WO2024094044A1 - 优化的pah基因和表达盒及其用途 - Google Patents

优化的pah基因和表达盒及其用途 Download PDF

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WO2024094044A1
WO2024094044A1 PCT/CN2023/128981 CN2023128981W WO2024094044A1 WO 2024094044 A1 WO2024094044 A1 WO 2024094044A1 CN 2023128981 W CN2023128981 W CN 2023128981W WO 2024094044 A1 WO2024094044 A1 WO 2024094044A1
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seq
promoter
nucleotide sequence
identity
virus
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French (fr)
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文圣梅
刘昳婷
郭玉峰
蒋立新
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苏州诺洁贝生物技术有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)

Definitions

  • the present disclosure belongs to the technical field of gene therapy, and particularly relates to an optimized PAH gene and expression cassette and uses thereof.
  • Phenylketonuria (PKU, OMIM 261600) is an autosomal recessive genetic disease caused by mutations in the phenylalanine hydroxylase (PAH) gene.
  • PAH phenylalanine hydroxylase
  • control standard recommended by the genetic variation classification standard and treatment guidelines in the United States is to control the concentration of peripheral blood phenylalanine between 120 ⁇ mol/L and 360 ⁇ mol/L through lifelong diet and drug treatment (Vockley, J., et al., Phenylalanine hydroxylase deficiency: diagnosis and management guideline. Genet Med, 2014. 16 (2): p. 188-200.).
  • the control standard of the treatment guidelines of the European Union is less than 360 ⁇ mol/L for women under 12 years old and women preparing for pregnancy and during pregnancy, and less than 600 ⁇ mol/L for other groups (van Wegberg, A.M.J., et al., The complete European guidelines on phenylketonuria: diagnosis and treatment.
  • a single treatment with Zolgensma can enable children with spinal muscular atrophy (SMA) to grow and develop basically like normal children.
  • a single treatment with Luxturna can restore the vision of patients with Leber congenital amaurosis (LCA) to a state where they can basically live, study and work normally.
  • LCA Leber congenital amaurosis
  • the purpose of the present disclosure is to provide an optimized PAH gene, expression cassette and viral vector to achieve effective, sustained and stable expression of human PAH in the liver at a relatively low dose for the treatment of phenylketonuria.
  • the present disclosure provides a polynucleotide molecule encoding a PAH protein, comprising a nucleotide sequence having 90% or more identity with a nucleotide sequence shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22 or SEQ ID NO.23. preferably a nucleotide sequence with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity; more preferably a nucleotide sequence with 98% or 99% or more identity.
  • the second aspect of the present disclosure provides an expression cassette, which comprises the polynucleotide molecule provided in the first aspect of the present disclosure, and a promoter operably linked to the polynucleotide molecule.
  • the expression cassette further comprises an expression control element, which is operably linked to the polynucleotide molecule.
  • the expression control element is selected from at least one of a transcription/translation control signal, an enhancer, an intron, a polyA signal, an ITR, an insulator, an RNA processing signal, and an element that enhances the stability of mRNA and protein.
  • the third aspect of the present disclosure provides an expression vector, which comprises the polynucleotide molecule provided by the first aspect of the present disclosure or the expression cassette provided by the second aspect of the present disclosure.
  • the expression vector is selected from a plasmid, a cosmid, a viral vector, an RNA vector, or a linear or circular DNA or RNA molecule.
  • the expression vector is an adeno-associated virus vector.
  • the fourth aspect of the present disclosure provides a virus particle, which comprises at least one of the polynucleotide molecule provided by the first aspect of the present disclosure, the expression cassette provided by the second aspect of the present disclosure, and the expression vector provided by the third aspect of the present disclosure.
  • the fifth aspect of the present disclosure provides a pharmaceutical composition for treating phenylketonuria, which comprises at least one of the polynucleotide molecule provided in the first aspect of the present disclosure, the expression cassette provided in the second aspect of the present disclosure, the expression vector provided in the third aspect of the present disclosure, and the virus particle provided in the fourth aspect of the present disclosure.
  • the sixth aspect of the present disclosure provides the use of at least one of the polynucleotide molecule provided in the first aspect of the present disclosure, the expression cassette provided in the second aspect of the present disclosure, the expression vector provided in the third aspect of the present disclosure, the virus particle mentioned in the fourth aspect of the present disclosure, and the pharmaceutical composition of the fifth aspect of the present disclosure in the preparation of a medicament for treating phenylketonuria.
  • the seventh aspect of the present disclosure provides the use of at least one of the polynucleotide molecule provided in the first aspect of the present disclosure, the expression cassette provided in the second aspect of the present disclosure, the expression vector provided in the third aspect of the present disclosure, the viral particle mentioned in the fourth aspect of the present disclosure, and the pharmaceutical composition of the fifth aspect of the present disclosure in the treatment of phenylketonuria.
  • An eighth aspect of the present disclosure provides a method for treating phenylketonuria, comprising administering to a subject an effective amount of at least one of the polynucleotide molecules, expression cassettes, expression vectors, viral particles and pharmaceutical compositions of the present disclosure.
  • the coding gene of PAH is optimized so that it can express human PAH more effectively, thereby achieving effective, long-lasting and stable expression of human PAH in the liver at a relatively low dose, so that the concentration of phenylalanine in the blood of the subject is maintained at a low level for a long time, and can be used for the treatment of phenylketonuria.
  • the polynucleotide molecules, expression cassettes, expression vectors, viral particles and/or pharmaceutical compositions disclosed in the present disclosure have a lower dose, thereby having lower potential liver toxicity.
  • FIG1 shows a schematic diagram of the structure of the PAH expression cassette of the recombinant AAV (rAAV) vector of the present disclosure.
  • FIG2 shows the in vitro expression of the codon-optimized PAH opt gene in HepG2 cells transfected by plasmids.
  • FIG. A is a representative image of PAH protein Western blot;
  • FIG. B shows the quantitative results of PAH protein Western blot to detect the expression levels of PAH and GAPDH (plasmid transfection control) in HepG2 cells, where WT or opt gene was expressed with pAAV8-ATT-PAH WT or pAAV8-ATT-PAH opt plasmids, respectively.
  • Cell lysates were harvested 2 days after equal amounts of plasmid transfection. Equal amounts of total protein were separated by SDS-PAGE and then immunoblotted.
  • the expression level of PAH was normalized with GAPDH and calculated as a ratio to the WT expression level.
  • Figure 3A shows the effect of reducing Phe in PKU model mice after high-pressure injection of PAH WT and optimized genes by tail vein plasmid. At 0h, 6h, 24h, 3 days, 5 days, 10 days, 16 days, and 19 days after injection, the blood of mice was collected to measure the Phe content in the blood; Figure 3B shows the Phe content in the blood of PKU model mice at 10 days after injection.
  • Figure 4 shows the effect of AAV8-ATT-PAH WT and optimized gene virus on reducing Phe in PKU model mice.
  • the blood of mice was collected weekly to measure the Phe content in the blood.
  • FIG. 5 is a schematic diagram of the structure of the PAH expression cassette of the recombinant AAV (rAAV) vector containing different promoters.
  • Figure 6 shows the effect of reducing Phe in PKU model mice after high-pressure injection of PAH WT expression cassettes from different promoters by plasmid tail vein. On the third day after injection, blood was collected from mice to measure the Phe content in the blood.
  • FIG. 7 is a schematic diagram of the structure of a recombinant AAV (rAAV) vector PAH expression cassette containing different expression regulatory elements.
  • rAAV recombinant AAV
  • Figure 8 shows the effect of reducing Phe in PKU model mice after high-pressure injection of a PAH expression cassette containing different expression regulatory elements via tail vein plasmid. Blood was collected from mice at the 3rd day after injection to measure the Phe content in the blood.
  • FIG. 9 is a schematic diagram of the structure of the PAH expression cassette of the optimized recombinant AAV (rAAV) vector containing different stuffer sequences.
  • Figure 10A shows the effect of AAV8-ATT-PAH opt9 virus with different filling sequences on the effect of reducing Phe in PKU model mice.
  • Figure 10B shows the effect of AAV8-ATT-PAH opt9 and AAV8-ATT-PAH opt-HPRT (4CpG) viruses on the effect of reducing Phe in PKU model mice.
  • the blood of mice was collected weekly to measure the content of Phe in the blood.
  • Figure 11 shows the functional test results of the expression product PAH of AAV8-ATT-PAH opt-HPRT (4CpG) virus in HepG2 cell line to reduce Phe concentration.
  • AAV8-ATT-PAH opt-HPRT (4CpG) virus was infected at MOI of 0, 5e4, 1e5, and 2e5, and the change in Phe concentration was calculated with the HepG2 cell line not infected with the virus as a reference.
  • Figure 12A shows the effect of different doses of AAV8-ATT-PAH opt9-HPRT (4CpG) virus on the effect of reducing Phe in male PKU model mice, wherein the blood of mice was collected weekly within 6 weeks after injection to determine the content of Phe in the blood.
  • Figure 12B shows the effect of different doses of AAV8-ATT-PAH opt9-HPRT (4CpG) virus on the effect of reducing Phe in female PKU model mice, wherein the blood of mice was collected weekly within 6 weeks after injection to determine the content of Phe in the blood.
  • Figure 13A shows the Tyr content in the brain tissue of PKU homozygous mice after 6 weeks of administration of AAV8-ATT-PAH opt9-HPRT (4CpG) virus at 3.0e10 and 3.0e11 vg/mouse, where the heterozygous mice that were not administered served as the normal mouse control group, and the homozygous mice that were administered with the vehicle served as the untreated control group.
  • Figure 13B shows the 5-hydroxyindoleacetic acid (5-HIAA) content in the brain tissue of mice.
  • Figure 13C shows the comparison of the coat color of PKU homozygous mice and the vehicle group after 3 weeks of administration at a dose of 3.0e10 vg/mouse.
  • references herein to "about” a value or parameter include (and describe) embodiments for the value or parameter itself. For example, a description referring to "about X” includes a description of "X.”
  • vector refers to a recombinant plasmid or virus containing a nucleic acid to be delivered into a host cell (in vitro or in vivo).
  • polynucleotide molecule or “nucleic acid” refers to a polymeric form of nucleotides of any length, which are ribonucleotides or deoxyribonucleotides. Therefore, the term includes, but is not limited to, single, double or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases.
  • the backbone of the nucleic acid may contain sugar and phosphate groups (as commonly found in RNA or DNA), or modified or substituted sugar or phosphate groups.
  • the backbone of the nucleic acid may contain synthetic subunits such as polymers of aminophosphorylation and may therefore be oligodeoxynucleoside aminophosphorylation (P-NH 2 ) or mixed aminophosphorylation ester-phosphodiester oligomers.
  • double-stranded nucleic acids may be obtained from single-stranded polynucleotide products chemically synthesized (by synthesizing complementary chains under appropriate conditions and annealing the chains, or using DNA polymerase to synthesize complementary chains de novo with appropriate primers).
  • Recombinant viral vector refers to a recombinant polynucleotide vector comprising one or more heterologous sequences (ie, nucleic acid sequences of non-viral origin).
  • heterologous sequences ie, nucleic acid sequences of non-viral origin.
  • the recombinant nucleic acid is flanked by at least one, preferably two, inverted terminal repeats (ITRs).
