇ 10 13 GC/kg (genome copies/kg). The mice in group 2 were injected with rscAAV9-CAR-Mut-Gluc through the tail vein at a dose of 1 ⁇ 10 13 GC/kg. The mice in group 3 were injected with 200 ⁇ L PBS into each tail vein as a control.
  • mice All mice were sacrificed 1 month after injection, and the brain tissue, heart, and liver of each mouse were dissected and separated. Tissues of equal quality were taken to extract tissue total protein. Pierce BCA Protein Aaasy Kit (ThermoFisher, USA) was used to measure the total protein concentration of each group, and the detailed process refers to the kit instructions. 50 ⁇ l protein was taken from all mouse tissues to detect the Gluc level with a Glomax96 microplate luminometer.
  • an AAV plasmid vector comprising the target gene GAA, target gene expression regulatory elements, and ITR sequence was constructed.
  • the pRDAAV-CMV-EGFP plasmid vector contains:
  • coGAA codon-optimized human GAA coding nucleotide sequence
  • the pUC57-coGAA vector and the pRDAAV-CAR-Mut-EGFP vector were digested with KpnI and EcoRI respectively, the coGAA fragment and the pRDAAV-CAR-Mut-EGFP vector fragment with the EGFP reporter gene removed were recovered, and the two fragments were ligated and transformed into E.coli DH5 ⁇ competent cells (Qingke Xinye, Beijing) were screened and identified to obtain the pRDAAV-CAR-Mut-coGAA vector ( Figure 4C).
  • the artificially synthesized miR-142-3pT fragment (comprising two miR-142-3p target sequences in series, see SEQ ID No.12 for sequence information) was cloned into the EcoRI and pRD.AAV-CAR-Mut-coGAA vectors.
  • the pRD.AAV-CAR-Mut-coGAA-2 ⁇ 142-3P vector was obtained between the SalI restriction sites ( FIG. 4D ).
  • the oligo primer containing MicroRNA 142-3pT was synthesized by Beijing Qingke Xinye Biotechnology Co., Ltd., and after annealing, the 142-3pT fragment with the upstream EcoRI restriction site and the downstream SalI restriction site was obtained.
  • the AAV virus was packaged using the Bac-to-AAV system. Briefly, the following operations were carried out: Sf9 cell culture, transfection preparation and identification of two baculoviruses encoding the target GAA gene and AAV-Rep2/Cap9 respectively, amplification of two baculoviruses, co-infection with two baculoviruses Sf9 cells, harvest Sf9 cell pellet, lyse the cells to release AAV virus, purify AAV virus by ultracentrifugation, desalt and concentrate AAV virus in membrane package, and sterilize and filter to obtain recombinant AAV virus rAAV9-CAR-Mut-coGAA-2 ⁇ 142-3P.
  • the packaging process can be adopted in Chen H.Intron Splicing-mediated Expression of AAV Rep and Cap Genes and Production of AAV Vectors in Insect Cells, [J]. Molecular Therapy, the Journal of the American Society of Gene Therapy, 2008,16(5 ): 924 and the method described in the patent US8945918 and CN101522903B.
  • the PCR method was used to specifically amplify the CAR-Mut promoter to obtain a DNA probe fragment with a length of 175 bp , using the pRDAAV-CAR-Mut-coGAA-2 ⁇ 142-3P plasmid and its 2-fold serial dilution as a standard, the 2-fold serial dilution of the rAAV sample was used as the detection sample.
  • the standard and detection samples are spotted on the hybridization membrane, and the membrane is hybridized with the probe.
  • the rAAV sample titers were calculated using ImigeJ software grayscale scan analysis.
  • GAA-KO mice Thirty-two 8- to 10-week-old GAA gene homozygous deletion model mice (GAA-KO mice, purchased from Jax lab, No. 004154) were randomly and equally divided into 4 groups.
  • One of them is the model control group, as a negative control, each single IV injection of 200uL PBS; the other three groups are low-dose group, middle-dose group and high-dose group, which are used as the experimental group with a single IV injection dose of 5E12vg/ kg, 1.1E13vg/kg, 3E13vg/kg rAAV9-CAR-Mut-coGAA-2 ⁇ 142-3P.
  • Another group was added as the wild control group, and eight 8-10-week-old 129 wild mice were used as controls. All mice were sacrificed 5 weeks after injection, and the heart, liver, spleen, lung, kidney, and muscle tissue of each mouse were dissected and separated.
  • Figure 7A shows the results of H&E staining of liver tissue 5 weeks after intravenous treatment with AAV9-CAR-Mut-coGAA-2 ⁇ 142-3P virus vector.
  • Gaa -/- model mice can clearly see extensive liver multifocal necrosis (black arrows) caused by the disease, mainly concentrated around the central vein.
  • Model mice given different doses (LD, low dose; MD, middle dose; HD, high dose) of AAV9-CAR-Mut-coGAA-2 ⁇ 142-3P showed that the area of liver necrosis was greatly reduced, and no obvious nucleus was observed. pyknosis, and showed a dose-dependent correlation. The results show that the liver treatment effect of the drug is remarkable.
  • FIG. 7B shows H&E staining of cardiomyocytes in Gaa -/- model mice after IV single injection administration.
  • a large area of vacuolar degeneration and myocardial wall vascular congestion black arrows
  • Cardiomyocytes of Gaa -/- model mice treated with low, medium and high doses were improved to varying degrees, and showed a dose-dependent relationship.
  • the middle and high doses improved more significantly, and there was no vacuolar degeneration of cardiomyocytes and obvious congestion of blood vessels in the myocardial arm.
  • the results show that the drug has a significant therapeutic effect on cardiomyopathy.
  • Figure 7C shows H&E staining of skeletal muscle cells in Gaa-/- model mice after IV single injection administration.
  • a large area of vacuolar degeneration (black arrow) can be seen on the tissue sections of Gaa -/- model mice in the PBS administration group.
  • the skeletal muscle cells of Gaa-/- model mice treated with low, medium and high doses all improved to varying degrees, and showed a dose-dependent relationship.
  • the middle and high doses improved more significantly, no significant inflammatory cell infiltration was seen, and normal muscle fibers (red arrows) appeared, and the effect of the high dose group was the most significant (Fig. 7C).
  • the results show that the drug can significantly improve skeletal muscle damage without toxic changes, and the drug safety is good.
  • Figure 7D shows the PAS staining results of Gaa -/- model mouse skeletal muscle cells and cardiomyocytes in the exploratory animal model experiments before this experiment. Large areas of vacuolar degeneration and deep staining of glycogen staining can be seen on the tissue sections of mice administered with PBS (upper left and lower left). After 3 months of intravenous injection of 5E12vg/kg recombinant AAV, the skeletal muscle and myocardium of Gaa -/- model mice showed obvious improvement in glycogen accumulation, and the vacuolar degeneration of muscle fibers was restored (upper and lower right figures ). Therefore, similar to the previous results shown in Figures 7A-7C, the results in Figure 7D also illustrate that the recombinant AAV drug of the present invention can significantly improve skeletal muscle and myocardial damage, and solve the cause of glycogen accumulation in Pompe disease.
  • mice with homozygous deletion of the GAA gene were treated. Briefly, the model mice were randomly and equally divided into 3 groups (5 mice in each group). One of the groups was used as the negative control group, each with a single IV injection of 200uL PBS; the other two groups were used as the experimental group with a single IV injection of 3E13vg/kg and 6.8E13vg/kg of rAAV9-CAR-Mut-coGAA-2 ⁇ 142 -3P. Another group of 129 wild mice aged 8-10 weeks was added as a control. All mice were sacrificed 5 weeks after injection, and the brain tissue, spinal cord and cerebellum tissue of each mouse were dissected.
  • the results of PAS staining of spinal cord tissues after a single intravenous injection of AAV9-CAR-Mut-coGAA-2 ⁇ 142-3P are shown in 8B.
  • the results show that there are more PAS glycogen-positive cells in the forefoot of the spinal cord of the Pompeii model animal, and the proportion of PAS-positive neurons is relatively higher (upper left figure), indicating that there is glycogen accumulation in the spinal cord of the model animal, which is consistent with the relevant literature on model animals.
  • the motoneurons of the anterior horn of the spinal cord of WT mice were only strongly positive for glycogen (upper right figure), which indicated that the motoneurons of the forefoot occasionally had cells with strong glycogen metabolism.
  • FIGS 8A-8C show that the recombinant AAV drug of the present invention can effectively improve pathological changes caused by diseases of the nervous system (including brain, spinal cord and cerebellum). This shows that the recombinant AAV drug of the present invention has a dose-dependent central nervous system glycogen scavenging ability after IV injection, can cross the blood-brain barrier, and correct intracellular glycogen metabolism disorders.
  • the 6.8E+13vg/kg dose group significantly increased the level of GAA enzyme activity in brain tissue.
  • the GAA enzyme activity in the brain tissue of Pompeii model mice was negative.
  • a control recombinant AAV9 virus AAV9-CAR-Mut-coGAA without miRNA-142 target sequence was constructed, and combined with a recombinant AAV9 virus AAV9-CAR-Mut-coGAA-2 ⁇ 142-3P with miRNA-142 target sequence, in Treatment effects and serum antibody titers were compared.
  • Serum anti-drug antibody titers were detected in the following manner. After 5 weeks after AAV administration, the mice were sacrificed and blood was taken, and the titers of anti-GAA antibodies in the mouse serum samples were detected by ELISA after the serum was separated.
  • GAA-KO mice Sixteen 8- to 10-week-old GAA gene homozygous deletion model mice (GAA-KO mice, purchased from Jax lab) were randomly divided into 2 groups. One group was used as the negative control group, and each animal received a single IV injection of 200uL PBS; the other group was used as the experimental group with a single IV injection of rAAV9-CAR-Mut-coGAA-2 ⁇ 142-3P at a dose of 1.1E13vg/kg. Observe their survival and record their survival curves. The results are shown in Figure 10.