  • a “recombinant AAV vector (rAAV vector)” refers to a polynucleotide vector containing one or more heterologous sequences (i.e., nucleic acid sequences not of AAV origin) flanked by at least one, preferably two, AAV inverted terminal repeats (ITRs).
  • the rAAV vector can replicate and be packaged into infectious viral particles when present in a host cell that has been infected with an appropriate helper virus (or expresses appropriate helper functions) and expresses the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins).
  • a rAAV vector When a rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid used for cloning or transfection), the rAAV vector can be referred to as a "pro-vector" that can be "rescued” by replication and encapsidation in the presence of AAV packaging functions and appropriate helper functions.
  • the rAAV vector can be in any of a variety of forms, including, but not limited to, a plasmid, a linear artificial chromosome, which can be complexed with, encapsulated within a liposome, and in embodiments, encapsulated within a viral particle, particularly an AAV particle.
  • the rAAV vector can be packaged into an AAV viral capsid to generate a "recombinant adeno-associated viral particle (rAAV particle)."
  • AAV helper functions i.e., functions that allow AAV to be replicated and packaged by host cells
  • helper viruses or helper virus genes that aid in AAV replication and packaging.
  • Other AAV helper functions are known in the art.
  • rAAV virus or "rAAV viral particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated rAAV vector genome.
  • Heterologous means derived from an entity that is genotypically different from the rest of the entity to which it is compared or into which it is introduced or incorporated.
  • a nucleic acid introduced into a different cell type by genetic engineering techniques is a heterologous nucleic acid (and when expressed, may encode a heterologous polypeptide).
  • a cellular sequence (e.g., a gene or portion thereof) incorporated into a viral vector is a heterologous nucleotide sequence relative to the vector.
  • genomic particles As used in reference to viral titer, the terms “genomic particles (gp)", “genomic equivalents” or “genomic copies” refer to the number of viral particles containing the recombinant AAV DNA genome, regardless of their infectivity or functionality. The number of genomic particles in a particular vector preparation can be measured by methods such as those described in the Examples herein or, for example, in Clark et al. (1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther., 6:272-278.
  • infectious unit iu
  • infectious particles infectious particles
  • replication unit refers to the number of recombinant AAV vector particles that are infectious and replication competent as measured by the infectious center assay, also known as the replication center assay, as described, for example, in McLaughlin et al. (1988) J. Virol., 62: 1963-1973.
  • transduction unit (tu) refers to the number of infectious recombinant AAV vector particles that result in the production of a functional transgene product, as measured in a functional assay, such as described in the Examples herein or, for example, in Xiao et al. (1997) Exp. Neurobiol., 144: 113-124; or Fisher et al. (1996) J. Virol., 70: 520-532 (LFU assay).
  • ITR sequence is a term well known in the art and refers to relatively short sequences found at the ends of viral genomes that are in a relatively Reverse direction.
  • AAV inverted terminal repeat (ITR) sequence is a term well known in the art, which is a sequence of about 145 nucleotides present at both ends of the native single-stranded AAV genome.
  • the outermost 125 nucleotides of the ITR can be present in either of two alternative orientations, resulting in heterogeneity between different AAV genomes and between the two ends of a single AAV genome.
  • the outermost 125 nucleotides also contain several shorter regions (called A, A', B, B', C, C' and D regions) that are self-complementary, allowing interstrand base pairing to occur within this ITR portion.
  • helper virus for AAV refers to a virus that allows AAV (which is a defective parvovirus) to replicate and be packaged by host cells.
  • helper viruses have been identified, including adenovirus, herpes virus, and poxviruses such as vaccinia.
  • Adenoviruses encompass a number of different subclasses, although adenovirus type 5 (Ad5) of subclass C is the most commonly used.
  • Ad5 adenovirus type 5
  • a variety of adenoviruses of human, non-human mammalian, and avian origin are known and can be obtained from depositories such as the ATCC.
  • the herpes virus family which can also be obtained from depositories such as the ATCC, includes, for example, herpes simplex viruses (HSV), Epstein-Barr viruses (EBV), cytomegaloviruses (CMV), and pseudorabies viruses (PRV).
  • HSV herpes simplex viruses
  • EBV Epstein-Barr viruses
  • CMV cytomegaloviruses
  • PRV pseudorabies viruses
  • stuffing sequence refers to a human non-coding sequence added to make the nucleotide sequence length of the recombinant AAV expression cassette close to the length of the wild-type AAV genome.
  • addition of a stuffing sequence can enable the expression cassette or the polynucleotide sequence carrying it to have a higher viral packaging yield when expressed using an AAV viral vector.
  • Percent (%) sequence identity relative to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to the amino acid residues or nucleotides in the reference polypeptide or nucleic acid sequence, after aligning the sequences and introducing gaps (if necessary to achieve maximum percent sequence identity and not considering any conservative substitutions as part of the sequence identity). Alignment for the purpose of determining percent amino acid or nucleic acid sequence identity can be achieved in a variety of ways within the art, such as using publicly available computer software programs such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. A preferred alignment software is ALIGN Plus (Scientific and Educational Software, Pennsylvania).
  • the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by a sequence alignment program in a program alignment of A and B, and where Y is the total number of amino acid residues in B.
  • the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D is calculated as follows: 100 times the fraction W/Z, where W is the number of nucleotides scored as identical matches by a sequence alignment program in a program alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not be equal to the % nucleic acid
  • an "effective amount” is an amount sufficient to affect a beneficial or desired outcome, including a clinical outcome (e.g., improving symptoms, achieving a clinical endpoint, etc.).
  • An effective amount can be administered once or multiple times.
  • an effective amount is an amount sufficient to improve, stabilize, or delay disease progression.
  • an effective amount of rAAV particles expresses a desired amount of a heterologous nucleic acid, such as a therapeutic polypeptide or therapeutic nucleic acid.
  • mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
  • domesticated animals e.g., cattle, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rats
  • rodents e.g., mice and rats.
  • the individual or subject is a human.
  • treatment is a means for obtaining a beneficial or expected clinical outcome.
  • a beneficial or expected clinical outcome includes, but is not limited to, improvement of symptoms, reduction in extent of disease, a stabilized (e.g., non-worsening) state of disease, prevention of disease spread (e.g., metastasis), delay or slowing of disease progression, improvement or alleviation of the disease state, and remission (whether partial or complete), whether detectable or undetectable.
  • Treatment may also mean prolonging survival compared to the expected survival rate if not receiving treatment.
  • the unit vg (Vector Genomes) represents the number of viral genome copies.
  • the virus infection multiplicity means the ratio of the number of viruses to bacteria during infection, that is, the average number of phages that infect each bacterium.
  • the present disclosure provides a polynucleotide molecule encoding a PAH protein, which comprises a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22 or SEQ ID NO.23; preferably a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity; more preferably a nucleotide sequence having 98% or more identity.
  • the polynucleotide molecule has a nucleotide sequence shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22 or SEQ ID NO.23.
  • the codon-optimized human PAH protein encoding gene has the nucleotide sequence shown in SEQ ID NO.17.
  • the second aspect of the present disclosure provides an expression cassette, which comprises the polynucleotide molecule provided in the first aspect of the present disclosure, and a promoter operably linked to the polynucleotide molecule.
  • the promoter is a specific or non-specific promoter.
  • the promoter comprises a core promoter.
  • the promoter may be a constitutive promoter; preferably, the constitutive promoter is selected from at least one of CMV promoter, EF1A promoter, EFS promoter, CAG promoter, CBh promoter, SFFV promoter, MSCV promoter, SV40 promoter, mPGK promoter, hPGK promoter, UBC promoter, etc.
  • the promoter is an inducible promoter; preferably, the inducible promoter comprises at least one of a tetracycline-regulated promoter, an alcohol-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, a pathogenicity-regulated promoter, a temperature/heat-inducible promoter, a light-regulated promoter, and an IPTG-inducible promoter.
  • the tetracycline-regulated promoter is selected from a Tet on promoter, a Tet off promoter, and a Tet Activator promoter.
  • the alcohol-regulated promoter is selected from a promoter of the alcohol dehydrogenase I (alcA) gene, and a promoter responsive to an alcohol transactivator protein (AlcR).
  • the steroid-regulated promoter is selected from a rat glucocorticoid receptor promoter, a human estrogen receptor promoter, a moth ecdysone receptor promoter, a retinoid promoter, and a thyroid receptor superfamily promoter.
  • the metal-regulated promoter is selected from metallothionein promoters of yeast, mouse, and human.
  • the pathogenicity-regulated promoter is selected from a salicylic acid-regulated promoter, an ethylene-regulated promoter, and a benzothiadiazole-regulated (BTH) promoter.
  • the temperature/heat-inducible promoter is selected from an HSP-70 promoter, an HSP-90 promoter, and a soybean heat shock promoter.
  • the light-regulated promoter is a light-responsive promoter of a plant cell.
  • the promoter is a liver-specific promoter; some non-limiting examples of liver-specific promoters include, but are not limited to, ApoA-I promoter, ApoA-II promoter, ApoA-IV promoter, ApoB promoter, ApoC-1 promoter, ApoC-II promoter, ApoC-III promoter, ApoE promoter, albumin promoter, alpha-fetoprotein promoter, phosphoenolpyruvate carboxykinase (PCK1) promoter, phosphoenolpyruvate carboxykinase 2 (PCK2) promoter, alpha-
  • the promoters used herein include transthyretin (TTR) promoter, ⁇ -antitrypsin (AAT or SerpinA1) promoter, TK (thymidine kinase) promoter, hemoglobin promoter, alcohol dehydrogenase 6 promoter, cholesterol 7 ⁇ -25 hydroxylase promoter, factor IX promoter, ⁇ -microglobin,
  • the liver-specific promoter is human ⁇ 1 antitrypsin promoter (hAAT or SERPINA1 promoter).
  • the core promoter comprises a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO.3, 24, 25, 30, 33 or 38, preferably a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence having 98% or more identity;
  • the core promoter has a nucleotide sequence as shown in SEQ ID NO.SEQ ID NO.3, 24, 25, 30, 33 or 38; more preferably, more preferably, the nucleotide sequence of the core promoter is as shown in SEQ ID NO.3.
  • the expression cassette further comprises an expression control element, which is operably linked to the polynucleotide molecule.
  • the expression control element is selected from at least one of a transcription/translation control signal, an enhancer, an intron, a polyA signal, an ITR, an insulator, an RNA processing signal, and an element that enhances the stability of mRNA and protein.
  • the expression cassette comprises a 5’ITR; preferably, the 5’ITR comprises a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO.1, preferably a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence having 98% or more identity; more preferably, the 5’ITR has a nucleotide sequence as shown in SEQ ID NO.1.
  • the expression cassette comprises a 3'ITR.
  • the 3'ITR comprises a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO. 8, preferably a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably a nucleotide sequence having 98% or more identity; more preferably, the 3'ITR has a nucleotide sequence as shown in SEQ ID NO.8.
  • the expression cassette further comprises an enhancer.
  • the enhancer is selected from ApoE HCR enhancer or an active fragment thereof, CRMSBS2 enhancer or an active fragment thereof, TTRm enhancer or an active fragment thereof, and CMV enhancer or an active fragment thereof; more preferably, the enhancer comprises a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO.2, 29, 32, 37 or 40, preferably a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence having 98% or more identity; preferably, the enhancer has a nucleotide sequence as shown in SEQ ID NO.2, 29, 32, 37 or 40; more preferably, the nucleotide sequence of the enhancer is as shown in SEQ ID NO.2.