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Abstract

提供一种组成型启动子CAR-Mut，以及包含所述启动子及与之功能性连接的GAA编码核苷酸序列的表达构建体和重组载体及宿主细胞，还提供使用所述重组载体向哺乳动物细胞或个体递送GAA编码多核苷酸、和治疗庞贝氏病或酸性葡萄糖苷酶缺陷受试者的组合物和方法。

Description

用于治疗庞贝氏病的基因治疗构建体、药物组合物和方法 技术领域
本发明涉及基因治疗，更具体地涉及用于庞贝氏病基因治疗的构建体、以及包含所述构建体的药物组合物和治疗庞贝氏病的方法。
背景技术
基因治疗
首个获得授权的基因治疗研究诞生于1989年，经历了三十多年的发展之后，基因治疗获得了里程碑式的突破，进入了一个新时代。在治疗以前无法治愈的遗传疾病方面，基因治疗取得了长足的进步。目前已有几种基因治疗药物获得FDA/EMA的批准被用于临床。针对神经肌肉疾病和血友病等更多遗传疾病的基因治疗药品也有望在将来获得更多的批准许可。此外，基因治疗现在正被广泛用于肿瘤、感染性疾病、心血管疾病和自身免疫性疾病的治疗研究中。
基因治疗药物的关键是采用适宜的载体材料将外源基因传递至受体细胞中，并通过转录表达达到疾病治疗的目的。目前常用的基因治疗载体主要包括病毒类和非病毒类两种。病毒载体凭借其天然特性可以高效的将外源基因导入受体细胞而被广泛应用，其中腺相关病毒(Adeno-associated virus，AAV)载体具有良好的安全性，以及对多种靶组织的高效转导，成为体内基因治疗的最活跃的载体之一。
启动子作为基因工程表达载体中的重要元件，在很大程度上可以决定克隆基因的表达效率和组织表达谱。因此，在基因治疗领域中，为了满足基因治疗需求，常常需要基于具体的治疗目的，构建符合需求的新启动子。同时，在该领域也客观地存在着提供多样的启动子选择的需求。
作为转基因递送工具的病毒载体的基因容量是有限的。传统的单链AAV病毒载体(ssAAV)的情况下，总包装容量大约为4.8kb。在双链自互补型AAV病毒载体(scAAV)的情况下，总包装容量大约为一半，约2.5kb。因此，在兼顾基因构建体大小的情况下，选择适宜的载体基因元件组合，以保证目的基因以合适的水平在期望的组织(或多种组织)中表达，就显得尤为重要。
在一些情况下，转基因在所有或大多数细胞类型中的组成型表达是被期望的，例如，当所治疗的疾病或疾患累及多种组织时。本领域已经提出了一些组成型启动子，例如人延伸因子1、巨细胞病毒启动子CMV、鸡肌动蛋白启动子CBA、以及包含CMV增强子的合成CAG启动子等。然而，组成型启动子的使用效果常会因具体应用的疾病或疾患组织、施用方式等因素的影响而表现各异，并且在一些情况下会带来较高的药物免疫原性和/或动物毒性，从而限制基因治疗药物构建体的应用。因此，在基因治疗中，持续需要提供适于更有效转导疾病相关组织且更安全的基因治疗构建体。
庞贝氏病
庞贝氏病又称为酸性α-葡萄糖苷酶缺乏症或糖原贮积症Ⅱ型(GSDⅡ)，是一种系统性溶酶体贮积病，主要累及肌肉，也影响中枢神经系统。在患病个体中，溶酶体内缺乏功能性酸性α-葡萄糖苷酶(GAA)，从而导致糖原不能转化为葡萄糖而被利用，致使糖原在患者体内细胞的溶酶体中，尤其是在骨骼肌、心肌等外周器官组织和中枢神经系统(包括脑和脊髓)的细胞内聚积而致病。庞贝氏病可以通过酶学活性检测，检测α-葡萄糖苷酶的活性来确诊。
根据起病年龄和严重程度，庞贝氏病可分为：婴儿型；和晚发型。婴儿发病型庞氏病(IOPD)个体具有极低的残留GAA酶活性，表现出呼吸困难、全身肌肉无力和心肺功能衰竭等比较严重的症状，并常具有致死性。儿童至成年发病型庞贝氏病个体，因残留GAA酶活性较高，病情进展较缓。这种较为温和形式的庞贝氏病也称作晚发型庞贝氏病(LOPD)。在LOPD个体中常常不存在心肌缺陷，但肌无力会导致严重呼吸问题和呼吸衰竭。
目前唯一获得批准的庞贝氏病治疗方法是酶替代治疗(ERT，enzyme replacement therapy)。ERT具有持续改善心脏功能异常和预防心力衰竭的优势。然而，对于受累的骨骼肌和CNS系统，ERT表现出局限性。接受ERT治疗的患者个体可以具有差别较大的骨骼肌反应。这种反应差异性的因素之一被认为可能与高滴度抗药抗体的形成相关。在动物和人体中的研究已经提示，针对GAA酶形成的抗体可以降低ERT的疗效。此外，ERT药物不能跨血脑屏障，不能治疗CNS病变和受累的呼吸运动神经元。在接受ERT的婴儿个体中已经报道了严重的进行性神经变性。在ERT治疗的长期存活者中的脑MRI研究，也揭示出缓慢进展的白质损伤。ERT的再一局限性是某些组织类型中完全缺乏或仅具有不充分的糖原清除，例如血管、眼部、胃肠道和呼吸系统的平滑肌。
在病理学上，庞贝氏病作为一种常染色隐性单基因病症，由酸性α-葡萄糖苷酶(GAA)基因的病理性突变(包括导致GAA酶活性丧失或减小的各种无义突变和错义突变)引起。因此，作为ERT的替代或补充，基因治疗方法被提出来克服患者个体的GAA基因缺陷。
Darin J Falk等(2013,Intrapleural Administration of AAV9 Improves Neural and Cardiorespiratory Function in Pompe Disease,doi:10.1038/mt.2013.96)使用AAV9携带在组成型启动子CMV和组织特异性启动子DES控制下的重组GAA基因，通过胸腔内注射来治疗庞贝氏病小鼠。结果显示，GAA酶活性在心脏中有增加，但在肝脏中几乎没有检测到GAA酶活性。
Enyu Deng等人(MOLECULAR THERAPY Vol.5,No.4,2002；doi:10.1006/mthe.2002.0563)使用携带组成型启动子CMV和重组GAA基因的AAV载体(Ad CMV-GAA)，通过静脉内注射小鼠来治疗庞贝氏病。结果显示，重组AAV注射后血浆中会出现瞬时高水平的GAA，但CMV启动子在载体注射数天中快速关 闭，且AAV基因治疗触发了快速的抗GAA抗体，导致血浆中GAA水平快速降低至完全无法检测。
为了解决AAV载体药物引起的免疫反应，Sang-oh Han等人(Molecular Therapy:Methods&Clinical Development Vol.4 March 2017,http://dx.doi.org/10.1016/j.omtm.2016.12.010.)提出使用肝特异性启动子LSP替代组成型启动子并采用肝靶向性AAV2/8，构建AAV载体AAV2/8-LSPhGAA用于治疗GAA KO小鼠。该研究评估了三种较低剂量AAV2/8-LSPhGAA在单独使用或与ERT结合使用时的功效。结果显示，尽管在组成型启动子CMV增强子/CB启动子控制下携带hGAA的AAV载体不诱导免疫耐受，但利用肝特异性启动子LSP的AAV8-LSP-hGAA可以有利于阻抑抗GAA抗体反应。然而，AAV2/8靶向肝脏，产生的蛋白无法跨越血脑屏障到达中枢神经系统，无法减轻庞贝氏病的中枢神经系统受累。也参见WO2009075815A1。
Allison M.Keeler等人和Jeong-A Lim等人提出通过改良病毒衣壳来提高AAV基因治疗对受累中枢神经系统CNS的疗效。在Allison M.Keeler的研究中(Systemic Delivery of AAVB1-GAA Clears Glycogenand Prolongs Survival in a Mouse Model of Pompe Disease,HUMAN GENE THERAPY,VOLUME 30 NUMBER 1,DOI:10.1089/hum.2018.016)，应用对肌肉和CNS具有高亲和力的AAVB1，构建了腺相关病毒(AAV)载体AAVB1-DES-h GAA及对照AAV9-DES-h GAA载体。在将载体注射到敲除了GAA基因的GAA KO小鼠的尾静脉中后，两种载体均能有效地转导心脏，导致糖原清除，并在组织切片上观察到膈肌和中枢神经系统的转导。然而，仅AAVB1处理的小鼠表现出稳定的增重和四肢力量的恢复。此外，受限于DES启动子的组织特异性和较弱的表达水平，经AAV处理的动物的肝脏GAA水平明显低于野生型，且在两个AAV治疗组的气管，髓质，颈，胸和腰脊髓中的GAA活性均低于酶测定的检测极限。
在Jeong-A Lim等人(Molecular Therapy:Methods&Clinical Development Vol.12 March 2019；https://doi.org/10.1016/j.omtm.2019.01.006.)的研究中，使用病毒衣壳PHP.B构建AAV病毒载体，在2周龄的GAA KO小鼠中单次静脉注射AAV-PHP.B-CB-GAA后，糖原含量在脑和心脏中降低至野生型水平，在骨骼肌中含量显著降低。PHP.B-CB-hGAA的转导效率足以阻止GAAKO小鼠大脑中糖原的积累并挽救相关的神经表型。但遗憾的是，PHP.B衣壳的这种异常高的中枢神经系统靶向性仅限于特定的转基因小鼠模型。
采用膈肌内递送AAV载体(rAAV1-CMV-hGAA)的基因治疗在庞贝氏病儿童患者中进行了临床试验。临床试验证实了AAV载体的安全性；但临床结果不显著，并在所有未接受免疫调节剂的患者中均观察到抗衣壳和抗转基因抗体反应。(Giuseppe Vita，2019，https://doi.org/10.1007/s10072-019-03764-z)。
因此，在庞贝氏病的基因治疗中，本领域仍持续需要提供新的治疗载体和药物，以实现疾病相关组织的有效转导和病变的改善，以及降低的抗药免疫反应。
发明概述
本发明人经过深入研究提出了一种可以用于在静脉注射后减轻庞贝氏病的中枢神经系统负担和纠正外周器官受累并同时具有低药物免疫原性的新型人工合成组成型启动子、基于所述启动子的新AAV病毒载体、及其用途。
因此，在一个方面，本发明提供了一种突变启动子，其包含SEQ ID NO:4或与SEQ ID NO:4具有至少95％同一性或一个或几个核苷酸改变的多核苷酸，且该多核苷酸在SEQ ID NO:4的位置562-572，优选地位置568中具有T到C或G或A,尤其是T到C的突变。本发明的突变启动子相对于未突变的参照启动子增加与之功能性连接的目的基因的表达，尤其是在哺乳动物细胞或组织中的表达。本发明突变启动子的强启动子活性使其尤其适用于庞贝氏病的治疗用途。
在再一方面，本发明提供了包含本发明突变启动子的表达构建体、载体、宿主细胞、以及其药物组合物。
在再一方面，本发明提供了包含本发明突变启动子和编码酸性α葡萄糖苷酶GAA的多核苷酸的重组AAV病毒载体。本发明的病毒载体可以是ssAAV或scAAV病毒载体。优选地，本发明的病毒载体包含具有肌肉和/或神经系统靶向性的AAV衣壳蛋白，例如AAV9血清型衣壳蛋白。
在再一方面，本发明提供了本发明的重组病毒载体在庞贝氏病或具有酸性葡萄糖苷酶缺陷的受试者中治疗或预防所述疾病或缺陷的应用方法，也提供在制备用于预防或治疗所述疾病或缺陷的药物中的用途。在优选实施方案中，本发明方法导致受试者的外周组织和中枢神经系统组织中GAA酶活性水平的增加和糖原贮积量减少。通过本发明方法，可以有利地在静脉注射后减轻庞贝氏病的中枢神经系统负担和纠正外周器官受累，并同时具有低药物免疫原性的优点。
附图说明
图1A-1D分别显示pscAAV-CAR-Gluc载体、pscAAV-CAR-MutC-Gluc载体、pscAAV-CAR-MutA-Gluc载体、和pscAAV-CAR-MutG-Gluc载体的结构示意图。
图2显示，在体外培养的细胞试验中，与未转染质粒的BHK-21细胞(即，空白对照)相比，在转染了pscAAV-CAR-Gluc载体以及pscAAV-CAR-MutC-Gluc载体、pscAAV-CAR-MutA-Gluc载体和pscAAV-CAR-MutG-Gluc载体的BHK-21细胞中，测定的Gluc水平变化。其中，**表示p<0.01。
图3A-3C分别显示，在以携带CAR及CAR-Mut启动子(SEQ ID NO:1)的重组AAV载体IV注射小鼠后，在解剖的小鼠脑组织(图3A)、心脏组织(图3B)和肝脏组织(图3C)中检测到的Gluc水平。其中，**表示p<0.01。
图4A-4D分别显示pRDAAV-CMV-EGFP载体、pRDAAV-CAR-Mut-EGFP载体、pRDAAV-CAR-Mut-coGAA载体、pRDAAV-CAR-Mut-coGAA-2×142-3P载体的结构示意图。
图5显示，在体外培养的细胞试验中，在感染与未感染病毒的细胞中测定的GAA酶活性水平。BHK细胞：未感染病毒的BHK-21空白细胞；rAAV9-CAR-Mut-coGAA-142-3p：感染了重组AAV9病毒rAAV9-CAR-Mut-coGAA-2×142-3P的BHK-21细胞；其中，**表示p<0.01。
图6显示，在GAA -/-模型小鼠体内评价实验中，在单次IV注射施用重组AAV9病毒rAAV9-CAR-Mut-coGAA-2×142-3P后，于解剖的小鼠心脏、肝脏、肌肉、肾、肺、和脾组织中检测的GAA酶活力。模型对照组：注射PBS的模型小鼠，作为阴性对照；低剂量组：注射5E12vg/kg重组AAV9病毒的模型小鼠；中剂量组：注射1.1E13vg/kg重组AAV9病毒的模型小鼠；高剂量组：注射3E13vg/kg重组AAV9病毒的模型小鼠；野生型对照组：129野生型小鼠。
图7A-7D显示，在GAA -/-模型小鼠体内评价实验1中，在单次IV注射施用重组AAV9病毒rAAV9-CAR-Mut-coGAA-2×142-3P后的组织病理学染色分析。图7A显示肝脏组织H&E染色结果(上排：放大100倍；下排：放大400倍)；图7B显示心肌细胞H&E染色结果；图7C显示骨骼肌细胞H&E染色结果；图7D显示骨骼肌和心肌细胞的PAS染色。其中，Gaa -/-：PBS给药组的模型小鼠组织切片；LD：给予低剂量重组AAV9病毒的模型小鼠组织切片；MD：给予中剂量重组AAV9病毒的模型小鼠组织切片；HD：给予高剂量重组AAV9病毒的模型小鼠组织切片；AAV treated：经重组AAV9病毒治疗的模型小鼠的组织切片。
图8A-8C显示，在GAA -/-模型小鼠体内评价实验2中，在单次IV注射施用重组AAV9病毒rAAV9-CAR-Mut-coGAA-2×142-3P后的组织病理学染色分析。图8A显示脑组织PAS染色结果；图8B显示脊髓组织PAS染色结果；图8C显示小脑组织PAS染色结果。其中，Gaa -/-：PBS给药组的模型小鼠组织切片；WT：129野生型小鼠组织切片；3E13vg/kg：注射3E13vg/kg重组AAV9病毒的模型小鼠组织切片；和6.8E13vg/kg：注射6.8E13vg/kg重组AAV9病毒的模型小鼠组织切片。
图9显示，在GAA -/-模型小鼠体内评价实验2中，在单次IV注射施用重组AAV9病毒rAAV9-CAR-Mut-coGAA-2×142-3P后，在脑组织中测定的GAA酶活力水平。其中，模型对照：PBS给药组的模型小鼠；6.8E13vg/kg：注射6.8E13vg/kg重组AAV9病毒的模型小鼠。
图10显示，在GAA -/-模型小鼠体内评价实验4中，在单次IV注射PBS或重组AAV9病毒rAAV9-CAR-Mut-coGAA-2×142-3P后，记录的小鼠生存曲线。其中，Gaa -/-：PBS给药组的模型小鼠；AAV治疗1.1E13vg/kg：注射1.1E13vg/kg重组AAV9病毒的模型小鼠。
发明详述
本发明公开了用于治疗庞贝氏病或酸性葡萄糖苷酶缺陷受试者的基因治疗构建体、药物组合物和方法，尤其是用于递送GAA的重组AAV载体的构建、制备及应用。
除非下文中另外定义，否则本说明书中所用的全部技术与科学术语具有如本发明所属领域的普通技术人员通常理解的相同含义。本文所提及的全部出版物、专利申请、专利和其他参考文献通过引用的方式完整地并入。此外，本文中所述的材料、方法和例子仅是说明性的并且不意在是限制性的。本发明的其他特征、目的和优点将从本说明书及附图并且从后附的权利要求书中显而易见。
定义
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。该术语也旨在涵盖在指定数字±1％,±0.5％，或±0.1％范围内的数值。
在本文中，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。
在本文中，表述“第一”、“第二”或“第三”等用于在所述及的要素之间进行区分，并且除非另有说明，这些表述并不指示要求所述要素具有特定的数量或以任何特定顺序或位置存在。
在本文中，表述“和/或”用于表示所列相关项目中的任何一个、或所列相关项目中的多个的任何和所有可能的组合。
在本文中，重组腺相关病毒可以用衣壳的来源AAV病毒血清型单独来表示，或用衣壳和基因组ITR序列的来源AAV病毒血清型来表示。在后一情况下，在本文中，采用标识符“/”进行分隔，标识符“/”之后为衣壳的来源血清型，标识符“/”之前为ITR的来源血清型。因此，例如，表述重组AAV9中的数字9表示所述重组腺相关病毒具有来自AAV9血清型的衣壳；而表述重组AAV2/9中标识符“/”前的数字表示所述重组腺相关病毒具有来自AAV2的野生型或变体ITR序列，而标识符“/”后的数字表示所述重组腺相关病毒具有来自AAV9的衣壳蛋白。
术语“酸性α-葡萄糖苷酶”或“酸性葡萄糖苷酶”或GAA在本文中可互换使用，指：能在麦芽糖和其他线性寡糖中水解α-1-4键从而降解溶酶体中过量糖原的溶酶体酶。当GAA编码基因在细胞中表达时，GAA多肽将在胞质中合成，并在ER中被糖基化，在N端连接上高甘露糖型的糖链。在高尔基体中，GAA上的高甘露糖糖链可以进一步被修饰而添加甘露糖-6-磷酸(M6P)。通过M6P与M6P受体相互作用，GAA被递送到溶酶体中，并在其中发挥糖原降解功能。
GAA的例子包括但不限于，具有全长野生型(天然)人GAA(如Unipro数据库登录号UniProtKB-P10253所示)的氨基酸序列的酶蛋白、其成熟形式、其变体(例如，具有保守氨基酸置换的变体)、及其片段。 人GGA在氨基酸残基516-521具有保守六肽WIDMNE，该六肽是GAA蛋白活性所必需的。在本文中，也可以应用GAA的变体和片段，只要所述变体或片段保留水解糖原的活性，并例如提供至少约50％、至少约60％、至少约70％、至少约75％、至少约80％、至少约90％、或大约相同、或大于100％的全长野生型(天然)人GAA的酶活性水平。
在本发明的一个实施方案中，GAA多肽包含SEQ ID NO:13的氨基酸序列、或SEQ ID N:13的残基70-952的氨基酸序列；SEQ ID NO:13的残基123-952的氨基酸序列，SEQ ID NO:13的残基204-952的氨基酸序列，或与前述序列之任一具有至少90％，或至少95％，96％，97％，98％，99％或更高同一性的氨基酸序列。人GAA多肽的头27个氨基酸是溶酶体蛋白和分泌蛋白的典型信号肽。GAA可以通过该信号肽靶向溶酶体。因此，在一个实施方案中，本发明GAA多肽包含靶向溶酶体的信号肽，例如来自人GGA多肽的天然信号肽序列。在另一实施方案中，本发明GAA多肽包含来自异源溶酶体靶向蛋白的信号肽。
在本发明的一些实施方案中，编码GAA多肽的多核苷酸序列包含野生型GAA核酸序列。在本发明的再一实施方案中，编码GAA多肽的多核苷酸序列是人密码子优化的(即，为了在人类细胞中表达而被密码子优化)，以用于例如增强所述多核苷酸在体内的表达和/或稳定性。优选地，编码GAA的多核苷酸序列包含SEQ ID NO:10的多核苷酸序列。
术语“ETR”或“酶替换治疗”在本文中是指，用于庞贝氏病或酸型葡萄糖苷酶缺陷治疗的一种治疗程序，其中重组GAA蛋白被施用于有需要的受试者。用于ETR的重组GAA蛋白可以在工程化哺乳动物细胞系例如CHO细胞中产生，或在转基因动物如转基因兔的奶中产生。
如本文中所用，术语“保守性”氨基酸或核苷酸改变是指，导致包含所述氨基酸或核苷酸改变的蛋白质或核酸分子实质性地保持原有功能的中性或近中性氨基酸或核苷酸改变。例如，保守性氨基酸置换是将氨基酸置换或取代成侧链具有相似生物化学性质(例如电荷、疏水性和大小)的不同氨基酸。这类保守性修饰的变体相对于多态性变体、物种间同源物和等位基因而言是附加的并且不排斥它们。以下8组含有互为保守替换的氨基酸：1)丙氨酸(A)、甘氨酸(G)；2)天冬氨酸(D)、谷氨酸(E)；3)天冬酰胺(N)、谷氨酰胺(Q)；4)精氨酸(R)、赖氨酸(K)；5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V)；6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)；7)丝氨酸(S)、苏氨酸(T)；和8)半胱氨酸(C)、甲硫氨酸(M)(参阅例如，Creighton,Proteins(1984))。本领域技术人员可以通过常规技术手段，例如功能性测定试验，容易地检测一个特定多肽序列或核苷酸序列中的氨基酸或核苷酸改变的保守性。
术语“功能性连接”也称作“有效连接”，意指指定的各组分处于一种允许它们以预期的方式起作用的关系中。
术语序列“同一性”用于描述两个氨基酸序列或多核苷酸序列之间的序列结构相似性。为确定两个氨 基酸序列或两个核酸序列的同一性百分数，可以将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在http://www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。在又一个优选的实施方案中，使用GCG软件包中的GAP程序(在http://www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum 62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4)，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
术语“宿主细胞”指已经向其中引入外源多核苷酸的细胞，包括这类细胞的子代。在一些实施方案中，宿主细胞是可以用来产生本发明重组AAV载体的任何类型的细胞系统，例如，哺乳动物细胞(例如适用于通过三质粒包装系统生产重组AAV的HEK 293细胞)和昆虫细胞(例如适用于通过杆状病毒包装系统生产重组AAV的sf9细胞)。
术语“调控序列”或“表达控制序列”是指这样的核酸序列，其诱导、抑制或以其它方式控制与之有效连接的编码核酸序列的蛋白质转录。调控序列可以是例如起始序列、增强子序列、内含子序列和启动子序列等。
描述核酸或蛋白质时所用的术语“外源的”或“异源的”可互换使用，是指核酸或蛋白质不是天然存在于其存在的染色体或宿主细胞的位置。外源核酸序列也指衍生自并插入相同宿主细胞或受试者但以非天然状态存在的序列，例如，所述序列以不同的拷贝数存在，或处于不同调控元件的控制下。
在本文中，“分离的”多核苷酸(例如，分离的DNA或分离的RNA)是指，多核苷酸至少部分地从包含其的天然生物体或病毒的至少一些其他组分中分离出来。在一些实施方案中，“分离的”核酸相对于起 始材料被富集了至少大约10倍、100倍、1000倍、10,000倍或更多。
在本文中，“分离的”多肽是指多肽至少部分地从包含其的天然生物体或病毒的至少一些其他组分中分离出来。在一些实施方案中，“分离的”多肽相对于起始材料被富集了至少大约10倍、100倍、1000倍、10,000倍或更多。
在本文中，“分离”或“纯化”病毒载体是指，病毒载体从包含其的起始材料的至少一些组分中被部分地分离出来。在一些实施方案中，“分离的”病毒载体相对于起始材料被富集了至少大约10倍、100倍、1000倍、10,000倍或更多。
在本文中，术语“病毒载体”是指，能够作为目的核酸的运载工具的病毒颗粒(例如AAV病毒颗粒)。通常，病毒载体包含衣壳和包装在其中的病毒基因组(例如，病毒DNA)，待递送的目的核酸插在病毒基因组中。在重组AAV病毒载体的情况下，为了产生可以将目的核酸递送至组织或细胞的重组病毒颗粒，通常仅需要在基因组中保留反向末端重复(ITR)顺式元件，而病毒包装所需的其余序列可以反式提供。因此，在一些实施方案中，本发明的重组AAV病毒载体包含衣壳和包装在其中的重组病毒基因组，其中所述重组病毒基因组包含或由位于两个AAV ITR序列之间的一个或多个外源核苷酸序列组成。位于重组病毒基因组5’和3’末端的两个ITR序列(即，5’ITR和3’ITR)可以相同或不同。
术语AAV“反向末端重复”(inverted terminal repeat,ITR)在本文中是指，来自AAV病毒基因组的顺式作用元件，在AAV病毒的整合、拯救、复制和基因组包装中发挥重要作用。天然AAV病毒的ITR序列中包含Rep蛋白结合位点(Rep binding site，RBS)和末端解链位点trs(terminal resolution site)，能够被Rep蛋白结合识别并在trs处产生切口。该ITR序列还可形成独特的“T”字母型二级结构，在AAV病毒的生活周期中发挥重要作用。最早分离到的AAV病毒，AAV2，具有位于基因组两端、长度145bp的呈回文-发卡结构的“反向末端重复序列”(ITR)。之后，在各种血清型的AAV病毒中发现不尽相同的ITR序列，但是都能形成发卡结构和存在Rep结合位点。基于这些野生型ITR序列的传统重组AAV病毒载体一般为单链AAV载体(ssAAV)，病毒基因组以单链形式包装在AAV衣壳中。与此类ssAAV不同，已经发现，通过改造ITR，删除AAV病毒的一侧ITR序列中的trs序列和任选地D序列，能够使包装得到的重组AAV病毒载体所携带的基因组自我互补，形成双链(Wang Z等人，Gene Ther.2003；10(26):2105-2111；McCarty DM等人，Gene Ther.2003；10(26):2112-2118)。由此包装得到的病毒为双链AAV病毒，即，scAAV(self-complementary AAV)病毒。scAAV病毒载体的包装容量更小，仅为ssAAV病毒载体包装容量的一半，约为2.2kb-2.5kb，但感染细胞后转导效率更高。