  • the expression cassette further comprises an intron; preferably, the intron is a truncated ⁇ 1 antitrypsin intron or an active fragment thereof, a ⁇ -globin 2 intron or an active fragment thereof, an SV40 intron or an active fragment thereof, and a minute virus of mice intron or an active fragment thereof; preferably, the intron comprises a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO.4, 26, 27, 28, 31 or 34, preferably a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence having 98% or more identity; preferably, the intron has a nucleotide sequence as shown in SEQ ID NO.4, 26, 27, 28, 31 or 34; more preferably, the nucleotide sequence of the intron is as shown in SEQ ID NO.4.
  • the promoter of the expression cassette is a combined promoter comprising an upstream regulatory element, a core promoter, and an intron.
  • the upstream regulatory element is an enhancer or an active fragment thereof.
  • the combined promoter comprises a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO.44, 45, 46, 47, 48 or 49, preferably a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably a nucleotide sequence having 98% or more identity; preferably, the combined promoter has a nucleotide sequence as shown in SEQ ID NO.44, 45, 46, 47, 48 or 49; more preferably, the nucleotide sequence of the combined promoter is as shown in SEQ ID NO.44.
  • the expression cassette further comprises a polyA signal; preferably, the polyA signal is at least one of bovine growth hormone poly A (BGH poly A), short poly A, SV40polyA, and human ⁇ -globin poly A; preferably, the polyA signal comprises a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO.7, preferably a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence having 98% or more identity; more preferably, the nucleotide sequence of the polyA signal is as shown in SEQ ID NO.7.
  • BGH poly A bovine growth hormone poly A
  • short poly A short poly A
  • SV40polyA short poly A
  • the polyA signal comprises a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO
  • the expression cassette comprises an optimized stuffer sequence; preferably, the stuffer sequence is selected from a partial intron sequence of hypoxanthine phosphoribosyltransferase (HPRT) and a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); preferably, the number of CpG sequences contained in the partial intron sequence does not exceed 100, 80, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1; preferably, the partial intron sequence does not contain a CpG sequence or Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); preferably, the stuffer sequence is a partial intron sequence of hypoxanthine phosphoribosyltransferase (HPRT); preferably, the stuffer sequence comprises a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO.39 or SEQ ID NO.43, preferably a nucleotide sequence having 91%, 9
  • the expression cassette comprises a Kozak start sequence;
  • the Kozak start sequence comprises a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO.5, preferably a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably a nucleotide sequence having 98% or more identity; more preferably, the Kozak start sequence has the nucleotide sequence shown in SEQ ID NO.5.
  • the expression cassette comprises 5'ITR, ApoE HCR enhancer, human ⁇ 1 antitrypsin promoter (SERPINA1 promoter), truncated ⁇ 1 antitrypsin intron (SerpinA1 intron), Kozak start sequence (GCCACC, SEQ ID NO.5), the polynucleotide molecule, BGH poly A, a partial intron sequence of hypoxanthine phosphoribosyltransferase (HPRT) (HPRT (4CpG)) and 3'ITR; preferably, the expression cassette comprises SEQ ID NO.80, SEQ ID NO.81, SEQ ID NO.82, SEQ ID NO.83, SEQ ID NO.84, SEQ ID NO.85, SEQ ID NO.86, SEQ ID NO.87, SEQ ID NO.89, SEQ ID NO.90, SE The nucleotide sequence shown in SEQ ID NO.91, SEQ ID NO.92 or SEQ ID NO.93 has a nucleot
  • the third aspect of the present disclosure provides an expression vector, which comprises the polynucleotide molecule provided by the first aspect of the present disclosure or the expression cassette provided by the second aspect of the present disclosure.
  • the expression vector further comprises a gene encoding a marker, preferably, the marker is selected from at least one of an antibiotic resistance protein, a toxin resistance protein, a colored or fluorescent or luminescent protein, and a protein that mediates enhanced cell growth and/or gene amplification.
  • the antibiotic is selected from at least one of ampicillin, neomycin, G418, puromycin and blasticidin.
  • the toxin is selected from at least one of anthrax toxin and diphtheria toxin.
  • the colored or fluorescent or luminescent protein is selected from at least one of green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein and luciferase.
  • the protein that mediates enhanced cell growth and/or gene amplification is dihydrofolate reductase (DHFR).
  • DHFR dihydrofolate reductase
  • the expression vector comprises a replication origin; preferably, the replication origin sequence is selected from at least one of f1 phage ori, RK2oriV, pUC ori and pSC101ori.
  • the expression vector is selected from a plasmid, a cosmid, a viral vector, an RNA vector, or a linear or circular DNA or RNA molecule.
  • the plasmid is selected from pCI, puc57, pcDNA3, pSG5, pJ603 or pCMV.
  • the viral vector is selected from a retrovirus, an adenovirus, a parvovirus (e.g., an adeno-associated virus), a coronavirus, a negative-strand RNA virus such as an orthomyxovirus (e.g., influenza virus), a rhabdovirus (e.g., rabies and vesicular stomatitis virus), a paramyxovirus (e.g., mammary gland and Sendai), a positive-strand RNA virus (such as a picornavirus and an alphavirus), or a double-stranded DNA virus selected from an adenovirus, a herpesvirus (e.g., herpes simplex virus type 1 and 2, Epstein-Barr virus, cytomegalovirus), a poxvirus (e.g., vaccinia virus, fowlpox virus, and canarypox virus), a Norwalk virus, a togavirus, a flaxa virus such
  • the retrovirus is selected from avian leukocytoproliferation-sarcoma, mammalian C-type, B-type virus, D-type virus, HTLV-BLV collection, lentivirus or foamy virus.
  • the lentiviral vector is selected from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or ovine demyelinating leukoencephalitis lentivirus.
  • the expression vector is an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • the adeno-associated virus is selected from AAV type 1, AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, avian AAV, bovine AAV, canine AAV, equine AAV or ovine AAV.
  • the expression cassette can be packaged in a rAAV vector with a capsid from any AAV serotype or a hybrid or variant thereof.
  • the expression vector comprises SEQ ID NO.53, SEQ ID NO.54, SEQ ID NO.55, SEQ ID NO.56, SEQ ID NO.57, SEQ ID NO.58, SEQ ID NO.59, SEQ ID NO.60, SEQ ID NO.62, SEQ ID NO.63, SEQ ID NO.64, SEQ ID NO.65, SEQ ID NO.66, SEQ ID NO.72, SEQ ID NO.73, SEQ ID NO.74, SEQ ID NO.75, SEQ ID NO.76, SEQ ID NO.77, SEQ ID NO.78, SEQ ID NO.96, SEQ ID NO.97, SEQ ID NO.98, SEQ ID NO.99, SEQ ID NO.100, SEQ ID NO.101, SEQ ID NO.102, SEQ ID NO.103, SEQ ID NO.105, SEQ ID NO.106, SEQ ID NO.107, SEQ ID NO.108 or SEQ ID NO.
  • the nucleotide sequence shown in NO.109 has a nucleotide sequence with 90% or more identity, preferably a nucleotide sequence with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence with 98% or 99% identity; more preferably, the expression vector has SEQ ID NO.53, SEQ ID NO.54, SEQ ID NO.55, SEQ ID NO.56, SEQ ID NO.57, SEQ ID NO.58, SEQ ID NO.59, SEQ ID NO.60, SEQ ID NO.62, SEQ ID NO.63, SEQ ID NO.64, SEQ ID NO.65, SEQ ID NO.66, SEQ ID NO.72, SEQ ID NO.73, SEQ ID NO.74, SEQ ID NO.75, SEQ ID NO.76, SEQ ID NO.77, SEQ ID NO.78, SEQ ID NO.96, SEQ ID NO.97, SEQ ID NO.
  • the expression vector has a nucleotide sequence as shown in SEQ ID NO.73.
  • the fourth aspect of the present disclosure provides a virus particle, which comprises at least one of the polynucleotide molecule provided by the first aspect of the present disclosure, the expression cassette provided by the second aspect of the present disclosure, and the expression vector provided by the third aspect of the present disclosure.
  • the fifth aspect of the present disclosure provides a pharmaceutical composition for treating phenylketonuria, which comprises at least one of the polynucleotide molecule provided by the first aspect of the present disclosure, the expression cassette provided by the second aspect of the present disclosure, the expression vector provided by the third aspect of the present disclosure, and the viral particle provided by the fourth aspect of the present disclosure.
  • the sixth aspect of the present disclosure provides the use of the polynucleotide molecule provided in the first aspect of the present disclosure, the expression cassette provided in the second aspect of the present disclosure, the expression vector provided in the third aspect of the present disclosure, the viral particle provided in the fourth aspect of the present disclosure, or the pharmaceutical composition of the fifth aspect of the present disclosure in the preparation of a drug for treating phenylketonuria.
  • the seventh aspect of the present disclosure provides the use of the polynucleotide molecule provided in the first aspect of the present disclosure, the expression cassette provided in the second aspect of the present disclosure, the expression vector provided in the third aspect of the present disclosure, the viral particle mentioned in the fourth aspect of the present disclosure, or the pharmaceutical composition of the fifth aspect of the present disclosure in the treatment of phenylketonuria.
  • An eighth aspect of the present disclosure provides a method for treating phenylketonuria, comprising administering to a subject an effective amount of at least one of the polynucleotide molecules, expression cassettes, expression vectors, viral particles and pharmaceutical compositions of the present disclosure.
  • the initial AAV shuttle plasmid vector was synthesized by Universal Genetics according to the sequence of SEQ ID NO.112.
  • the wild-type AAV2 ITR sequence was recombined between the vector BamHI and AleI restriction sites to repair the mutated ITR sequence in the vector;
  • the Amp resistance gene between the initial shuttle plasmid vector ApaLI restriction sites was replaced with Kan; then it was recombined between the vector HindIII and NheI restriction sites to increase the length of the vector to facilitate the packaging of the AAV virus;
  • the CAG promoter sequence was PCR amplified from the pCAGGS vector (Golden Weichi) and recombined between the SpeI and KpnI restriction sites of the shuttle plasmid vector to obtain the final shuttle plasmid vector (SEQ NO ID.113).
  • the CAG promoter contains the CMV enhancer (SEQ ID NO.40), the chicken ⁇ -actin promoter (SEQ ID NO.41) and the chimeric intron (S
  • AAV vector adopts a three-plasmid system, that is, a shuttle plasmid containing the PAH target gene, a pRepCap plasmid with the AAV vector repcap gene, and a helper plasmid Phelper (pRepCap plasmid, synthesized by GeneWei according to SEQ ID NO.110 sequence and a helper plasmid pHelper, synthesized by GeneWei according to SEQ NO ID.111 sequence), using PEI as a transfection reagent, co-transfecting HEK293 cells to recombinantly package the AAV virus vector.
  • Harvest 48-72 hours after transfection, and the harvest fluid is purified to obtain a recombinant AAV virus vector of a certain purity.
  • the purification method is as follows:
  • the harvested fluid was pretreated: HEK293 cells were fully lysed to release the AAV viral vector in the cells, and nuclease was added to digest the free nucleic acid. After digestion, deep filtration was used to remove large molecular impurities and cell fragments. After deep filtration, the filtrate was filtered twice to obtain a clarified liquid for affinity loading.