在本文中，与AAV相关的该术语ITR涵盖野生型ITR和变体IRT。野生型ITR可以来自任何天然AAV病毒，例如AAV2病毒。野生型ITR中包含Rep蛋白结合位点(Rep binding site，RBS)和末端解链位 点trs(terminal resolution site)，能够被Rep蛋白结合识别并在trs处产生切口。野生型ITR序列可形成独特的“T”字母型二级结构，在AAV病毒的生活周期中发挥重要作用。在本文中，变体ITR是非天然的ITR序列，其可以例如来自任何野生型AAV ITR序列，并相对于野生型ITR包含一个或多个核苷酸的缺失、替代、和/或添加，和/或截短，但仍具有功能性，即，能够用于产生ssAAV病毒载体或scAAV病毒载体。在一些实施方案中，变体ITR是缺失了功能性trs位点和任选地D区序列的AAV ITR序列(在本文中，也称作ΔITR)。在一些实施方案中，野生型ITR与ΔITR组合用于产生自我互补型重组AAV病毒载体(scAAV)。在另一些实施方案中，两个野生型ITR组合用于产生单链重组AAV病毒载体(ssAAV)。
AAV蛋白VP1,VP2和VP3是衣壳蛋白，其相互作用以形成AAV衣壳。不同血清型的AAV病毒具有不同的组织感染嗜性，可以通过选择重组AAV病毒载体衣壳的来源血清型，将外源基因转运至特定的器官和组织(Wu Z等人，Mol Ther.2006；14(3):316-327)。在本发明中，重组AAV病毒载体可以通过选择衣壳的来源血清型，而具有不同的靶向性。在一些实施方案中，重组AAV病毒的衣壳来自对神经元细胞具有靶向性的AAV血清型。在一个实施方案中，重组AAV病毒载体包含来自AAV9的衣壳。在再一个实施方案中，重组AAV病毒载体包含来自AAV9的衣壳和来自AAV2的ITR。
术语“免疫相关miRNA”是优先在免疫系统细胞，例如抗原提呈细胞中表达的miRNA。在一些实施方案中，免疫相关miRNA在免疫细胞中的表达水平，相对于其在非免疫细胞(例如参照细胞，例如HEK293细胞)中的表达水平高，尤其是高至少2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、或10倍。在一些实施方案中，表达免疫相关miRNA的免疫系统细胞是B细胞、T细胞、T杀伤细胞、T辅助细胞、树突细胞、巨噬细胞、单核细胞、血管内皮细胞、或其他免疫细胞。在一些实施方案中，免疫相关miRNA是miR-142-3P。miR-142-3p是一种miRNA，其在造血干细胞系来源细胞中高表达。免疫细胞均分化来源于造血干细胞系，因此利用miRNA抑制基因表达的原理，携带miR-142-3p靶序列的基因表达会在免疫细胞中受到明显抑制，从而降低机体产生针对基因表达产物免疫反应的概率。
术语“治疗”指意欲改变正在接受治疗的个体中疾病之天然过程的临床介入。想要的治疗效果包括但不限于防止疾病出现或复发、减轻症状、减小疾病的任何直接或间接病理学后果、防止转移、降低病情进展速率、改善或缓和疾病状态，以及缓解或改善预后。在一些实施方案中，本发明重组AAV病毒在施用给庞贝氏病或GAA缺陷受试者后，优选在全身施用后，降低受试者的多个受累组织(尤其是，骨骼肌、心肌、膈肌和中枢神经系统)中的溶酶体糖原贮积量。在一些实施方案中，本发明重组AAV病毒在施用给庞贝氏病或GAA缺陷受试者后，优选在全身施用后，改善受试者的中枢神经系统损伤。在一些实施方案中，本发明重组AAV病毒在施用给庞贝氏病或GAA缺陷受试者后，优选在全身施用后，改善受试者的骨骼肌、心肌损伤。在一些实施方案中，本发明重组AAV病毒在施用给庞贝氏病或GAA缺陷受试者后， 优选在全身施用后，改善受试者的神经系统(包括大脑、脊髓和/或小脑组织)中疾病所致的病理变化。在一些实施方案中，改善脑组织中胶质细胞的糖原累积。在另一实施方案中，本发明重组AAV病毒在施用给庞贝氏病或GAA缺陷受试者后，优选在全身施用后，延长受试者的生存期。
在本文中，“预防”包括对疾病或特定疾病的症状的发生或发展的抑制。在一些实施方式中，具有庞贝氏病发生倾向的受试者是预防性方案的候选者。通常，术语“预防”是指在疾病的至少一个症状发生前实施的医院干预。因此，在一个实施方案中，预防包括在具有GAA基因缺陷的受试者中于庞贝氏病的症状发生前的本发明基因治疗药物的施用，以延缓疾病发展或阻止疾病的出现。
以下对本发明的各个方面进行描述。
I.用于基因治疗的构建体
组成型CAR-Mut启动子
启动子(promoter)是RNA聚合酶(RNA polymerase)识别、结合、开始转录的一段特定DNA序列。真核生物类别Ⅱ(classⅡ)启动子参与蛋白质编码基因的转录控制，通常位于基因编码区的上游，通过与转录因子(transcription factors,TFs)的相互作用调控基因转录的时机和部位。该类启动子包含5类作用元件：基本启动子、起始子、上游元件、下游元件和应答元件。这些元件的不同组合及序列的变化，赋予了对启动子功能活性的多重影响(汤方,涂慧珍.真核启动子研究进展[J].林业科技开发.2015,29(2):7-12.)。
在本发明的一个方面，提供了一种人工合成的突变组成型启动子CAR-Mut。本发明组成型启动子可以在多种组织中有效启动外源基因表达，因而尤其适用在本发明治疗方法中应用以纠正庞贝氏病的外周器官受累并减少中枢神经系统负担。
在一个实施方案中，本发明提供了一种突变启动子，其包含选自以下的多核苷酸：
(i)SEQ ID NO:4的多核苷酸，
(ii)与SEQ ID NO.4具有至少95％、96％、97％、98％、99％、99.5％同一性的多核苷酸，
(iii)在SEQ ID NO.4的多核苷酸中经取代、缺失或添加一个或几个核苷酸得到的多核苷酸，
且其中，所述多核苷酸在SEQ ID NO:4的核苷酸第562-572位或与之相应的位置具有突变，优选地所述突变为核苷酸568位或相应位置上的T突变为C或G或A，更优选为T突变为C。
在一个优选实施方案中，本发明的突变启动子，相对于由不具有所述突变的相应多核苷酸组成的参照启动子，增加与其功能性连接的目的基因的表达，例如，使所述目的基因表达增加1％-70％，例如，至少5％，10％，20％，30％，40％，或至少50％，60％。
在再一优选实施方案中，本发明的突变启动子，相对于参照启动子，增加与之功能性连接的目的基因 在哺乳动物细胞或组织中的表达，例如，增加所述目的基因在哺乳动物外周组织和/或中枢神经组织，尤其是选自心脏、肝脏和/或脑的哺乳动物组织中的表达。优选地，所述哺乳动物为人或非人哺乳动物，例如，小鼠、大鼠和非人灵长类动物。
在再一些实施方案中，所述启动子包含选自SEQ ID NOs:1至3之任一的核苷酸序列、或与之相差一个或几个核苷酸取代、缺失和/或添加且具有同等启动子活性的核苷酸序列。优选地，所述启动子包含或由SEQ ID NO:1的核苷酸序列组成。
本领域技术人员可以采用本领域已知的任何启动子功能性测定试验(例如实施例1的荧光素酶报告基因表达测定试验)，来确定任何两个启动子是否具有同等启动子活性。在一个实施方案中，在相同的测试条件下，与参考启动子(例如SEQ ID NO：1-3)相比，如果待测启动子具有相同或基本上相同的活性，例如参考启动子活性±10％、优选地±5％，或更优选±1％的活性，则可以认为待测启动子具有同等启动子活性。
在一些方面，本发明也涵盖包含所述启动子及与之功能性连接的编码核苷酸序列的表达盒、重组载体和宿主细胞、以及使用所述表达盒、载体或宿主细胞向哺乳动物细胞或个体递送编码多核苷酸的组合物和方法。
表达构建体
在一个方面，本发明提供了表达构建体。本发明的表达构建体包含本发明的启动子，并可以有利地用于GAA编码核酸序列在庞贝氏病患者或酸性葡萄糖苷酶缺陷患者的期望组织或细胞中的表达。
在一个实施方案中，本发明的表达构建体以转录方向彼此功能性连接的如下元件：
-本发明的任何CAR-Mut启动子，尤其是SEQ ID NO：1的启动子，
-任选地，Kozak序列，
-编码目的基因的多核苷酸，例如，编码α酸性葡萄糖苷酶(GAA)的多核苷酸序列，优选地人密码子优化的人GAA多肽编码序列，更优选地SEQ ID NO:10的序列，
-任选地，至少一个(例如2-4个)免疫相关的miRNA结合位点，尤其是miR-142结合位点，例如包含至少一个(例如一个或两个)SEQ ID NO:11序列的miR-142结合位点，
-任选地，转录终止子，例如polyA信号序列，优选地选自SV40晚期polyA序列、兔β-珠蛋白polyA序列、牛生长激素polyA序列、或其任何变体，更优选包含SEQ ID NO:13或与其具有至少95％同一性的牛生长激素polyA序列。
在一些实施方案中，表达构建体还包括两个ITR序列。例如，从5’末端到3’末端，表达构建体可以包含如下排列的元件：5’ITR-启动子-GAA编码序列-miRNA结合位点-polyA-3’ITR。在一些实施方案中， 5’ITR和3’ITR相同。在另一实施方案中，5’ITR和3’ITR不同，且其一(优选3’ITR)为缺少功能性trs位点的ΔITR。在一个实施方案中，表达构建体中的5’ITR和3’ITR相同，均包含或由SEQ ID NO:5序列组成。在再一实施方案中，表达构建体中的5’ITR和3’ITR不同，其中5’ITR包含或由SEQ ID NO:5序列组成，且3’ITR包含或由SEQ ID NO:6序列组成。
用于本发明表达构建体中的启动子可以是本发明上述任何实施方案中描述的CAR-Mut启动子。在一个优选的实施方案中，所述启动子包含或由SEQ ID No:1的核苷酸序列组成。在另一优选的实施方案中，所述启动子包含或由SEQ ID NO:2的核苷酸序列组成。在另一优选的实施方案中，所述启动子包含或由SEQ ID NO:3的核苷酸序列组成。
本发明的表达构建体在一个实施方案中可以包含位于GAA编码核酸序列的起始密码子上游的Kozak序列，以促进GAA的翻译。用于本发明的Kozak序列可以是定义为GCCRCC的共有序列，其中R是嘌呤(即A或G)，且其中所述序列位于起始密码子上游。在一个优选的实施方案中，在本发明表达构建体的核酸序列中，所述Kozak序列具有5’-GCCACC-3’序列。也可以使用其他不同的Kozak序列。可通过序列文库筛选Kozak序列，并且对翻译效率的增强作用可以使用本领域已知的常规方式进行评估。例如，可以构建包含具有不同Kozak序列的报告基因或重组GAA基因的重组核酸，将重组核酸引入宿主细胞，例如BHK细胞中，经一段时间后检测细胞或培养上清液中的报告基因表达水平或GAA酶活性水平，并与具有参考Kozak序列的重组核酸进行比较，以确定所测试的Kozak序列的翻译增强效率。
在一些实施方案中，本发明的表达构建体还包含一个或多个免疫相关的miRNA结合位点，即，miRNA靶序列，位于目的GAA编码核酸序列的3’UTR中。不受任何具体理论的约束，包括miRNA结合位点在表达构建体中允许对目的基因在产生相应miRNA的细胞和组织中的表达进行调节(例如，抑制)。由此，在一个实施方案中，本发明表达构建体包含一个或多个miRNA结合位点，从而可以以细胞类型特异性方式下调GAA的表达。在一个实施方案中，本发明表达构建体包含一个或多个miRNA结合位点，其中所述miRNA在抗原呈递细胞中表达，由此降低了本发明表达构建体在所述抗原呈递细胞中表达GAA的效率。在一些实施方案中，一个或多个miRNA结合位点位于GAA编码基因的3’非翻译区(3’UTR)中，例如，编码GAA的核苷酸序列的最后一个密码子和polyA序列之间。
在一些实施方案中，表达构建体包含一个或多个(例如,1,2,3,4,5或更多个)miRNA结合位点，所述miRNA结合位点下调GAA基因从免疫细胞(例如，抗原呈递细胞APC,例如巨噬细胞和树突细胞等)的表达。不受具体理论的约束，此类免疫相关miRNA结合位点在表达构建体中并入可以导致减少目的GAA基因在具有该miRNA的抗原呈递细胞中的表达，并由此减少或抑制受试者产生抗GAA免疫反应。
在一些优选的实施方案中，表达构建体包含一个或多个miR-142结合位点(在本文中也称作miR-142 靶序列)，例如SEQ ID NO:11的miR-142-3P靶序列，或其串联重复序列，例如2,3,4,5,6个串联重复,优选地2个串联重组，如SEQ ID NO：12的miR-142-3P靶序列。在一些实施方案中，所述miRNA结合位点可以降低重组AAV载体在抗原提呈细胞中的表达。在一些实施方案中，所述miRNA结合位点可以降低重组AAV载体的免疫原性。在一些实施方案中，包含所述miRNA结合位点的重组AAV载体在受试者中引发低的免疫反应。在另一些实施方案中，相对于不含miRNA结合位点的重组AAV载体对照，包含所述miRNA结合位点的重组AAV载体在施用后受试者中引发低的抗GAA血清效价。优选地，所述施用为静脉内施用。在一个实施方案中，在施用1-6周，例如5周后测量，确定抗GAA血清效价，优选地，相对于对照，该血清效价降低大约1至10倍，例如，大约2倍、3倍、4倍、5倍、6倍、7倍或8倍。