  • Affinity chromatography uses the specific adsorption of ligands and proteins to capture the AAV viral vector in the harvested fluid and remove most of the process-related impurities to achieve the effect of concentration and impurity removal.
  • the collected eluate is mixed and neutralized with neutralization buffer, and stored in a sterile storage bottle as anion chromatography loading solution.
  • Anion chromatography uses the difference in isoelectric points of different components to separate solid and empty AAV viruses, while continuing to remove residual impurities.
  • the eluate is collected in a new sterile storage bottle, and then the buffer is replaced by ultrafiltration and concentration to a preparation-stable buffer.
  • the virus titer is concentrated to about 1 ⁇ 10 13 vg/mL, and finally sterilized, filtered, and packaged for later use.
  • the genome titer is the most classic test item to characterize the physical titer of AAV.
  • the most common method for genome titer detection is to design primer probes for the rAAV genome sequence and then perform Q-PCR detection.
  • the standard curve should be established first.
  • the positive standard plasmid is diluted to 2 ⁇ 10 7 , 2 ⁇ 10 6 , 2 ⁇ 10 5 , 2 ⁇ 10 4 , 2 ⁇ 10 3 , and 2 ⁇ 10 2 copies/ ⁇ l with sample diluent as the standard curve template.
  • the standard curve needs to control its linearity and amplification efficiency. Generally, R 2 >0.99 is required, and the amplification efficiency is between 90% and 110%.
  • the pre-treated rAAV sample is diluted and QPCR detection is performed to ensure that the sample detection Ct value is within the range of the standard curve.
  • the rAAV sample genome titer is calculated according to the sample Ct value substituted into the standard curve to identify the content of the product.
  • Lipofectamine 3000 and P3000 (Thermo, L3000015) transfection reagents were pre-mixed with plasmids, and 60ng/well PAH-opt/WT expression plasmid, 90nL/well P3000 and 90nL/well Lipo 3000 (Thermo, L3000015) were added to 96-well HepG2 cells and placed in a CO 2 constant temperature incubator for 48h.
  • a suitable container Place the mouse in a suitable container, place it under an infrared lamp, turn on the switch, irradiate for several minutes, take out the mouse, wipe the tail with an alcohol cotton ball, and fully expand the tail vein. Place the mouse in a mouse holder, exposing the tail.
  • a suitable syringe and needle to extract the sample to be injected.
  • For injection use a 1mL syringe needle and a matching 1mL syringe syringe. After diluting the virus sample according to the virus dosage, extract 200 ⁇ L of the dilution of the sample to be injected and inject it through the tail vein.
  • the injection should be completed in more than 10 seconds. The injection should be slow and uniform. After the injection is completed, press the injection site with a dry cotton ball to stop bleeding.
  • Blood was collected from the periorbital area of mice, and 20 ⁇ L was dripped onto a blood collection card and allowed to air dry.
  • the phenylalanine concentration in the blood was quantified using a phenylalanine assay kit (FENGHUA, AN302) and calibrated using a standard blood card.
  • FENGHUA phenylalanine assay kit
  • the cells to be tested were cultured in a 24-well plate. During the test, the cells to be tested were collected, rinsed with PBS, and 150 ⁇ L of reaction solution (containing 0.25% NP-40, 50 mM Hepes, 150 mM KCL, 800 mM L-Phe, 100 ⁇ g/mL Catalase, 400 ⁇ M FeNH 4 (SO 4 ) 2 , 400 ⁇ M BH 4 , 2 mM DTT) was added to each well and incubated at 37°C for 3 hours. 15 ⁇ L of the reaction solution was added dropwise to the blood collection card and air-dried naturally. The phenylalanine concentration in the blood was quantified using a phenylalanine assay kit (FENGHUA, AN302) to calculate the reduction in Phe concentration.
  • FENGHUA phenylalanine assay kit
  • mice After the mice were killed, brain tissues were obtained and the 5-HIAA content was detected by mass spectrometry by Suzhou Panomik Biotechnology Co., Ltd.
  • the mixture was centrifuged at 12000 rpm and 4°C for 10 minutes, and the supernatant was taken for testing; an XDB-C18analytical 4.6*150mm 5-Micron chromatographic column and an electrospray ionization source were used for scanning detection using multiple reaction monitoring (MRM); the content of 5-HIAA in the sample to be tested was calculated according to the standard curve quantitative method.
  • MRM multiple reaction monitoring
  • Example 1 Construction and purification of adeno-associated virus vector
  • the structure of the PAH expression cassette is shown in Figure 1.
  • the PAH expression cassette includes 5'ITR, ApoE HCR enhancer, SerpinA1 promoter, truncated SerpinA1 intron, Kozak sequence, target gene: wild-type human PAH gene hPAH WT or optimized PAH gene hPAH opt, BGH polyA, HPRT (4CpG) filling sequence and 3'ITR from the 5' end to the 3' end.
  • the nucleotide sequences of the PAH expression cassettes containing different target genes are shown in SEQ ID NO.79-93. Among them:
  • the nucleotide sequence of 5’ITR is shown in SEQ ID NO.1.
  • ApoE HCR enhancer is the hepatocyte control region of human apolipoprotein E, and its nucleotide sequence is shown in SEQ ID NO.2.
  • SerpinA1 promoter is human ⁇ 1 antitrypsin promoter, and its nucleotide sequence is shown in SEQ ID NO.3.
  • the SerpinA1 intron is a truncated ⁇ 1 antitrypsin intron with a length of 261bp, and its nucleotide sequence is shown in SEQ ID NO.4.
  • the Kozak sequence was inserted before the PAH gene sequence, and its sequence is shown in SEQ ID NO.5.
  • the hPAH WT gene is derived from the wild-type human PAH gene (GeneID: 5053), and its gene sequence is shown in SEQ ID NO.6.
  • the NCBI accession number of the raw hPAH WT protein is NP_000268.1.
  • the optimized PAH gene hPAH opt is the codon-optimized gene opt1 to 15 encoding the wild-type human PAH protein, and its nucleotide sequences are SEQ ID NO.9 to 23, respectively.
  • BGH polyA is the bovine growth hormone polyadenylation signal, and its nucleotide sequence is shown in SEQ ID NO.7.
  • HPRT (4CpG) filling sequence is a partial intron sequence of hypoxanthine phosphoribosyltransferase, and its nucleotide sequence is shown in SEQ ID NO.43.
  • the nucleotide sequence of 3’ITR is shown in SEQ ID NO.8.
  • the expression cassette containing the wild-type human PAH gene hPAH WT was constructed as a shuttle plasmid pAAV8-ATT-PAH-WT-HPRT.
  • SEQ ID NO.51 shows the sequence of the shuttle plasmid containing the expression cassette with the wild-type human PAH gene hPAH WT.
  • the expression cassette containing the optimized PAH genes opt 1 to 15 was constructed as a shuttle plasmid pAAV8-ATT-PAH-opt1 to 15-HPRT.
  • SEQ ID NO. 52 to 66 respectively show the shuttle plasmid sequences containing the expression cassette with the codon-optimized genes opt 1 to 15.
  • AAV vectors uses a three-plasmid system, that is, the shuttle plasmid pAAV8-ATT-PAH-WT-HPRT or pAAV8-ATT-PAH-opt1 ⁇ 15-HPRT containing the PAH target gene, the pRepCap plasmid with the AAV vector repcap gene, and the auxiliary plasmid pHelper are used to co-transfect HEK293 cells with PEI as a transfection reagent to recombinantly package the AAV virus vectors, which are named AAV8-ATT-PAH-WT-HPRT and AAV8-ATT-PAH-opt1 ⁇ 15-HPRT.
  • the harvested fluid is harvested 48-72 hours after transfection, purified by affinity chromatography, further purified by anion chromatography, ultrafiltration and concentration, and buffer replacement.
  • the genome titer of the purified recombinant AAV virus vector is measured, and it is sterilized, filtered and aliquoted for use.
  • the in vitro expression level of codon-optimized PAH opt was evaluated in the HepG2 cell line (purchased from the cell bank of the Chinese Academy of Sciences Type Culture Collection Committee, catalog number: TCHu72).
  • the shuttle plasmid pAAV8-ATT-PAH WT containing the wild-type human PAH gene hPAH WT driven by the ATT promoter was constructed, and its nucleotide sequence is as shown in SEQ ID NO.94; and the shuttle plasmid pAAV8-ATT-PAH opt 1 ⁇ 15 containing the optimized PAH gene hPAH opt1-15, and its nucleotide sequence is as shown in SEQ ID NO.95 ⁇ 109, and the target gene was introduced into the cell by the aforementioned "in vitro cell plasmid transfection experiment" method.
  • plasmid pAAV8-ATT-PAH WT expressing PAH WT or plasmid pAAV8-ATT-PAH opt1-15 expressing PAH opt were transiently transfected, and protein expression in cell lysates was evaluated by Western blot analysis, and the results are shown in Figure 2.
  • Figure A shows a representative image of Western blot of PAH protein;
  • Figure B shows the quantitative results.
  • the expression levels of codon-optimized PAH genes opt2-9 and opt11-15 in HepG2 cells are significantly higher than those of wild-type human PAH gene (WT), indicating that the codon-optimized PAH gene disclosed in the present invention has a higher expression level.
  • Example 3 In vivo efficacy evaluation of codon-optimized PAH opt in a PKU mouse model
  • PKU model mice purchased from Beijing Chengtian Biotechnology Co., Ltd., strain BTBR-Pah ⁇ enu2>/J
  • mice were injected with 40 ⁇ g of pAAV8-ATT-PAH WT or pAAV8-ATT-PAH opt2, opt9, opt14 plasmids by tail vein high pressure injection.
  • Blood was collected from mice at 0h, 6h, 24h, 3 days, 5 days, 10 days, 16 days, and 19 days after injection to determine the Phe content in the blood.
  • the effect of PAH opt plasmid on reducing Phe was compared and analyzed, and the results are shown in Figures 3A and 3B.
  • the shuttle plasmid of Example 2 was used to recombinantly package AAV viral vectors by the same method as 1.2 of Example 1, and they were named AAV8-ATT-PAH WT and AAV8-ATT-PAH opt2, 9, 11, and 14, respectively.
  • AAV8-ATT-PAH WT or AAV8-ATT-PAH opt2, opt9, opt11, opt14 viruses were injected intravenously into PKU model mice at a dose of 1e10 vg/mouse. Blood was collected from mice every week after injection to measure the Phe content in the blood until 4 weeks, and the effect of AAV8-ATT-PAH opt virus on reducing Phe was compared and analyzed. The results are shown in Figure 4. As can be seen from Figure 4, the blood Phe concentration of model mice injected with AAV8-ATT-PAH opt2, 9, and 14 viruses was lower than that of AAV8-ATT-PAH WT, indicating that AAV8-ATT-PAH opt2, 9, and 14 viruses carrying codon optimization have better Phe reduction effects.
  • ApoE HCR enhancer is the hepatocyte control region of human apolipoprotein E, and its nucleotide sequence is shown in SEQ ID NO.2; Core ApoE HCR enhancer is the hepatocyte control region of human apolipoprotein E, and its nucleotide sequence is shown in SEQ ID NO.37; CRMSBS2 enhancer is a modified Serpin1 enhancer, and its nucleotide sequence is shown in SEQ ID NO.29; TTRm enhancer is a mutated transthyretin enhancer region, and its nucleotide sequence is shown in SEQ ID NO.32; SerpinA1 promoter is human ⁇ 1 antitrypsin promoter, and its nucleotide sequence is shown in SEQ ID NO.3; Core SerpinA1 promoter (218bp) is the core region of human ⁇
  • the combined promoter was used to construct an expression cassette, and the structure of the expression cassette is shown in Figure 5.