在一些实施方案中，本发明表达构建体包含至少一个polyA尾位于编码GAA和miRNA结合位点的多核苷酸下游。任何合适的polyA序列均可以使用，包括但不限于hGHpolyA,BGHpolyA,SV40晚期polyA序列、兔β-珠蛋白polyA序列、或其任何变体。在一个优选的实施方案中，polyA是BGHpolyA，例如SEQ ID NO:7所示的polyA，或与SEQ ID NO:7具有至少80％,85％,90％,95％,96％,97％,98％或99％的核苷酸序列同一性的polyA多核苷酸序列。
包含在本发明表达构建体中的GAA编码核酸可以是任何编码功能性GAA酶活性的多核苷酸。在一个实施方案中，所述核酸编码人全长GAA序列例如SEQ ID NO：13的序列，或其片段，例如起始于SEQ ID NO:14的残基1-204之间且终止于残基952、或相应位置的GAA酶片段。优选地，所述GAA包含靶向溶酶体的天然信号肽(即，在SEQ ID NO：13的情况下，氨基酸1-27的信号肽)。或者，所述GAA可以包含来自异源信号肽，例如来自人溶酶体靶向蛋白或分泌蛋白的信号肽。可在本发明中使用的异源信号肽的例子包括但不限于：来自免疫球蛋白(例如IgG)、细胞因子(例如IL-2)、胰岛素的信号肽。参见例如WO2018046774。
在一些实施方案中，本发明的表达构建体包含GAA编码核酸序列，其中所述核酸序列编码具有GAA酶活性的多肽，其中所述多肽包含：与SEQ ID NO：13的序列、或与SEQ ID NO:13的氨基酸70-952的序列、与SEQ ID NO:13的氨基酸123-952的序列、或与SEQ ID NO:13的氨基酸204-952的序列，具有至少95％，至少97％，至少98％，或至少99％或更高序列同一性的氨基酸序列。优选地，所述多肽与SEQ ID NO:13的参照GAA蛋白相比具有大约相同的糖原水解活性，例如所述多肽的GAA酶活性是参照GAA蛋白酶活性的至少大约95％、大约96％，大约97％、98％、99％或更高。用于测定GAA酶活性的测定试验是本领域已知的。本领域技术人员可以采用任何这样的测定试验确定可以用于本发明表达构建体、重组AAV病毒载体、以及方法和用途中的适宜GAA多肽。
为了有利于在人类细胞中的表达，用于编码GAA多肽优选地进行密码子优化。在一个实施方案中， 用于本发明表达构建体中的GAA编码核酸包含SEQ ID NO:13的多核苷酸序列，或与之具有至少大约95％、大约96％，大约97％、98％、99％或更高核苷酸序列同一性的多核苷酸序列。
在一些方面，本发明也提供包含本发明表达构建体的载体。在一些实施方案中，所述载体是质粒(例如用于重组病毒颗粒生产的质粒)。在另一些实施方案中，所述载体是病毒载体，例如重组AAV载体或杆状病毒载体。在一些实施方案中，重组AAV载体的基因组是单链的(例如单链DNA)。在一些实施方案中，重组AAV载体的基因组是自互补的。在再一些实施方案中，载体是杆状病毒载体(例如，苜蓿银纹夜蛾(Autographa californica)核多角体病毒(AcNPV)载体)。
在再一方面，本发明也提供了包含本发明的表达构建体或载体的宿主细胞，例如哺乳动物细胞或昆虫细胞。在一些实施方案中，所述细胞可以用于生产重组AAV病毒。
重组AAV载体
在一个方面，本发明提供了重组AAV载体。本发明的重组AAV载体尤其可用于治疗庞贝氏病或酸性葡萄糖苷酶缺陷。在一个实施方案中，重组AAV载体包含衣壳和位于衣壳中的核酸，在本文中也称作“重组AAV载体的基因组”。重组AAV载体的基因组包含多个元件，包括但不限于两个反向末端重复(ITR，即，5’-ITR和3’-ITR)，以及位于两个ITR之间的其它元件，包括启动子、异源基因、和polyA尾。优选地，两个ITR之间还可以包含至少一个免疫相关的miRNA结合位点。
在本文中，腺相关病毒(AAV)包括但不限于，任何血清型的AAV，例如1,2,3,4,5,6,7,8,9,10,11型AAV，及具有人工改变的衣壳蛋白的AAV。各种血清型和人工AAV的基因组序列及其天然反向末端重复(ITR)序列、Rep蛋白和衣壳cap蛋白是本领域已知的。这些序列可以在公开数据库例如GenBank或文献中找到。
在一些实施方案中，本发明提供包含衣壳的重组AAV病毒载体，其中所述衣壳由能够跨血脑屏障的衣壳蛋白，例如AAV9、AAVPHP.B、AAVPHP.eB衣壳蛋白构成。在一些实施方案中，本发明的重组AAV载体转导中枢神经系统(CNS)的神经元细胞，也转导外周非神经元细胞。在另一些实施方案中，重组AAV载体在全身给药后能靶向和转导肌细胞和神经元细胞。在另一实施方案中，重组AAV载体在全身给药后能靶向和转导受试者的外周器官和中枢神经系统。在再一实施方案中，重组AAV载体在全身给药后能靶向和转导受试者的多数组织(例如，脑、脊髓、骨骼肌、心脏、和肝脏)，且优选地，以未接受重组AAV载体施用的对照受试者相比，重组AAV载体在所述靶向和转导的组织中导致更高的目的外源基因(在本申请中GAA)的表达和/或酶活性。
在一些实施方案中，本发明的重组AAV载体具有来自AAV9血清型的衣壳(也本文中也称作AAV9载体)；优选地，所述重组AAV载体在其基因组具有来自AAV2的野生型或变体ITR序列(也在本文中也称作AAV2/9载体)。
在一些实施方案中，本发明的重组AAV载体的两个ITR序列均是全长ITR(例如长度为约125-145bp，并含有功能性的Rep结合位点(RBS)和末端解链位点(trs))。在一些实施方案中，全长功能性ITR被用于生产单链重组AAV载体(ssAAV)。在再一些实施方案中，所述重组AAV载体的ITR之一是截短的。在一些实施方案中，截短的ITR缺少功能性末端解链位点trs并被用于生产自我互补型重组AAV载体(scAAV载体)。
在一些实施方案中，本发明的重组AAV载体包含野生型AAV ITR，例如野生型AAV2 ITR，例如SEQ ID NO:5所示的ITR序列。在另一些实施方案中，本发明的重组AAV载体包含相对于野生型AAV ITR具有一个或多个修饰，例如核苷酸添加、缺失和/或替代的变体ITR，例如相对于野生型AAV2 ITR发生截短而缺失功能性trs位点的ΔITR，例如SEQ ID NO：6所示的ΔITR序列。
因此，在一个方面，本发明提供了一种重组腺相关病毒(AAV)载体，其中所述重组AAV载体在其基因组中包含：
a.5’和3’AAV反向末端重复(ITR)序列，和
b.位于5’和3’ITR之间的表达构建体，其中所述表达构建体包含以转录方向彼此功能性连接的如下元件：
-根据本发明的任何CAR-Mut启动子，尤其是SEQ ID NO：1的启动子，
-任选地，Kozak序列，
-编码人α酸性葡萄糖苷酶(GAA)的多核苷酸，
-任选地，至少一个(例如2-8个)免疫相关的miRNA结合位点，尤其是miR-142结合位点，例如包含至少一个(例如1个或2个)SEQ ID NO:11序列的miR-142结合位点，
-转录终止子，例如polyA信号序列，优选地选自SV40晚期polyA序列、兔β-珠蛋白polyA序列、牛生长激素polyA序列、或其任何变体。
在一些实施方案中，所述重组AAV载体中，编码GAA的多核苷酸为人密码子优化的，优选地所述密码子优化用于增强所述多核苷酸的体内表达效率和/或稳定性，更优选地，所述多核苷酸包含SEQ ID NO:10的序列。
在一些实施方案中，所述重组AAV病毒载体的两个ITR均为野生型AAV2 ITR序列，或所述ITR之一是缺少功能性末端解链位点(trs)的AA2ΔITR序列。
在一些实施方案中，所述重组AAV载体为ssAAV载体。在另一些实施方案中，所述重组AAV载体 是scAAV载体。
在一些实施方案中，所述重组AAV载体包含来自AAV9血清型的衣壳蛋白，优选地，所述重组AAV载体是AAV2/9载体。
II.重组AAV载体的制备
现有技术中对AAV载体具有相对成熟的包装系统，这便于规模化生产AAV载体。
目前常用的AAV载体包装系统主要包括三质粒共转染系统、腺病毒作为辅助病毒的系统、单纯疱疹病毒(Herpes simplex virus type 1，HSV1)作为辅助病毒的包装系统、以及基于杆状病毒的包装系统。每种包装系统都各具特点，本领域技术人员可以根据需要做出合适的选择。
三质粒转染包装系统因无需辅助病毒，安全性高，是应用最为广泛的AAV载体包装系统，也是目前国际上主流的生产系统。略显不足的是，高效大规模转染方法的缺失限制了三质粒转染系统在AAV载体大规模制备中的应用。
Yuan等建立以腺病毒为辅助病毒的AAV大规模包装系统(Yuan Z等人，Hum Gene Ther.2011；22(5):613-624)，该系统生产效率高，但包装系统中腺病毒在最后AAV成品中的痕量存在，影响了AAV成品的安全性。
HSV1作为辅助病毒的包装系统是另一类应用较为广泛的AAV载体包装系统。伍志坚和Conway等几乎同时在国际上提出了以HSV1为辅助病毒的AAV2载体包装策略(伍志坚，吴小兵等，科学通报，1999，44(5)：506-509；Conway JE等人，Gene Ther.1999，6：986-993)。随后Wustner等提出了以HSV1为辅助病毒的AAV5载体包装策略(Wustner JT等人，Mol Ther.2002，6(4)：510-518)。在此基础上，Booth等利用两个HSV1分别携带AAV的rep/cap基因和AAV的反向末端序列(Inverted terminal repeat，ITR)/外源基因表达框，然后用这两个重组HSV1病毒共同感染生产细胞，包装产生AAV病毒(Booth MJ,et al.Gene Ther.2004；11:829-837)。Thomas等进一步建立双HSV1病毒AAV生产的悬浮细胞系统(Thomas DL等人，Gene Ther.2009；20:861-870)，使更大规模的AAV病毒生产成为可能。
Urabe等利用三个杆状病毒分别携带AAV的结构基因、非结构基因和ITR/外源基因表达框，构建了AAV载体的杆状病毒包装系统。考虑到杆状病毒携带外源基因的不稳定性，随后减少了生产系统中所需杆状病毒的个数，逐渐从最开始的需要三个杆状病毒到需要两个或一个杆状病毒(Chen H.，Mol Ther.2008，16(5)：924-930；Galibert L.et al.，J Invertebr Pathol.2011；107Suppl:S80-93)以及一个杆状病毒组合一株诱导细胞株策略(Mietzsch M等人，Hum Gene Ther.2014；25:212-222，Mietzsch M等人，Hum Gene Ther.2015；26(10):688-697)。
本发明的重组AAV病毒载体可以使用本领域已知的任何合适的方法来生产。在一个实施方案中，本发明重组AAV病毒采用三质粒包装系统进行生产。在另一实施方案中，本发明重组AAV病毒采用杆状病毒包装系统进行生产。
因此，在一个方面，本发明提供了一种细胞，其包含：(i)编码一种或多种腺相关病毒rep蛋白和/或一种或多种腺相关病毒cap蛋白的第一载体；和(ii)包含本文中所述的本发明任何表达构建体的第二载体。本发明的细胞可以用于生产本发明重组AAV病毒载体的生产。
在再一方面，本发明也提供了一种生产重组AAV病毒载体的方法，其中所述方法包括步骤：
(i)提供细胞，其中所述的细胞包含：(i)编码一种或多种腺相关病毒rep蛋白和/或一种或多种腺相关病毒cap蛋白的第一载体；和(ii)包含本发明任何表达构建体的第二载体；
(ii)在允许包装重组AAV的条件下培养所述细胞；和
(iii)收获培养的宿主细胞或培养基以收集所述重组AAV病毒载体。
在上述细胞和生产方法的一个实施方案中，第一载体是质粒，且第二载体是质粒；所述细胞是哺乳动物细胞，任选地其中所述哺乳动物细胞是HEK293细胞。根据情况，细胞可以提供感染性重组AAV病毒粒子生产所需的其它功能，或部分功能。在细胞仅提供部分功能的情况下，在一些实施方案中，所述细胞还包含第三辅助质粒载体。通过瞬时共转染第一质粒载体、第二质粒载体，和/或第三辅助质粒，可以容易地制备本发明的细胞。在一些实施方案中，感染性AAV粒子生产所需的功能由腺病毒基因提供，其中第三辅助质粒提供腺病毒基因VA,E2A和E4；其余生产所需的腺病毒基因产物由稳定表达腺病毒E1基因的宿主细胞提供。参见例如T Matsushita等,Adeno-associated virus vectors can be efficientlyproduced without helper virus.Gene Therapy(1998)5,938–945.