  • the expression cassette was constructed into a shuttle plasmid to obtain plasmid vectors ATT-PAH-WT (SEQ ID NO.94), 100-AT-PAH-WT (SEQ ID NO.67), ATG-PAH-WT (SEQ ID NO.68), ATS-PAH-WT (SEQ ID NO.69), CTS-PAH-WT (SEQ ID NO.70) and TTM-PAH-WT (SEQ ID NO.71).
  • Example 5 Effects of other expression regulatory elements on in vivo drug efficacy in PKU mouse model
  • the expression cassettes shown in Figure 7 were constructed into shuttle plasmids to obtain plasmid vectors ATT-PAH-opt9-HPRT(47CpG)(SEQ ID NO.73), U6-ATT-PAH-opt9-HPRT(47CpG)(SEQ ID NO.75), ATT-PAH-opt9-WPRE(SEQ ID NO.74), U6-ATT-PAH-opt9-WPRE(SEQ ID NO.72), CAG-PAH-opt9(SEQ ID NO.77), and ATT-PAH-opt9(SEQ ID NO.76).
  • PKU model mice were injected with 40 ⁇ g of the above plasmids by tail vein high pressure injection.
  • Example 6 Effect of filler sequence optimization on in vivo efficacy in PKU mouse model
  • the expression cassettes containing different stuffer sequences were constructed into shuttle plasmids respectively, and the recombinant viral vector was obtained using the three-plasmid system of Example 1.
  • AAV8-ATT-PAH-opt9 (wherein the shuttle plasmid sequence is shown as SEQ ID NO.76), AAV8-ATT-PAH-opt9-HPRT(47CpG) (wherein the shuttle plasmid sequence is shown as SEQ ID NO.73), AAV8-ATT-PAH-opt9-HPRT(4CpG) (wherein the shuttle plasmid sequence is shown as SEQ ID NO.60), AAV8-HPRT(4CpG)-ATT-PAH-opt9 (wherein the shuttle plasmid sequence is shown as SEQ ID NO.78).
  • the viral vector was injected into PKU model mice by intravenous injection at a dose of 2e11vg/mouse. Blood was collected from mice every week after injection to measure the Phe content in the blood until 8 weeks, and the effects of optimization of different filling sequences on the effect of reducing Phe were compared and analyzed. As shown in Figure 10A, after the PKU model mice were injected with the viruses of the above four expression cassettes, the Phe content in the blood of the mice reached the optimal therapeutic effect ( ⁇ 120 ⁇ M).
  • an expression cassette with a filler sequence with a smaller number of CpGs was selected to further study the therapeutic effect of AAV8-ATT-PAH-opt9-HPRT (4CpG) on PKU model mice at a lower dose of 1e10 vg/mouse.
  • the blood of the mice was collected every week after injection, and the Phe content in the blood was measured for up to 4 weeks.
  • the results are shown in Figure 10B.
  • the effect of AAV8-ATT-PAH-opt9-HPRT (4CpG) in reducing Phe is better than that of AAV8-ATT-PAH-opt9.
  • Example 7 In vitro PAH-Phe-reducing function test of the optimized expression cassette AAV8-ATT-PAH-opt9-HPRT (4CpG) virus expression product
  • This example evaluates the Phe-lowering function of the codon-optimized AAV8-ATT-PAH-opt9-HPRT (4CpG) virus expression product in the HepG2 cell line.
  • a plasmid expressing the adeno-associated virus receptor AAVR was transiently transfected in HepG2 (the AAVR gene was synthesized by GeneWeizhi according to the sequence of SEQ ID NO.117, and then inserted into the pcDNA3.1(+) plasmid between the NheI and XbaI restriction sites). Overexpression of AAVR will increase the efficiency of AAV8 in infecting HepG2.
  • the AAV8-ATT-PAH-opt9-HPRT (4CpG) virus of Example 1 was used to infect the cells at MOIs of 0, 5e4, 1e5, and 2e5, respectively. After 48 hours of culture, the Phe content was detected. The HepG2 cell line not infected with the virus was used as a reference, and the Phe-reducing activity of PAH was evaluated by calculating the change in Phe concentration. The results are shown in Figure 11, indicating that the AAV8-ATT-PAH-opt9-HPRT (4CpG) virus expression product has the function of reducing Phe, and the virus infection MOI has a dose effect.
  • Example 8 In vivo efficacy of the optimized expression cassette in the PKU mouse model: low dose, high activity
  • the AAV8-ATT-PAH-opt9-HPRT (4CpG) virus of Example 1 was injected into male PKU model mice by intravenous injection at a dose of 3.0e11, 1.0e11, 3.3e10, 1.5e10, 1.1e10, and 3.0e9 vg/mouse, respectively. Blood of mice was collected every week after injection, and the content of Phe in the blood was measured until 6 weeks. The effects of different viral administration doses on the effect of reducing Phe were compared and analyzed, and the results are shown in FIG12A.
  • the Phe concentration in the blood of mice was lower than 120 ⁇ M at a dose of 3.0e11, 1.0e11, and 3.3e10 vg/mouse, achieving a therapeutic effect. Therefore, it is believed that the minimum effective dose of AAV8-ATT-PAH-opt9-HPRT (4CpG) virus for treating PKU in male mice is about 3.3e10 vg/mouse.
  • the AAV8-ATT-PAH-opt9-HPRT (4CpG) virus of Example 1 was injected into female PKU model mice at a dose of 4.0e11, 2.0e11, 1.0e11, 5.0e10, and 2.5e10 vg/mouse, respectively, by intravenous injection.
  • the blood of the mice was collected every week after injection, and the content of Phe in the blood was measured until 6 weeks.
  • the effects of different viral administration doses on the effect of reducing Phe were compared and analyzed, and the results are shown in FIG12B.
  • the blood Phe concentration of the mice was lower than 120 ⁇ M at a dose of 4.0e11, 2.0e11, 1.0e11, and 5.0e10 vg/mouse, and the therapeutic effect was achieved. Therefore, it is believed that the minimum effective dose of AAV8-ATT-PAH-opt9-HPRT (4CpG) virus for treating PKU in female mice is about 5.0e10 vg/mouse.
  • Example 9 Other efficacy of the optimized expression cassette in the PKU mouse model
  • FIG. 13B shows the content of 5-hydroxyindoleacetic acid (5-HIAA, a pharmacodynamic marker for PKU patients) in the brain tissue of PKU homozygous mice, and heterozygous mice that were not given the drug were used as normal mouse controls.
  • 5-HIAA 5-hydroxyindoleacetic acid
  • FIG. 13C shows the hair color of PKU homozygous mice compared with the vehicle group after 3 weeks of administration at a dose of 3.0e10vg/mouse.
  • the present disclosure achieves the effective, long-lasting and stable suppression of peripheral blood phenylalanine concentration in PKU mice at low doses and improves other PKU phenotypes by optimizing and screening genes and expression regulatory elements.
  • the present disclosure can effectively reduce the dose and possible side effects of gene therapy drugs used to treat PKU and improve the treatment effect.

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Abstract

本公开涉及优化的PAH基因和表达盒及其用途，其中披露了一种编码PAH蛋白的多核苷酸分子。采用本公开所提供的多核苷酸分子、表达盒、表达载体、病毒颗粒和/或药物组合物，能够实现用相对较低的剂量在肝脏中有效持久且稳定地表达人PAH，使受试者血液中苯丙氨酸的浓度长期稳定维持在较低水平，从而可以用于苯丙酮尿症治疗。

Description

优化的PAH基因和表达盒及其用途 技术领域
本公开属于基因治疗技术领域，具体涉及一种优化的PAH基因和表达盒及其用途。
背景技术
苯丙酮尿症(phenylketonuria PKU，OMIM 261600)是由苯丙氨酸羟化酶(PAH)基因突变导致的常染色体有隐形遗传性疾病，美国的发病率1/13500，中国的发病率约为1/15,924。新生儿和儿童可以通过低苯丙氨酸奶粉与饮食来较好地控制外周血苯丙氨酸的浓度以便神经系统的发育。目前有口服的四氢生物蝶呤(tetrahydrobiopterin)，又称沙丙蝶呤(Sapropterin，商品名Kuvan)和皮下注射的长效化的苯丙氨酸氨解酶(pegvaliase-pqpz，商品名Palynziq，一种PEG化的苯丙氨酸氨解酶)可供成人使用。但这两种药物都有其缺陷和局限性，主要表现为：只有部分病人适用，免疫反应的副作用，见效慢等。
目前美国的遗传变异分类标准与治疗指南推荐的控制标准是终身通过饮食与药物治疗，使外周血苯丙氨酸的浓度在120μmol/L至360μmol/L之间(Vockley,J.,et al.,Phenylalanine hydroxylase deficiency:diagnosis and management guideline.Genet Med,2014.16(2):p.188-200.)。欧盟的治疗指南的控制标准是12岁以下和备孕及孕期妇女小于360μmol/L，其他人群小于600μmol/L(van Wegberg,A.M.J.,et al.,The complete European guidelines on phenylketonuria:diagnosis and treatment.Orphanet J Rare Dis,2017.12(1):p.162.)。然而由于成年患者很难坚持饮食控制，大约只有19％的患者坚持不超过9个月，因此大部分成年患者会失去随访，且处于高苯丙氨酸血症的状态。有调查显示在成年PKU患者中，67％的患者苯丙氨酸的浓度Phe>360μmol/L，45％的高于600μmol/L，18％高于1200μmol/L；只有约24％的成年患者可以控制在≤360μmol/L(Brown,C.S.and U.Lichter-Konecki,Phenylketonuria(PKU):A problem solved？Mol Genet Metab Rep,2016.6:p.8-12.)。
即使早期得到良好的治疗，成年患者如果不控制外周血苯丙氨酸，则很可能出现神经系统方面的症状，比如：震颤、深腱反射活跃、运动协调性差、白质异常等。此外成年PKU患者也会面临很多生活质量的问题，比如工作能力低下，自治力不足，容易绝望，缺乏取得成就的动力，抑郁与焦虑类的症状，朋友之间的关系难以维持足够长时间、晚年容易离家出走(Murphy,G.H.,et al.,Adults with untreated phenylketonuria:out of sight,out of mind.Br J Psychiatry,2008.193(6):p.501-2.；Hoeks,M.P.,M.den Heijer,and M.C.Janssen,Adult issues in phenylketonuria.Neth J Med,2009.67(1):p.2-7.)。对于晚期诊断发现和早期没有得到充分治疗的青少年和成年病人，外周血苯丙氨酸的高浓度会导致癫痫、痉挛，严重的行为问题如改善行为：攻击性行为、自伤、多动、烦躁、易怒、睡眠障碍、焦虑、刻板行为，以及神经和认知问题(van Vliet,D.,et al.,Can untreated PKU patients escape from intellectual disability？A systematic review.Orphanet J Rare Dis,2018.13(1):p.149.；Ashe,K.,et al.,Psychiatric and Cognitive Aspects of Phenylketonuria:The Limitations of Diet and Promise of New Treatments.Front Psychiatry,2019.10:p.561.；Romani,C.,et al.,Adult cognitive outcomes in phenylketonuria:explaining causes of variability beyond average Phe levels.Orphanet J Rare Dis,2019.14(1):p.273.；Trepp,R.,et al.,Impact of phenylalanine on cognitive,cerebral,and neurometabolic parameters in adult patients with phenylketonuria(the PICO study):a randomized,placebo-controlled,crossover,noninferiority trial.Trials,2020.21(1):p.178.；Altman,G.,et al.,Mental health diagnoses in adults with phenylketonuria:a retrospective systematic audit in a large UK single centre.Orphanet J Rare Dis,2021.16(1):p.520.；Trefz,F.,et al.,Health economic burden of patients with phenylketonuria(PKU)-A retrospective study of German health insurance claims data.Mol Genet Metab Rep,2021.27:p.100764.；Yamada,K.,et al.,Long-Term Neurological Outcomes of Adult Patients with Phenylketonuria before and after Newborn Screening in Japan.Int J Neonatal Screen,2021.7(2).)。
因此成年期苯丙酮尿症的治疗仍然具有极大的临床需求。研究证明通过控制外周血苯丙氨酸的浓度治疗成年病人，能减少或停止癫痫发作，改善行为和神经认知问题(van Spronsen,F.J.,et al.,Phenylketonuria.Nat Rev Dis Primers,2021.7(1):p.36.)。
基因治疗经过近60年的发展已经取得了显著的成就。Zolgensma一次治疗可以使脊髓性肌萎缩(SMA)患儿基本可以向正常儿童一样生长发育。Luxturna一次治疗，可以使莱伯先天性黑蒙(LCA)患者的视力恢复到基本可以正常生活、学习及工作的状态。针对PKU，目前国际上有两项基因治疗项目正处于临床I阶段。两项都采用高剂量，尚没有实现有效的治疗效果的报告，但存在高剂量带来的潜在的肝脏毒性的隐患。
发明内容
本公开的目的在于提供一种优化的PAH基因、表达盒及病毒载体，以实现用相对较低的剂量在肝脏中有效持久且稳定地表达人PAH并用于苯丙酮尿症治疗。
本公开为解决上述技术问题，提出了如下技术方案：
本公开第一方面提供了一种编码PAH蛋白的多核苷酸分子，其包含与SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22或SEQ ID NO.23所示核苷酸序列具有90％或以上同一性的核苷酸序 列；优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸列；更优选具有98％或99％以上同一性的核苷酸序列。
本公开第二方面提供了一种表达盒，其包含本公开第一方面所提供的多核苷酸分子，以及与所述多核苷酸分子可操作连接的启动子。
在一些实施方式中，所述表达盒还包含表达控制元件，所述表达控制元件与所述多核苷酸分子可操作地连接。
在一些实施方式中，所述表达控制元件选自转录/翻译控制信号、增强子、内含子、polyA信号、ITR、绝缘子、RNA加工信号、增强mRNA和蛋白质的稳定性的元件中的至少一种。
本公开第三方面提供了一种表达载体，其包含本公开第一方面所提供的多核苷酸分子或本公开第二方面所提供的表达盒。
在一些实施方式中，所述表达载体选自质粒、粘粒、病毒载体、RNA载体或线性或圆形DNA或RNA分子。
在一些实施方式中，所述的表达载体为腺相关病毒载体。
本公开第四方面提供了一种病毒颗粒，其包含本公开第一方面所提供的多核苷酸分子、本公开第二方面所提供的表达盒和本公开第三方面所提供的表达载体中的至少一种。
本公开第五方面提供了一种治疗苯丙酮尿症的药物组合物，其包含本公开第一方面所提供的多核苷酸分子、本公开第二方面所提供的表达盒和、本公开第三方面所提供的表达载体和本公开第四方面所提的病毒颗粒中的至少一种。
本公开第六方面提供了本公开第一方面所提供的多核苷酸分子、本公开第二方面所提供的表达盒、本公开第三方面所提供的表达载体、本公开第四方面所提的病毒颗粒和本公开第五方面的药物组合物中的至少一种在制备治疗苯丙酮尿症的药物中的用途。
本公开第七方面提供了本公开第一方面所提供的多核苷酸分子、本公开第二方面所提供的表达盒、本公开第三方面所提供的表达载体、本公开第四方面所提的病毒颗粒和本公开第五方面的药物组合物中的至少一种在治疗苯丙酮尿症的中的用途。
本公开第八方面提供了一种治疗苯丙酮尿症的方法，其包括向有受试者施用有效量的本公开的多核苷酸分子、表达盒、表达载体、病毒颗粒和药物组合物中的至少一种。
采用本公开所提供的多核苷酸分子、表达盒、表达载体、病毒颗粒和/或药物组合物，通过对PAH的编码基因进行优化，使得其能够更有效地表达人PAH，进而能够实现用相对较低的剂量在肝脏中有效持久且稳定地表达人PAH，使受试者血液中苯丙氨酸的浓度长期稳定维持在较低水平，从而可以用于苯丙酮尿症治疗。进一步的，采用本公开的多核苷酸分子、表达盒、表达载体、病毒颗粒和/或药物组合物具有较低的剂量，从而具有更低的潜在的肝脏毒性。
附图说明
图1示出了本公开的重组AAV(rAAV)载体PAH表达盒的结构示意图。
图2示出了在由质粒转染的HepG2细胞中密码子优化的PAH opt基因的体外表达。其中，A图为PAH蛋白质Western印迹的代表性图像；B图显示了PAH蛋白质Western印迹的定量结果，以检测HepG2细胞中PAH和GAPDH(质粒转染对照)的表达水平，其中WT或opt基因分别用pAAV8-ATT-PAH WT或pAAV8-ATT-PAH opt质粒表达。等量的质粒转染后2天收获细胞裂解物。通过SDS-PAGE分离等量的总蛋白，然后进行免疫印迹。PAH的表达水平用GAPDH标准化，并计算为与WT表达水平的比率。
图3A示出了在由质粒尾静脉高压注射PAH WT和优化基因后，对PKU模型小鼠中降低Phe效果的影响。在注射后0h、6h、24h、3天、5天、10天、16天、19天的时间点，采集小鼠血液，测定血液中Phe的含量；图3B示出注射后10天时间点PKU模型小鼠血液中Phe的含量。
图4示出了AAV8-ATT-PAH WT和优化基因病毒对PKU模型小鼠中降低Phe效果的影响。在注射后4周时间内，每周采集小鼠血液，测定血液中Phe的含量。
图5为含有不同启动子的重组AAV(rAAV)载体PAH表达盒的结构示意图。
图6示出在由质粒尾静脉高压注射不同启动子的PAH WT表达盒后，对PKU模型小鼠中降低Phe效果的影响。在注射后第3天，采集小鼠血液，测定血液中Phe的含量。
图7为含有不同表达调控元件的重组AAV(rAAV)载体PAH表达盒的结构示意图。