在上述细胞和生产方法的另一个实施方案中，第一载体是杆状病毒载体且第二载体是杆状病毒载体；所述细胞是昆虫细胞，任选地其中所述昆虫细胞是sf9细胞。在一些实施方案中，AAV的Rep和Cap蛋白分别由分开的两个第一杆状病毒载体提供；在另一些实施方案中，AAV的Rep和Cap蛋白由一个第一杆状病毒载体同时提供。在一些实施方案中，可以通过例如Bac-to-AAV系统，产生分别编码目的GAA基因和AAV的Rep和Cap蛋白的两个杆状病毒，使用两个杆状病毒共感染草地贪夜蛾(Sf9)昆虫细胞而产生本发明的细胞。参见例如，Galibert L.et al.，J Invertebr Pathol.2011；107 Suppl:S80-93。
III.药物组合物
再一方面，本发明提供了包含本发明的重组AAV病毒载体的药物组合物。本发明的药物组合物优选地包含可药用赋形剂、稀释剂或载体。本发明的药物组合物可以配制为任何合适的制剂形式。
用于配制的合适可药用赋形剂、稀释剂或载体的实例在本领域中是众所周知的，包括例如，磷酸盐缓冲盐溶液，水，乳液，例如油/水乳液，各种类型的润湿剂，无菌溶液等。制剂可以通过常规方法配制，并以合适的剂量向受试者给药。合适配制的组合物的施用可以通过不同的方式来实现，例如。通过静脉内，腹膜内，皮下，肌肉内，局部或皮内给药。具体施用途径尤其取决于药物组合物中包含的载体的类型。剂量方案将由主治医师和其他临床因素决定。如医学领域众所周知的，任何一名患者的剂量取决于许多因素，包括患者的体型，体表面积，年龄，性别，所要施用的特定活性剂，所用的时间和途径，所用药物的种类和阶段。感染或疾病，一般健康状况、以及其他药物的联用。
在一些实施方案中，本发明的药物组合物可以包含第二活性剂。在一些实施方案中，第二活性剂是用于ERT的重组GAA蛋白，例如来自转基因动物奶或生产性哺乳动物细胞系的重组GAA蛋白。在一些实施方案中，第二活性剂是支气管扩张剂。
在另一些实施方案中，本发明的药物组合物可以包含能够降低药物施用时副反应(例如抗药免疫反应)的组分。在一些情况下，所述组分可以是免疫抑制剂。
本发明的药物组合物可以通过任何合适途径给药，包括全身给药和局部给药。在一个优选方案中，本发明药物组合物用于全身给药方式给药，尤其是静脉注射给药。因此，在一个实施方案中，本发明提供了包含本发明重组AAV载体的药物组合物，其中所述药物组合物是静脉注射制剂，或适用于配制为静脉注射制剂的冻干稳定制剂。在另一些实施方案中，本发明药物组合物适用于局部给药，例如直接施用予受试者的待治疗器官或组织中或附近。
IV.治疗方法
在另一方面，本发明涉及使用本发明的重组AAV载体或包含其的药物组合物治疗疾病的方法。在一个的实施方案中，所述疾病是庞贝氏病。在另一实施方案中，所述疾病是酸性α葡萄糖苷酶缺陷。在一个实施方案中，所述方法包括：将本发明的任何重组AAV载体或药物组合物施用给有需要的受试者。所述重组AAV载体或药物组合物可以通过任何适宜的途径施用，包括但不限于，肌内，皮下，脊髓内，脑室内，鞘内，静脉内，膈肌内，胸腔内，腹膜内。优选地，本发明的重组AAV载体或药物组合物通过全身给药方式，尤其是静脉内给药方式递送给受试者。在一些实施方案中，所述治疗是治疗性的。在另一些实施方案中，所述治疗是预防性的。在一些实施方案中，受试者是哺乳动物，其中所述哺乳动物尤其是人、灵长类、狗、马、牛，特别是人类受试者。
在涉及治疗庞贝氏病受试者的方法中，在一些实施方案中，治疗包括以下之任一项或多项：(1)阻止或延迟庞贝氏病的发作；(2)减轻庞贝氏病的严重程度；(3)减轻或阻止庞贝氏病的至少一个症状的出现和/ 或恶化；(4)改善庞贝氏病相关的神经变性和/或受试者行为；和(5)延长受试者的生存期。可以接受治疗的庞贝氏病受试者包括IOPD和LOPD患者。在一些实施方案中，受试者是IOPD患者。在再一些实施方案中，受试者是LOPD患者。
因此，在一个方面，本发明提供了本发明重组AAV病毒载体用于驱动编码α酸性葡萄糖苷酶(GAA)的多核苷酸在哺乳动物细胞(尤其是人细胞)中表达的用途，或在制备用于驱动编码α酸性葡萄糖苷酶(GAA)的多核苷酸在哺乳动物细胞或哺乳动物(尤其人)体内一种或多种组织或器官中表达的药物中的用途，
优选地，所述药物用于在哺乳动物的心脏、肝脏、肌肉、中枢神经系统(包括脑和脊髓)中表达GAA，
优选地，所述药物全身给药，例如腹膜内(i.p.)、肌内(i.m.)、动脉内或静脉内(i.v.)注射给药，优选地静脉内注射。
在再一方面，本发明提供了一种用于治疗庞贝氏病受试者或具有酸性葡萄糖苷酶缺陷的受试者的方法，和本发明的重组AAV载体在制备用于治疗庞贝氏病受试者或具有酸性葡萄糖苷酶缺陷的受试者的药物中的用途。所述治疗包括向所述受试者施用本发明的任何一个或多个重组AAV载体，优选地，通过全身给药，例如腹膜内(i.p.)、肌内(i.m.)、动脉内或静脉内(i.v.)注射给药，优选地静脉内注射，施用所述重组AAV载体。
在本发明治疗方法和用途的一些实施方案中，在施用本发明重组AAV载体后，GAA多肽在受试者的心脏、肝脏、肌肉、中枢神经系统(包括脑和脊髓)中表达。在再一些实施方案中，重组AAV载体施用导致受试者的骨骼肌、心肌、膈肌和中枢神经系统中的溶酶体糖原贮积量减少，且优选地不诱导或诱导低的免疫原性。在一些实施方案中，施用本发明重组AAV载体可以改善受试者的心脏、呼吸和/或骨骼肌功能。在再一些实施方案中，施用本发明重组AAV载体可以预防或改善受试者的中枢神经系统，例如脑、脊髓和/或神经元因糖原贮积而致的病变，例如进行性神经变性。在一些实施方案中，施用本发明重组AAV载体可以延长受试者的生存期。
因此，本发明也提供了如下方法和本发明重组AAV载体在制备用于如下方法的药物中的用途：
(1)在患有或有风险患有庞贝氏病风险或酸性葡萄糖苷酶缺陷的受试者中用于预防或减少受试者体内细胞的病理学溶酶体糖原过量贮积的方法；
(2)在患有或有风险患有庞贝氏病风险或酸性葡萄糖苷酶缺陷的受试者中用于预防或改善因溶酶体糖原过量贮积所致的心脏、呼吸和/或骨骼肌功能损伤的方法；
(3)在患有或有风险患有庞贝氏病风险或酸性葡萄糖苷酶缺陷的受试者中用于预防或改善因溶酶体糖原过量贮积所致的神经系统损伤的方法；
(4)在患有或有风险患有庞贝氏病风险或酸性葡萄糖苷酶缺陷的受试者中用于减轻因溶酶体糖原过 量贮积所致的中枢神经系统负担和纠正外周器官受累的方法；
(5)在患有或有风险患有庞贝氏病风险或酸性葡萄糖苷酶缺陷的受试者中用于延长受试者生存期的方法。
在本发明治疗方法和用途的一些实施方案中，本发明的重组AAV病毒载体与另一治疗药物或治疗程序组合施用。可以与本发明重组AAV载体组合施用的治疗药物或治疗程序可以选自免疫调节剂、支气管扩张剂、乙酰胆碱酯酶抑制剂、呼吸肌强度训练(RMST)、酶替换治疗(ERT)、和/或膈肌起搏治疗。
实施例
下面对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。如无特殊说明，实施例中涉及的各种反应试剂均可以通过商业渠道购买得到。
实施例1：CAR-Mut启动子构建和表征
1. CAR-Mut启动子构建
在由人CMV病毒的增强子序列和鸡β-actin蛋白的基础启动子组成的CA启动子上，在序列3’端引入人TATA盒结合蛋白相关因子1基因(GenBank:NG_012771.2)中第62804位至62890位的内含子序列，得到命名为CAR启动子。对CAR启动子进行改造，将启动子末端第568位的T突变为非T核苷酸，获得CAR-Mut启动子，即，CAR-MutC(具有突变T568C，序列如SEQ ID NO:1所示)，CAR-MutA(具有突变T568A，序列见SEQ ID NO:2所示),和CAR-MutG(具有突变T568G，序列见SEQ ID NO:3所示)。
2.CAR-Mut启动子体外活性的表征
为表征启动子CAR-Mut，构建了图1A所示的pscAAV-CAR-Gluc质粒载体，包含：
i)来自AAV2基因组(GenBank No.AF043303)3’端的ITR，序列如SEQ ID NO:5所示；
ii)CAR启动子，序列如SEQ ID NO:4所示；
iii)Gluc，编码荧光素酶报告基因的核苷酸序列；
iv)牛生长激素的多聚核苷酸加尾信号，也缩写为BGH polyA；
v)以AAV2基因组(GenBank No.AF043303)3’端ITR序列为基础，缺失该序列中的trs序列和D序列，得到的ΔITR，序列如SEQ ID NO:6所示。
以pscAAV-CAR-Gluc质粒(图1A)为基础，用CAR-Mut启动子(SEQ ID No.1,2,或3)替换pscAAV载体中的CAR启动子，得到pscAAV-CAR-Mut-Gluc(图1B-1D)载体。简言之，合成CAR-Mut启动子序列并在两端分别添加XhoI和KpnI酶切位点。将合成的序列克隆入pUC57simple载体(金斯瑞生物科技，南京)，得到pUC57-CAR-Mut。用XhoI和KpnI分别双酶切消化pUC57-CAR-Mut载体和pscAAV-CAR-Gluc载体，回收CAR-Mut片段和切去了CAR启动子的pscAAV载体片段，连接后转化E.coli DH5α感受态细胞(擎科新业，北京)，筛选、鉴定后得到含有CAR-Mut启动子的AAV质粒载体pscAAV-CAR-Mut-Gluc。
将生长良好的BHK-21细胞传代至24孔板，待密度达到60％时，利用Lipofectamine2000(Invitrogen,美国)，根据厂商说明书，转染pscAAV-CAR-Gluc、pscAAV-CAR-MutC-Gluc、pscAAV-CAR-MutA-Gluc和pscAAV-CAR-MutG-Gluc各3孔。转染48小时后每孔取上清液100μL，用Glomax96微孔板光度计(Promega)检测Gluc水平，并使用检测仪软件进行数据分析。
结果(图2)比较得出，与空白(未转染质粒的BHK-21细胞)相比，在转染质粒pscAAV-CAR-Gluc与pscAAV-CAR-Mut-Gluc后，细胞Gluc表达水平极显著的增加，且转染pscAAV-CAR-MutC-Gluc后Gluc水平相比转染pscAAV-CAR-Gluc提升29.8％。pscAAV-CAR-MutA-Gluc、pscAAV-CAR-MutG-Gluc与pscAAV-CAR-MutC-Gluc无明显差异。
这说明，CAR启动子经568位碱基替换后具有增加的功能。
3.CAR-Mut启动子体内活性的表征
以CAR-Mut启动子CAR-MutC为代表，检测了包含CAR-Mut启动子的重组AAV病毒在动物体内的功能活性。
(1)重组AAV病毒制备
应用三质粒包装系统包装和纯化重组AAV病毒，得到rscAAV9-CAR-Mut-Gluc和rscAAV9-CAR-Gluc重组病毒。
首先，构建AAV的Rep和Cap蛋白表达质粒pAAV-R2C9。以AAV Helper Free System(Agilent Technologies，目录号#240071)中的pAAV-RC质粒为基本骨架，采用标准的分子克隆方法，用合成的AAV9基因组中的衣壳蛋白编码序列(也称为Cap9)替换pAAV-RC质粒中HindIII至PmeI限制性酶切位点之间的序列，获得了pAAV-R2C9质粒。所述pAAV-R2C9质粒包含完整的AAV9的cap基因和AAV2的rep基因，在三质粒共转染包装制备重组AAV9病毒时提供包装所必须的4种Rep蛋白(Rep78、Rep68、Rep52和Rep40)和AAV9衣壳蛋白。
将之前构建的AAV载体质粒(pscAAV-CAR-Gluc与pscAAV-CAR-Mut-Gluc)、辅助质粒(pHelper,来自AAV Helper Free System，Agilent Technologies)和AAV的Rep及Cap蛋白表达质粒pAAV-R2C9，按照1:1:1的摩尔比混匀后，采用磷酸钙方法转染HEK293细胞。转染48h后，收获细胞和培养上清，应用氯化铯密度梯度离心法分离纯化重组AAV病毒，得到rscAAV9-CAR-Gluc与rscAAV9-CAR-Mut-Gluc。
(2)重组AAV病毒的滴度检测
采用点杂交方法测定制备得到的重组AAV病毒(rAAV)的基因组滴度。具体过程如下：
在CAR-Mut启动子中设计两条引物CAR-Mut-F与CAR-Mut-R：
CAR-Mut-F：5’-GTTCCCATAGTAACGCCAATAGGG-3’(SEQ ID NO:8)
CAR-Mut-R：5’-CCCATAAGGTCATGTACTGGGCAT-3’(SEQ ID NO:9)