图8示出在由质粒尾静脉高压注射含有不同表达调控元件的PAH表达盒后，对PKU模型小鼠中降低Phe效果的影响。在注射后第3天的时间点，采集小鼠血液，测定血液中Phe的含量。
图9为含有不同填充序列的优化重组AAV(rAAV)载体PAH表达盒的结构示意图。
图10A示出了具有不同填充序列的AAV8-ATT-PAH opt9病毒对PKU模型小鼠中降低Phe效果的影响，在注射后8周时间内，每周采集小鼠血液，测定血液中Phe的含量。图10B示出了AAV8-ATT-PAH opt9与AAV8-ATT-PAH opt-HPRT(4CpG)病毒对PKU模型小鼠中降低Phe效果的影响，在注射后4周时间内，每周采集小鼠血液，测定血液中Phe的含量。
图11示出了AAV8-ATT-PAH opt-HPRT(4CpG)病毒在HepG2细胞系中的表达产物PAH降低Phe浓度的功能检测结果。在瞬转AAVR的HepG2细胞内，以0、5e4、1e5、2e5的MOI侵染AAV8-ATT-PAH opt-HPRT(4CpG)病毒，以未感染病毒的HepG2细胞系为参照，计算Phe浓度的变化量。
图12A示出了不同剂量的AAV8-ATT-PAH opt9-HPRT(4CpG)病毒对PKU模型雄性小鼠中降低Phe效果的影响，其中，在注射后6周时间内，每周采集小鼠血液，测定血液中Phe的含量。图12B示出了不同剂量的AAV8-ATT-PAH opt9-HPRT(4CpG)病毒对PKU模型雌性小鼠中降低Phe效果的影响，在注射后6周时间内，每周采集小鼠血液，测定血液中Phe的含量。
图13A显示了PKU纯合子小鼠在接受3.0e10和3.0e11vg/只小鼠的AAV8-ATT-PAH opt9-HPRT(4CpG)病毒，给药6周后，小鼠脑组织中Tyr的含量，其中未给药的杂合子小鼠作为正常小鼠对照组，给药溶媒的纯合子小鼠作为未治疗对照组。图13B显示小鼠脑组织中的5-羟基吲哚乙酸(5-HIAA)的含量。图13C展示了PKU纯合子小鼠，在3.0e10vg/只小鼠剂量下给药3周后与溶媒组的毛色对比。
具体实施方式
为了更清楚地说明本公开实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本公开的一种实施方式，对于本领域普通技术人员来讲，还可以根据这些附图获得其他的实施方式。
定义
在本公开中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本公开，下面提供相关术语的定义和解释。
如本文所用，术语“一个”和“一种”以及“所述”和类似的指代物指示单数和复数，除非本文另外指明或上下文明显矛盾。
如本文所用，术语“约”、“基本上”和“类似于”是指在本领域普通技术人员所确定的特定值的可接受误差范围内，所述误差范围可部分取决于该值的测量或确定方式，或取决于测量系统的局限性。如本文使用本文提及“约”某个值或参数包括(和描述)针对值或参数本身的实施方式。例如，提及“约X”的描述包括“X”的描述。
如本文使用的“载体”指包含待递送入宿主细胞(体外或体内)的核酸的重组质粒或病毒。
如本文使用的术语“多核苷酸分子”或“核酸”指任何长度的聚合形式的核苷酸，其为核糖核苷酸或脱氧核糖核苷酸。因此，该术语包括但不限于单、双或多链DNA或RNA、基因组DNA、cDNA、DNA-RNA杂交物或包含嘌呤和嘧啶碱基或其他天然、化学或生化修饰的、非天然或衍生的核苷酸碱基的聚合物。核酸的骨架可包含糖和磷酸基团(如通常在RNA或DNA中所发现)，或修饰或取代的糖或磷酸基团。可替换地，核酸的骨架可包含合成的亚单元如氨基磷酸的聚合物且因此可以是寡脱氧核苷氨基磷酸(P-NH2)或混合的氨基磷酸酯-磷酸二酯低聚物。此外，双链核酸可从化学合成(通过在适当的条件下合成互补链并退火该链，或使用DNA聚合酶以适当的引物从头开始合成互补链)的单链多核苷酸产物获得。
“重组病毒载体”指包含一种或多种异源序列(即非病毒来源的核酸序列)的重组多核苷酸载体。在重组AAV载体的情况中，重组核酸侧翼为至少一个，优选两个反向末端重复序列(ITR)。
“重组AAV载体(rAAV载体)”指含有一个或多个异源序列(即非AAV来源的核酸序列)的多核苷酸载体，其侧翼为至少一个，优选两个AAV反向末端重复序列(ITR)。当存在于已经使用适当的辅助病毒(或表达适当的辅助功能)感染并表达AAV rep和cap基因产物(即AAV Rep和Cap蛋白)的宿主细胞中时，该rAAV载体可复制并包装入感染的病毒颗粒。当rAAV载体并入较大多核苷酸(例如，在染色体中或在用于克隆或转染的另一载体如质粒中)时，可将rAAV载体称为“原载体(pro-vector)”，其在AAV包装功能和适当的辅助功能存在下可通过复制和壳体化“援救”。rAAV载体可以是多种形式中的任一种，包括但不限于质粒、线性人工染色体，其可与脂质体复合、包封在脂质体内，且在实施方式中，包封在病毒颗粒特别是AAV颗粒中。可将rAAV载体包装入AAV病毒衣壳以生成“重组腺伴随病毒颗粒(rAAV颗粒)”。AAV辅助功能(即允许AAV由宿主细胞复制和包装的功能)可以多种形式中的任一种提供，包括但不限于帮助AAV复制和包装的辅助病毒或辅助病毒基因。其他AAV辅助功能为本领域已知。
“rAAV病毒”或“rAAV病毒颗粒”指由至少一种AAV衣壳蛋白和壳体化的rAAV载体基因组组成的病毒颗粒。
“异源”意为源自与其所比较的或其所导入或并入的实体的其余部分在基因型上不同的实体。例如，由基因工程技术引入不同细胞类型的核酸是异源核酸(且当表达时，可编码异源多肽)。相似地，并入病毒载体的细胞序列(例如基因或其部分)相对于载体是异源核苷酸序列。
如指代病毒滴度中使用，术语“基因组颗粒(gp)”、“基因组等价物”或“基因组拷贝”指包含重组AAV DNA基因组的病毒粒数目，不论其感染性或功能性。在特定载体制备中基因组颗粒的数目可通过如本文实施例或例如Clark等人(1999)Hum.Gene Ther.,10:1031-1039；Veldwijk等人(2002)Mol.Ther.,6:272-278中所述的方法测量。
如指代病毒滴度中使用，术语“感染单位(iu)”、“感染颗粒”或“复制单位”指如通过感染中心测定所测量的具有感染和复制能力的重组AAV载体颗粒的数目，也称为复制中心测定，如例如McLaughlin等人(1988)J.Virol.,62:1963-1973中所述。
如指代病毒滴度中使用，术语“转导单位(tu)”指导致功能性转基因产物产生的感染性重组AAV载体颗粒的数目，如在功能性测定中所测量，如本文实施例或例如Xiao等人(1997)Exp.Neurobiol.,144:113-124中；或Fisher等人(1996)J.Virol.,70:520-532(LFU测定)中所述。
“反向末端重复”或“ITR”序列是本领域熟知的术语且指代在病毒基因组的末端发现的相对短的序列，其处于相 反方向。
“AAV反向末端重复(ITR)”序列是本领域熟知的术语，其为在天然单链AAV基因组两末端存在的约145个核苷酸的序列。ITR的最外面125个核苷酸可以两种可替换的方向中的任一种存在，导致不同AAV基因组间和单AAV基因组的两末端间的异质性。最外面125个核苷酸还包含自身互补的数个较短区(称为A、A'、B、B'、C、C'和D区)，允许在该ITR部分内发生链间碱基配对。
对于AAV的“辅助病毒”指允许AAV(为有缺陷的细小病毒)通过宿主细胞复制并包装的病毒。已识别多种这样的辅助病毒，包括腺病毒、泡疹病毒和痘类病毒如牛痘。腺病毒涵盖多个不同的亚类，尽管亚类C的腺病毒5型(Ad5)是最常用的。已知人、非人哺乳动物和鸟类来源的多种腺病毒且可从保藏机构如ATCC获得。也可从保藏机构如ATCC获得的泡疹病毒家族包括例如单纯泡疹病毒(herpes simplex viruses，HSV)、EB病毒(Epstein-Barr viruses，EBV)、巨细胞病毒(cytomegaloviruses，CMV)和伪狂犬病病毒(pseudorabies viruses，PRV)。
本公开中“填充序列”是指为了使重组AAV表达盒的核苷酸序列长度接近野生型AAV基因组长度而添加的人非编码序列。在本公开的一些实施方式中，加入填充序列能够使该表达盒或携带其的多核苷酸序列在使用AAV病毒载体进行表达时，具有更高的病毒包装产量。
相对于参照多肽或核酸序列的“百分比(％)序列同一性”定义为对齐序列并引进缺口后(若需要，以实现最大百分比序列同一性，且不将任何保守取代考虑为序列同一性的部分)，候选序列中与参照多肽或核酸序列中的氨基酸残基或核苷酸相同的氨基酸残基或核苷酸的百分比。出于确定百分比氨基酸或核酸序列同一性的目的的比对可以多种本领域内的方式实现，例如使用公共可获得的计算机软件程序，例如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。优选的比对软件是ALIGN Plus(Scientific and Educational Software,Pennsylvania)。本领域的技术人员可确定用于测量比对的适当的参数，包括任何在比较的序列全长上需要实现最大比对的算法。就本文而言，给定氨基酸序列A对、与或针对给定氨基酸序列B的％氨基酸序列同一性(其可替换地可称为具有或包含对、与或针对给定氨基酸序列B具有一定％氨基酸序列同一性的氨基酸序列A)如下计算：100乘以分数X/Y，其中X是在A和B的程序比对中通过序列比对程序评分为相同匹配的氨基酸残基的数目，且其中Y是B中氨基酸残基的总数。可以理解的是其中氨基酸序列A的长度与氨基酸序列B的长度不等，A对B的％氨基酸序列同一性将不等于B对A的％氨基酸序列同一性。就本文而言，给定核酸序列C对、与或针对给定核酸序列D的％核酸序列同一性(其可替换地可称为对、与或针对给定核酸序列D具有或包含一定％核酸序列同一性的给定核酸序列C)如下计算：100乘以分数W/Z，其中W是在C和D的程序比对中通过序列比对程序评分为相同匹配的核苷酸的数目，且其中Z是D中核苷酸的总数。可以理解的是其中核酸序列C的长度不等于核酸序列D的长度，C对D的％核酸序列同一性将不等于D对C的％核酸序列同一性。
“有效量”是足以影响有益的或预期的结果，包括临床结果(例如改善症状、实现临床终点等)的量。有效量可以一次或多次施用。就疾病状态而言，有效量是足以改善、稳定或延迟疾病进展的量。例如，有效量的rAAV颗粒表达预期量的异源核酸如治疗性多肽或治疗性核酸。
“个体”或“受试者”是哺乳动物。哺乳动物包括但不限于驯养动物(例如，牛、绵羊(sheep)、猫、狗和马)、灵长类动物(例如，人和非人灵长类动物如猴)、兔和啮齿类动物(例如小鼠和大鼠)。在一些实施方式中，个体或受试者是人。
如本文使用的“治疗”是用于获得有益或预期的临床结果的方式。就本公开而言，有益或预期的临床结果包括但不限于症状改善、疾病程度的减少、疾病的稳定化(例如未恶化)状态、疾病扩散(例如转移)的防止、疾病进展的延迟或减缓、疾病状态的改善或缓和以及缓解(无论部分或全部)，无论是可检测的或不可检测的。“治疗”还可意为与未接受治疗的预期生存率相比延长生存率。
单位vg(Vector Genomes)代表病毒基因组拷贝数。
病毒感染复数(MOI)的含义是感染时病毒与细菌的数量比值，也就是平均每个细菌感染噬菌体的数量。
本公开第一方面提供了一种编码PAH蛋白的多核苷酸分子，其包含与SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22或SEQ ID NO.23所示核苷酸序列具有90％或以上同一性的核苷酸序列；优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸列；更优选具有98％或99％以上同一性的核苷酸序列。
在一些实施方式中，所述多核苷酸分子具有SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22或SEQ ID NO.23所示的核苷酸序列。
在一些实施方式中，所述密码子优化的人源PAH蛋白编码基因具有SEQ ID NO.17所示的核苷酸序列。
本公开第二方面提供了一种表达盒，其包含本公开第一方面所提供的多核苷酸分子，以及与所述多核苷酸分子可操作连接的启动子。
在一些实施方式中，所述启动子为特异性或非特异性启动子。
在一些实施方式中，所述启动子包含核心启动子。
在一些实施方式中，所述启动子可以是组成型启动子；优选地，所述组成型启动子选自CMV启动子、EF1A启动子、EFS启动子、CAG启动子、CBh启动子、SFFV启动子、MSCV启动子、SV40启动子、mPGK启动子、hPGK启动子、UBC启动子等的至少一种。
在一些实施方式中，所述启动子是可诱导启动子；优选地，所述可诱导启动子包含四环素调控启动子、醇调控启动子、类固醇调控启动子、金属调控启动子、致病调控启动子、温度/热诱导型启动子和光调控启动子、IPTG诱导型启动子中的至少一种。在一些实施方式中，所述四环素调控启动子选自Tet on启动子、Tet off的启动子和Tet Activator启动子。在一些实施方式中，所述醇调控启动子选自醇脱氢酶I(alcA)基因启动子、响应于醇反式激活蛋白(AlcR)的启动子。在一些实施方式中，所述类固醇调控启动子选自大鼠糖皮质激素受体启动子、人雌激素受体启动子、蛾蜕皮激素受体启动子，类视黄醇启动子和甲状腺受体超家族启动子。在一些实施方式中，所述金属调控启动子选自酵母、小鼠和人的金属硫蛋白启动子。在一些实施方式中，所述致病调控启动子选自由水杨酸调控启动子、乙烯调控启动子和苯并噻二唑调控(BTH)启动子。在本公开的一些实施方式中，所述温度/热诱导型启动子选自HSP-70启动子、HSP-90启动子和大豆热激启动子。在本公开的一些实施方式中，所述光调控启动子是植物细胞的光应答型启动子。