以CAR-Mut-F与CAR-Mut-R为引物利用PCR法特异性地扩增CAR-Mut启动子得到长度为175bp的DNA探针片段，使用pscAAV-CAR-Mut-Gluc质粒及其2倍比梯度稀释液为标准品，将rAAV样品2倍比梯度稀释为检测样品。将标准品和检测样品点在杂交膜上，用探针对膜进行杂交。操作过程详见分子克隆实验指南(第四版)。使用ImigeJ软件灰度扫描，比较样品点与系列标准点的杂交信号，分析计算rAAV样品滴度。
(3)重组AAV病毒的体内功能活性表征
将携带CAR及CAR-Mut启动子的重组AAV载体注射小鼠后检测Gluc水平，以对启动子的功能活性进行表征。具体而言，6周龄C57BL/6J野生小鼠共18只，随机分为3组。第1组小鼠尾静脉注射rscAAV9-CAR-Gluc，剂量为1×10 13GC/kg(genome copies/kg)。第2组小鼠尾静脉注射rscAAV9-CAR-Mut-Gluc，剂量为1×10 13GC/kg。第3组小鼠每只尾静脉注射200μL PBS作为对照。注射后1个月处死全部小鼠，解剖分离每只小鼠的脑组织、心脏、肝脏。取等质量的组织，提取组织总蛋白。利用Pierce BCA Protein Aaasy Kit(ThermoFisher,美国)分别测定各组的总蛋白浓度，详细过程参考试剂盒说明书。所有小鼠的组织均取50μl蛋白用Glomax96微孔板光度计检测Gluc水平。
结果(图3A,B,C)显示在注射两种AAV载体后，小鼠各组织能够有效的表达Gluc，注射rscAAV9-CAR-MutC-Gluc的小鼠组织Gluc水平相比注射rscAAV9-CAR-Gluc在多种组织内表达水平均显著提升，其中心脏提升34.7％(图3A)，肝脏提升47.6％(图3B)，脑提升48.0％(图3C)。
实施例2.用于庞贝氏病治疗的重组AAV的构建
1.AAV质粒载体的构建
在本实施例中构建了包含目的基因GAA和目的基因表达调控元件、以及ITR序列的AAV质粒载体。
首先以pRDAAV-CMV-EGFP(图4A)为基础，用CAR-MutC启动子(SEQ ID No.1)替换pRDAAV载体中的CMV启动子，得到pRDAAV-CAR-Mut-EGFP载体(图4B)。pRDAAV-CMV-EGFP质粒载体包含：
i)来自AAV2基因组的ITR，序列如SEQ ID NO:5所示；
ii)组成型CMV启动子；
iii)表达增强型绿色荧光蛋白EGFP的核苷酸序列；
iv)牛生长激素的多聚核苷酸加尾信号BGH polyA；
v)来自AAV2基因组的ITR，序列如SEQ ID NO:5所示。
在CAR-MutC启动子序列(SEQ ID No.1)的两端分别添加XhoI和KpnI酶切位点。添加酶切位点后序列由金斯瑞生物科技有限公司合成，合成序列克隆入pUC57simple载体(金斯瑞生物科技，南京)，得到pUC57-CAR-Mut。用XhoI和KpnI分别双酶切消化pUC57-CAR-Mut载体和pRDAAV-CMV-EGFP载体，回收CAR-Mut启动子片段和切去了CMV启动子的pRDAAV-CMV-EGFP载体片段(约6.9kb)，两片段连接后转化E.coli DH5α感受态细胞(擎科新业，北京)，筛选、鉴定后得到含有CAR-Mut启动子的AAV质粒载体pRDAAV-CAR-Mut-EGFP(图4B)。
接下来，将人工合成的密码子优化的人类GAA编码核苷酸序列(以下简称coGAA)克隆入pRDAAV-CAR-Mut-EGFP载体的KpnI和EcoRI酶切位点之间，得到pRDAAV-CAR-Mut-coGAA载体(图4C)。具体而言，由金斯瑞生物科技有限公司合成密码子优化的人类GAA基因的cDNA序列(coGAA，序列见SEQ ID No.10)，并在合成序列的coGAA序列上游加入KpnI酶切位点与Kozak序列5’-GCCACC-3’，在下游加入taa终止密码子及EcoRI酶切位点。合成后的序列克隆入pUC57simple载体(金斯瑞生物科技，南京)，得到pUC57-coGAA载体。KpnI和EcoRI分别双酶切消化pUC57-coGAA载体和pRDAAV-CAR-Mut-EGFP载体，回收coGAA片段和去除了EGFP报告基因的pRDAAV-CAR-Mut-EGFP载体片段，两片段连接后转化E.coli DH5α感受态细胞(擎科新业，北京)，筛选、鉴定后得到pRDAAV-CAR-Mut-coGAA载体(图4C)。
接着，将人工合成的miR-142-3pT片段(包含串联的两个miR-142-3p靶序列，序列信息见SEQ ID No.12)克隆入pRD.AAV-CAR-Mut-coGAA载体的EcoRI和SalI酶切位点之间得到pRD.AAV-CAR-Mut-coGAA-2×142-3P载体(图4D)。简言之，由北京擎科新业生物技术有限公司合成含有MicroRNA 142-3pT的oligo引物，退火后得到上游为EcoRI酶切位点，下游为SalI酶切位点的142-3pT片段。使用EcoRI和SalI消化pRDAAV-CAR-Mut-coGAA载体使其线性化，回收载体骨架与142-3pT片段连接后转化E.coli DH5α感受态细胞(擎科新业，北京)，筛选、鉴定后得到pRDAAV-CAR-Mut-coGAA-2×142-3P载体(图4D)。
2.重组AAV病毒的制备与检定
(1)重组AAV病毒的包装
采用Bac-to-AAV系统包装AAV病毒。简要地，实施如下操作：Sf9细胞培养、转染制备及鉴定分别编码目的GAA基因和AAV-Rep2/Cap9的两个杆状病毒、扩增两个杆状病毒、以两个杆状病毒共感染Sf9细胞、收获Sf9细胞沉淀、裂解细胞释放AAV病毒、超速离心纯化AAV病毒、膜包脱盐浓缩AAV病毒及除菌过滤，以得到重组AAV病毒rAAV9-CAR-Mut-coGAA-2×142-3P。
包装过程可以采用在Chen H.Intron Splicing-mediated Expression of AAV Rep and Cap Genes and Production of AAV Vectors in Insect Cells，[J].Molecular Therapy,the Journal of the American Society of Gene Therapy,2008,16(5):924和专利US8945918和CN101522903B中描述的方法进行。
(2)重组AAV病毒的滴度检测
采用实施例1中描述的点杂交方法，测定制备得到的rAAV基因组滴度。具体过程如下：
以CAR-Mut-F(SEQ ID NO:8)与CAR-Mut-R(SEQ ID NO:9)为引物利用PCR法特异性地扩增CAR-Mut启动子得到长度为175bp的DNA探针片段，使用pRDAAV-CAR-Mut-coGAA-2×142-3P质粒及其2倍比梯度稀释液为标准品，将rAAV样品2倍比梯度稀释为检测样品。将标准品和检测样品点在杂交膜上，用探针对膜进行杂交。使用ImigeJ软件灰度扫描分析计算rAAV样品滴度。
3.庞贝氏病基因治疗重组AAV病毒的体外表达
将BHK-21细胞铺到6孔板中。细胞汇合度约80％时消化计数，根据计数结果以每个细胞50000病毒颗粒计算所需病毒量，将病毒与新鲜培养基混匀后加至对应孔板。37℃孵育。感染后6-8h用含1％血清的新鲜培养基替换含有病毒的培养基。继续培养48h后，消化收集细胞。采用反复冻融后离心的方式提取细胞总蛋白。利用Pierce BCA Protein Aaasy Kit(ThermoFisher,美国)分别测定转染了rAAV9-CAR-Mut-coGAA-2×142-3P的细胞及空白细胞的总蛋白浓度，详细过程参考试剂盒说明书。
在提取细胞总蛋白后，将上述提取的蛋白分别取10ul，使用4-MUG为底物在酸性条件下反应1h后检测荧光值(激发365nm，发射450nm)，根据标准曲线计算生成的4-MU浓度，进一步计算得到样品GAA蛋白酶活。(参见，邱文娟等，2010，干血滤纸片和白细胞酸性α-葡萄糖苷酶活性测定平台的建立及临床应用)
结果见图5。在BHK-21细胞中，GAA酶活性为65.62±7.49nmol/h/mg protein。而转染了rAAV9-CAR-Mut-coGAA-2×142-3P质粒的BHK-21细胞的GAA酶活性为113.60±4.19nmol/h/mg protein，是空细胞的1.73倍(**,p<0.01)。证明庞贝氏病基因治疗重组AAV可以在转导细胞后表达具有活性的GAA蛋白。
实施例3.庞贝氏病基因治疗重组AAV病毒在模型小鼠体内的有效性评价
实验1：
8-10周龄的GAA基因纯合缺失的模型小鼠(GAA-KO小鼠，购自Jax lab，编号004154)32只，随机平均分为4组。其中1组为模型对照组，作为阴性对照，每只单次IV注射200uL PBS；另外三组为低剂量组、中剂量组和高剂量组，其作为实验组分别单次IV注射剂量为5E12vg/kg、1.1E13vg/kg、3E13vg/kg的rAAV9-CAR-Mut-coGAA-2×142-3P。另加入1组为野生对照组，以8只8-10周龄的129野生小鼠作为对照。注射后5周处死全部小鼠，解剖分离每只小鼠的心脏、肝脏、脾、肺、肾、及肌肉组织等。
GAA活性测定试验
取适量的不同组织，提取组织总蛋白。利用Pierce BCA Protein Aaasy Kit(ThermoFisher,美国)分别测定各组的总蛋白浓度，详细过程参考试剂盒说明书。所有小鼠的不同组织均取5ul总蛋白用于测定GAA酶活力，结果见图6。
从图6可见，注射PBS的模型小鼠由于缺乏GAA蛋白，酶活力极低；而在重组AAV给药组中，经过IV单次注射后，rAAV9-CAR-Mut-coGAA-2×142-3P能够广泛转导小鼠的外周组织并表达具有活性的GAA蛋白，且随注射剂量的升高，各组织的酶活力呈剂量依赖性提高。
组织病理学染色分析
取部分组织，切成合适尺寸后用4％多聚甲醛浸泡固定，做好标记送至龙麦达斯有限公司用作病理分析。结果见图7。
图7A显示了，经AAV9-CAR-Mut-coGAA-2×142-3P病毒载体静脉治疗后5周，肝脏组织H&E染色结果。Gaa -/-模型小鼠能够明显的看到因疾病影响造成了广泛的肝脏多灶性坏死(黑色箭头)，主要集中于中央静脉周围。给予不同剂量(LD，低剂量；MD，中剂量；HD，高剂量)AAV9-CAR-Mut-coGAA-2×142-3P的模型小鼠，可见肝脏坏死面积有极大地减少，未见明显核固缩,并且显示出剂量依赖的相关性。结果说明药物的肝脏治疗效果显著。
图7B显示Gaa -/-模型小鼠在IV单次注射给药后的心肌细胞H&E染色。在接受PBS给药的Gaa -/-模型小鼠组织切片上，可见大面积空泡样变性以及心肌壁血管淤血(黑色箭头)。经低、中、高剂量治疗的Gaa -/-模型小鼠的心肌细胞均有不同程度改善，并呈现剂量依赖关系。中、高剂量改善更为显著，未见心肌细胞空泡样变性和心肌臂血管明显淤血。结果说明药物对心肌病变的治疗效果显著。
图7C显示Gaa-/-模型小鼠在IV单次注射给药后的骨骼肌细胞H&E染色。PBS给药组的Gaa -/-模型小鼠组织切片上可见大面积空泡样变性(黑色箭头)。经低、中、高剂量治疗的Gaa-/-模型小鼠骨骼肌细胞 均有不同程度改善，并呈现剂量依赖关系。中、高剂量改善更为显著，未见显著的炎细胞浸润，并出现正常的肌纤维(红色箭头)，高剂量组的效果最为显著(图7C)。结果说明药物可以显著改善骨骼肌损伤，且无毒性改变，药物安全性良好。
图7D显示了在本实验前的探索性动物模型实验中Gaa -/-模型小鼠骨骼肌细胞、心肌细胞的PAS染色结果。PBS给药组的小鼠组织切片上可见大面积空泡样变性和糖原着色深染(左上图和左下图)。经静脉注射5E12vg/kg重组AAV治疗3个月后的Gaa -/-模型小鼠的骨骼肌、心肌均可见明显的糖原累积改善，并且肌纤维空泡样变性得以恢复(右上图和右下图)。因此，与前面图7A-7C的结果显示，图7D的结果也说明了本发明的重组AAV药物可以显著改善骨骼肌、心肌损伤，解决庞贝病的糖原累积病因。
实验2
按照以实验1基本相似的方式，处理8-10周龄的GAA基因纯合缺失的模型小鼠。简言之，模型小鼠随机平均分为3组(每组5只)。其中1组作为阴性对照组，每只单次IV注射200uL PBS；另外两组作为实验组分别单次IV注射剂量为3E13vg/kg和6.8E13vg/kg的rAAV9-CAR-Mut-coGAA-2×142-3P。另加入1组8-10周龄的129野生小鼠作为对照。注射后5周处死全部小鼠，解剖分离每只小鼠的大脑组织、脊髓及小脑组织。
组织病理学染色分析
取部分组织，切成合适尺寸后用4％多聚甲醛浸泡固定，做好标记送至龙麦达斯有限公司用作病理分析。
AAV9-CAR-Mut-coGAA-2×142-3P单次静脉注射后的大脑组织PAS染色结果显示在图8A中。结果表明，庞贝模型动物的脑组织中，胶质细胞出现广泛的糖原累积，导致大面积出现PAS染色阳性的区域(左上图，黑色箭头)，野生型小鼠未见此类现象(右上图)。经重组AAV药物干预的庞贝模型小鼠随剂量的提升，有效的改善了胶质细胞糖原累积的现象，于6.8E+13vg/kg剂量时检测不到糖原累积的胶质细胞(左下图和右下图)。
AAV9-CAR-Mut-coGAA-2×142-3P单次静脉注射后的脊髓组织PAS染色结果显示在8B中。结果表明，庞贝模型动物脊髓前脚存在较多PAS糖原强阳性细胞，PAS阳性神经元比例相对较多(左上图)，说明模型动物的脊髓存在糖原累积的现象，这与模型动物相关文献结果相符。WT小鼠的脊髓前角运动神经元仅个别存在糖原强阳性(右上图)，说明前脚运动神经元偶有糖原代谢旺盛的细胞个体。重组AAV治疗后，3E+13vg/kg剂量组PAS阳性神经元比例出现小幅度改变，剂量提升至6.8E+13vg/kg时，脊髓运动神经元PAS强阳性数量明显下降，仅个别神经元存在PAS糖原阳性现象，与WT的特征近似(左下图和右下图)。
AAV9-CAR-Mut-coGAA-2×142-3P单次静脉注射后的小脑组织PAS染色结果显示在8C中。结果表明，庞贝模型动物小脑浦肯野细胞糖原累积不明显，但是浦肯野细胞周围的胶质细胞存在较明显的糖原颗粒累积情况，小脑髓质区有明显的糖原累积特征(左上图，白色箭头)。施用重组AAV干预后，3E+13vg/kg的小脑组织中，浦肯野周围糖原阳性细胞数量有下降趋势(左下图)。剂量提升至6.8E+13vg/kg后，未检测到糖原的累积情况(右下图)。
图8A-8C的结果显示，本发明重组AAV药物能有效改善神经系统(包括大脑、脊髓和小脑组织)的疾病所致的病理变化。这说明，本发明重组AAV药物在IV注射后具有剂量依赖的中枢神经糖原清除能力，可以穿越血脑屏障，纠正细胞内糖原代谢障碍。
GAA活性测定试验
取适量的不同组织，提取组织总蛋白。利用Pierce BCA Protein Aaasy Kit(ThermoFisher,美国)分别测定各组的总蛋白浓度，详细过程参考试剂盒说明书。所有小鼠的不同组织均取5ul总蛋白用于测定GAA酶活力，结果见图9。
如图9所示，6.8E+13vg/kg剂量组显著提升脑组织GAA酶活力水平。庞贝模型小鼠脑组织中的GAA酶活力阴性。AAV9-CAR-Mut-coGAA-2×142-3P单次静脉注射后，能够在脑组织中检测到高于模型对照7～8倍的GAA酶活力水平(n＝5,p＜0.001)。说明，重组AAV病毒能够经血液系统穿越血脑屏障，将GAA表达载体递送至中枢神经系统，成功表达具有活性的GAA酶。因此，本发明重组AAV药物对于改善庞贝中枢神经系统的酶缺乏特征具有针对性的纠正作用。
实验3
构建不带有miRNA-142靶序列的对照重组AAV9病毒AAV9-CAR-Mut-coGAA，并与带有miRNA-142靶序列的重组AAV9病毒AAV9-CAR-Mut-coGAA-2×142-3P，在治疗效果和血清抗体效价上进行了比较。
基本按照类似于实施例1和2的方式，在重组AAV9病毒给药后，检查了治疗效果。
血清抗药抗体效价采用如下方式检测。在AAV施用后5周后，处死小鼠并取血，分离血清后使用ELISA检测小鼠血清样品中抗GAA抗体的效价。
结果显示，带与不带miRNA-142靶序列对于治疗效果无显著差异；但携带miRNA-142靶序列的重组AAV9病毒在IV施用后，抗药抗体滴度有所下降，其中携带mi142靶序列的重组AAV病毒试验组的抗体滴度为1:800，而不携带mi142靶序列的对照组的抗体滴度大于1：6400，表明mi142靶序列的加入削弱了药物相关抑制物的水平，有助于诱导免疫耐受。
实验4
8-10周龄的GAA基因纯合缺失的模型小鼠(GAA-KO小鼠，购自Jax lab)16只，并随机平均分为2组。其中1组作为阴性对照组，每只单次IV注射200uL PBS；另外1组作为实验组单次IV注射剂量为1.1E13vg/kg的rAAV9-CAR-Mut-coGAA-2×142-3P。观察其生存情况，记录生存曲线。结果见图10。
如图10所示，Gaa -/-模型小鼠以1.1E+13vg/kg治疗后，显著改善了庞贝模型小鼠的自然病史，中位存活期极大延长，相比模型动物中位生存期，治疗组存活率100％，结果说明药物发挥了预期的治疗作用，消除了疾病影响，使模型小鼠的生存状态与野生型正常小鼠无差异。
以上描述了本发明的示例性实施方案。本领域技术人员可以借鉴本文内容，适当改进工艺参数来实施本申请的发明。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明的范围内。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明的技术。
序列表：
Figure PCTCN2022095637-appb-000001
Figure PCTCN2022095637-appb-000002
Figure PCTCN2022095637-appb-000003

Claims (20)

  1. 一种突变启动子，其包含选自以下的多核苷酸：
    (i)SEQ ID NO:4的多核苷酸，
    (ii)与SEQ ID NO:4具有至少95％、96％、97％、98％、99％、99.5％同一性的多核苷酸，
    (iii)在SEQ ID NO:4的多核苷酸中经取代、缺失或添加一个或几个核苷酸得到的多核苷酸，
    且其中，所述多核苷酸在SEQ ID NO:4的核苷酸第562-572位或与之相应的位置具有突变，优选地所述突变为核苷酸568位或相应位置上的T突变为C或G或A，更优选为T突变为C，
    优选地，相对于由不具有所述突变的相应多核苷酸组成的参照启动子，所述突变启动子增加与其功能性连接的目的基因的表达，例如，使所述目的基因表达增加1％-70％，例如，至少5％，10％，20％，30％，40％，或至少50％，60％，
    再优选地，所述突变启动子为组成型启动子，其中相对于参照启动子，所述突变启动子增加所述目的基因在哺乳动物细胞或组织中的表达，例如，增加所述目的基因在哺乳动物外周组织和/或中枢神经组织，例如选自心脏、肝脏和/或脑的哺乳动物组织中的表达，优选地，所述哺乳动物为人或非人哺乳动物。
  2. 权利要求1的启动子，其中所述启动子包含选自SEQ ID NO:1至3之任一的核苷酸序列、或与之相差一个或几个核苷酸取代、缺失和/或添加且具有同等启动子活性的核苷酸序列，
    优选地，所述启动子包含或由SEQ ID NO:1的核苷酸序列组成。
  3. 一种表达构建体，其包含以转录方向彼此功能性连接的如下元件：
    -权利要求1或2任一项的启动子，
    -编码α酸性葡萄糖苷酶(GAA)的多核苷酸序列，优选地密码子优化的人GAA多肽编码序列，更优选的地SEQ ID NO:10的序列。
  4. 权利要求3的表达构建体，其在编码核酸序列之后还包含至少一个(例如2-6个，优选2个)免疫相关的miRNA靶序列，尤其是miR-142靶序列，例如包含至少一个(例如，2个或4个)SEQ ID NO:11序列的miR-142-3p靶序列，例如，SEQ ID NO:12所示的miR-142-3p靶序列，
    任选地，所述表达构建体还包含选自以下之一或多项：
    (1)位于编码核酸序列之前的Kozak序列，
    (2)转录终止子，例如polyA信号序列，优选地选自SV40晚期polyA序列、兔β-珠蛋白polyA序列、牛生长激素polyA序列，更优选牛生长激素polyA序列。
  5. 权利要求4的表达构建体，其中所述表达构建体还包含位于启动子上游的5’腺相关病毒反向末端重复(ITR)序列和位于转录终止子下游的3’腺相关病毒反向末端重复(ITR)序列，优选地，所述ITR序列是野生型ITR序列，或所述ITR之一是野生型ITR序列且所述ITR之另一是缺失了功能性末端解链位点(trs)和任选地D序列的ΔITR序列。
  6. 一种载体，其包含权利要求3-5任一项的表达构建体，其中所述载体为质粒或病毒载体，例如重组AAV病毒载体或杆状病毒载体。
  7. 一种重组腺相关病毒(AAV)载体，其中所述重组AAV载体在其基因组中包含：
    a.5’和3’AAV反向末端重复(ITR)序列，和
    b.位于5’和3’ITR之间的表达构建体，其中所述表达构建体包含以转录方向彼此功能性连接的如下元件：
    -权利要求1或2任一项的启动子，
    -任选地，Kozak序列，
    -编码人α酸性葡萄糖苷酶(GAA)的多核苷酸，
    -任选地，至少一个(例如2-6个)免疫相关的miRNA靶序列，尤其是miR-142靶序列，例如包含至少一个(例如2个或4个)SEQ ID NO:11序列的miR-142-3p靶序列，
    -转录终止子，例如polyA信号序列，优选地选自SV40晚期polyA序列、兔β-珠蛋白polyA序列、和牛生长激素polyA序列，更优选地牛生长激素polyA序列。
  8. 权利要求7的重组AAV病毒载体，其中，所述编码GAA的多核苷酸为人密码子优化的，更优选地，所述多核苷酸包含SEQ ID NO:10的序列。
  9. 权利要求7或8的重组AAV病毒载体，其中所述ITR为野生型AAV2 ITR序列，或所述ITR之一是缺少功能性末端解链位点(trs)和任选地D序列的AA2ΔITR序列。
  10. 权利要求7-9任一项的重组AAV病毒载体，其中所述载体为ssAAV载体或scAAV载体。
  11. 权利要求7-10任一项的AAV病毒载体，其中所述重组AAV载体包含来自AAV9血清型的衣壳蛋白，优选地，所述重组AAV载体是AAV2/9载体。
  12. 包含权利要求1-2任一项的启动子或权利要求3-5任一项的表达构建体或权利要求6的载体的宿主细胞，
  13. 权利要求7-11的重组AAV病毒载体用于驱动编码α酸性葡萄糖苷酶(GAA)的多核苷酸在哺乳动物细胞中表达的用途，或在制备用于驱动编码α酸性葡萄糖苷酶(GAA)的多核苷酸在哺乳动物细胞或哺乳动物体内一种或多种组织或器官中表达的药物中的用途，
    优选地，所述药物用于在哺乳动物的心脏、肝脏、肌肉、中枢神经系统(包括脑和脊髓)中表达GAA，
    优选地，所述药物全身给药，例如腹膜内(i.p.)、肌内(i.m.)、动脉内或静脉内(i.v.)注射给药，优选地静脉内注射。
  14. 一种用于预防或治疗庞贝氏病受试者或具有酸性葡萄糖苷酶缺陷的受试者的方法，包括向所述受试者施用权利要求7-11的重组AAV载体，
    优选地，通过全身给药，例如腹膜内(i.p.)、肌内(i.m.)、动脉内或静脉内(i.v.)注射给药，优选地静脉内注射，施用所述重组AAV载体。
  15. 权利要求14的方法，其中重组AAV载体施用增加GAA多肽在受试者的外周组织(优选地，心脏、肝脏、肌肉)和中枢神经系统(包括脑和脊髓)中的表达。
  16. 权利要求14的方法，其中重组AAV载体施用导致受试者的外周组织(优选地，心脏、肝脏、肌肉)和中枢神经系统(包括脑和脊髓，优选地胶质细胞)中的溶酶体糖原贮积量减少和优选地改善由所述糖原贮积所致的组织病损，且优选地不诱导或诱导低的免疫原性。
  17. 权利要求14的方法，其中所述重组AAV病毒载体与另一活性剂组合施用，优选地，所述活性剂用于酶替代治疗(ERT)的重组GAA蛋白。
  18. 一种药物组合物，其包含权利要求6的载体或权利要求7-11任一项的重组AAV病毒载体和药物可接受载体。
  19. 一种细胞，其包含：(i)编码一种或多种腺相关病毒rep蛋白和/或一种或多种腺相关病毒cap蛋白的第一载体；和(ii)包含权利要求3-5任一项的表达构建体的第二载体，
    优选地，第一载体是质粒，且第二载体是质粒；所述细胞是哺乳动物细胞，任选地其中所述哺乳动物细胞是HEK293细胞；或者
    优选地，第一载体是杆状病毒载体且第二载体是杆状病毒载体；所述细胞是昆虫细胞，任选地其中所述昆虫细胞是sf9细胞。
  20. 一种生产权利要求7-11任一项的重组AAV病毒载体的方法，其中所述方法包括步骤：
    (i)提供权利要求19的细胞；
    (ii)在允许包装重组AAV的条件下培养所述细胞；和
    (iii)收获培养的宿主细胞或培养基以收集所述重组AAV病毒载体。
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