在一些优选的实施方式中，所述启动子为肝脏特异性启动子；肝脏特异性启动子的一些非限制性实例包括但不限于ApoA-I启动子、ApoA-II启动子、ApoA-IV启动子、ApoB启动子、ApoC-1启动子、ApoC-II启动子、ApoC-III启动子、ApoE启动子、白蛋白启动子、甲胎蛋白启动子、磷酸烯醇丙酮酸羧化激酶(PCK1)启动子、磷酸烯醇丙酮酸羧化激酶2(PCK2)启动子、甲状腺素转运蛋白(transthyretin，TTR)启动子、α-抗胰蛋白酶(AAT或SerpinA1)启动子、TK(胸苷激酶)启动子、血色素结合蛋白启动子、醇脱氢酶6启动子、胆固醇7α-25羟化酶启动子、IX因子启动子、a-微球蛋白启动子、SV40启动子、CMV启动子、劳斯肉瘤病毒-LTR启动子、HBV启动子、ALB启动子和TBG启动子，当然也可使用来源于这些启动子的最小启动子。更优选地，所述肝脏特异性启动子为人α1抗胰蛋白酶启动子(hAAT或SERPINA1启动子)，优选地，所述核心启动子包含与SEQ ID NO.3、24、25、30、33或38所示核苷酸序列具有90％或以上同一性的核苷酸序列，优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列；优选地，所述核心启动子具有如SEQ ID NO.SEQ ID NO.3、24、25、30、33或38所示的核苷酸序列；更优选地，更优选地，所述核心启动子的核苷酸序列如SEQ ID NO.3所示。
在一些实施方式中，所述表达盒还包含表达控制元件，所述表达控制元件与所述多核苷酸分子可操作地连接。
在一些实施方式中，所述表达控制元件选自转录/翻译控制信号、增强子、内含子、polyA信号、ITR、绝缘子、RNA加工信号、增强mRNA和蛋白质的稳定性的元件中的至少一种。
在一些实施方式中，所述表达盒包含5’ITR；优选地，所述5’ITR包含与SEQ ID NO.1所示核苷酸序列具有90％或以上同一性的核苷酸序列，优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列；更优选地，所述5’ITR具有如SEQ ID NO.1所示的核苷酸序列。
在一些实施方式中，所述表达盒包含3’ITR。在一些优选的实施方式中，所述3’ITR包含与SEQ ID NO 8所示核苷酸序列具有90％或以上同一性的核苷酸序列，优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列；更优选地，所述3’ITR具有如SEQ ID NO.8所示的核苷酸序列。
在一些实施方式中，所述表达盒还包含增强子。在一些优选的实施方式中，所述增强子选自ApoE HCR增强子或其活性片段、CRMSBS2增强子或其活性片段、TTRm增强子或其活性片段以及CMV增强子或其活性片段；更优选地，所述增强子包含与SEQ ID NO.2、29、32、37或40所示核苷酸序列具有90％或以上同一性的核苷酸序列，优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列；优选地，所述增强子具有如SEQ ID NO.2、29、32、37或40所示的核苷酸序列；更优选地，所述增强子的核苷酸序列如SEQ ID NO.2所示。
在一些实施方式中，所述表达盒还包含内含子；优选地，所述内含子为截短的α1抗胰蛋白酶内含子或其活性片段、β-珠蛋白2号内含子或其活性片段、SV40内含子或其活性片段、以及小鼠微小病毒内含子或其活性片段；优选地，所述内含子包含与SEQ ID NO.4、26、27、28、31或34所示核苷酸序列具有90％或以上同一性的核苷酸序列，优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列；优选地，所述内含子具有如SEQ ID NO.4、26、27、28、31或34所示的核苷酸序列；更优选地，所述内含子的核苷酸序列如SEQ ID NO.4所示。
在一些实施方式中，所述表达盒的启动子为包含上游调控元件、核心启动子和内含子的组合启动子。
在一些实施方式中，所述上游调控元件为增强子或其活性片段。
在一些实施方式中，所述组合启动子包含与SEQ ID NO.44、45、46、47、48或49所示核苷酸序列具有90％或以上同一性的核苷酸序列，优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列；优选地，所述组合启动子具有如SEQ ID NO.44、45、46、47、48或49所示的核苷酸序列；更优选地，所述组合启动子的核苷酸序列如SEQ ID NO.44所示。
在一些实施方式中，所述表达盒还包含polyA信号；优选地，所述polyA信号为牛生长激素poly A(BGH poly A)、短poly A、SV40polyA、和人β珠蛋白poly A中的至少一种；优选地，所述polyA信号包含与SEQ ID NO.7所示核苷酸序列具有90％或以上同一性的核苷酸序列，优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列；更优选地，所述polyA信号的核苷酸序列如SEQ ID NO.7所示。
在一些实施方式中，所述表达盒包含优化的填充序列；优选地，填充序列选自次黄嘌呤磷酸核糖基转移酶(HPRT)的部分内含子序列和土拨鼠肝炎病毒转录后调控元件(Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element，WPRE)；优选地，所述部分内含子序列中含有的CpG序列个数不超过100、80、60、50、40、30、20、15、10、9、8、7、6、5、4、3、2或1；优选地，所述部分内含子序列中不含CpG序列或土拨鼠肝炎病毒转录后调控元件(WPRE)；优选地，所述填充序列为次黄嘌呤磷酸核糖基转移酶(HPRT)的部分内含子序列；优选地，所述填充序列包含与SEQ ID NO.39或SEQ ID NO.43所示核苷酸序列具有90％或以上同一性的核苷酸序列，优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列；更优选地，所述填充序列的核苷酸序列如SEQ ID NO.39或SEQ ID NO.43所示。
在一些实施方式中，所述表达盒包含Kozak起始序列；所述Kozak起始序列包含与SEQ ID NO.5所示核苷酸序列具有90％或以上同一性的核苷酸序列，优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列；更优选地，所述Kozak起始序列具有如SEQ ID NO.5所示的核苷酸序列。
在一些实施方式中，所述表达盒包含5’ITR、ApoE HCR增强子、人α1抗胰蛋白酶启动子(SERPINA1启动子)、截短的α1抗胰蛋白酶内含子(SerpinA1内含子)、Kozak起始序列(GCCACC，SEQ ID NO.5)、所述多核苷酸分子、BGH poly A、次黄嘌呤磷酸核糖基转移酶(HPRT)的部分内含子序列(HPRT(4CpG))和3’ITR；优选地，所述表达盒包含与SEQ ID NO.80、SEQ ID NO.81、SEQ ID NO.82、SEQ ID NO.83、SEQ ID NO.84、SEQ ID NO.85、SEQ ID NO.86、SEQ ID NO.87、SEQ ID NO.89、SEQ ID NO.90、SEQ ID NO.91、SEQ ID NO.92或SEQ ID NO.93所示核苷酸序列具有90％或以上同一性的核苷酸序列，优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列；更优选地，所述表达盒具有如SEQ ID NO.80、SEQ ID NO.81、SEQ ID NO.82、SEQ ID NO.83、SEQ ID NO.84、SEQ ID NO.85、SEQ ID NO.86、SEQ ID NO.87、SEQ ID NO.89、SEQ ID NO.90、SEQ ID NO.91、SEQ ID NO.92或SEQ ID NO.93所示的核苷酸序列。
本公开第三方面提供了一种表达载体，其包含本公开第一方面所提供的多核苷酸分子或本公开第二方面所提供的表达盒。
在一些实施方式中，所述表达载体还包含编码标志物的基因，优选地，所述标志物选自抗生素抗性蛋白质、毒素抗性蛋白质、有色的或荧光的或发光的蛋白质和介导增强的细胞生长和/或基因扩增的蛋白质中的至少一种。
在一些实施方式中，所述抗生素选自氨苄青霉素、新霉素、G418、嘌呤霉素和杀稻瘟素的至少一种。
在一些实施方式中，所述毒素选自炭疽毒素和白喉毒素的至少一种。
在一些实施方式中，所述有色的或荧光的或发光的蛋白质选自绿色荧光蛋白、增强型绿色荧光蛋白、红色荧光蛋白和萤光素酶的至少一种。
在一些实施方式中，所述介导增强的细胞生长和/或基因扩增的蛋白质为二氢叶酸还原酶(DHFR)。
在一些实施方式中，所述表达载体包含复制起点；优选地，所述复制起点序列选自f1噬菌体ori、RK2oriV、pUC ori以及pSC101ori的至少一种。
在一些实施方式中，所述表达载体选自质粒、粘粒、病毒载体、RNA载体或线性或圆形DNA或RNA分子。
在一些实施方式中，所述质粒选自pCI、puc57、pcDNA3、pSG5、pJ603或pCMV。
在一些实施方式中，所述病毒载体选自逆转录病毒、腺病毒、细小病毒(例如，腺伴随病毒)、冠状病毒、负链RNA病毒诸如正粘病毒(例如，流感病毒)、弹状病毒(例如，狂犬病和水疱性口炎病毒)、副粘病毒(例如，麻疼和仙台)、正链RNA病毒(诸如小RNA病毒和甲病毒)，或双链DNA病毒，所述双链DNA病毒选自腺病毒、疱疹病毒(例如，单纯疱疹病毒1和2型、愛泼斯坦-巴尔病毒、巨细胞病毒)、痘病毒(例如，牛痘病毒、鸡痘病毒和金丝雀痘病毒)、诺沃克病毒、披膜病毒、黄病毒、呼肠孤病毒、乳多泡病毒、嗜肝DNA病毒、杆状病毒或肝炎病毒。
在一些实施方式中，所述逆转录病毒选自禽造白细胞组织增生-肉瘤、哺乳动物C-型、B-型病毒、D-型病毒、HTLV-BLV集合、慢病毒或泡沫病毒。
在一些实施方式中，所述慢病毒载体选自HIV-1、HIV-2、SIV、FIV、BIV、EIAV、CAEV或绵羊脱髓鞘性脑白质炎慢病毒。
在一些实施方式中，所述表达载体为腺相关病毒(AAV)载体。
在一些实施方式中，所述腺相关病毒选自AAV 1型、AAV 2型、AAV 3型、AAV 4型、AAV 5型、AAV 6型、AAV 7型、AAV 8型、AAV 9型、AAV 10型、禽AAV、牛AAV、犬AAV、马AAV或绵羊AAV。
在一些实施方式中，所述表达盒可以包装在rAAV中具有来自任何AAV血清型或其杂交体或变体的衣壳的载体。
在一些实施方式中，所述表达载体包含与SEQ ID NO.53、SEQ ID NO.54、SEQ ID NO.55、SEQ ID NO.56、SEQ ID NO.57、SEQ ID NO.58、SEQ ID NO.59、SEQ ID NO.60、SEQ ID NO.62、SEQ ID NO.63、SEQ ID NO.64、SEQ ID NO.65、SEQ ID NO.66、SEQ ID NO.72、SEQ ID NO.73、SEQ ID NO.74、SEQ ID NO.75、SEQ ID NO.76、SEQ ID NO.77、SEQ ID NO.78、SEQ ID NO.96、SEQ ID NO.97、SEQ ID NO.98、SEQ ID NO.99、SEQ ID NO.100、SEQ ID NO.101、SEQ ID NO.102、SEQ ID NO.103、SEQ ID NO.105、SEQ ID NO.106、SEQ ID NO.107、SEQ ID NO.108或SEQ ID NO.109所示核苷酸序列具有90％或以上同一性的核苷酸序列，优选具有91％、92％、93％、94％、 95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列；更优选地，所述表达载体具有如SEQ ID NO.53、SEQ ID NO.54、SEQ ID NO.55、SEQ ID NO.56、SEQ ID NO.57、SEQ ID NO.58、SEQ ID NO.59、SEQ ID NO.60、SEQ ID NO.62、SEQ ID NO.63、SEQ ID NO.64、SEQ ID NO.65、SEQ ID NO.66、SEQ ID NO.72、SEQ ID NO.73、SEQ ID NO.74、SEQ ID NO.75、SEQ ID NO.76、SEQ ID NO.77、SEQ ID NO.78、SEQ ID NO.96、SEQ ID NO.97、SEQ ID NO.98、SEQ ID NO.99、SEQ ID NO.100、SEQ ID NO.101、SEQ ID NO.102、SEQ ID NO.103、SEQ ID NO.105、SEQ ID NO.106、SEQ ID NO.107、SEQ ID NO.108或SEQ ID NO.109所示的核苷酸序列；更优选地，表达载体具有如SEQ ID NO.76、SEQ ID NO.73、SEQ ID NO.60或SEQ ID NO.78所示的核苷酸序列。
在一些实施方式中，表达载体具有如SEQ ID NO.73所示的核苷酸序列。
本公开第四方面提供了一种病毒颗粒，其包含本公开第一方面所提供的多核苷酸分子、本公开第二方面所提供的表达盒和本公开第三方面所提供的表达载体的至少一种。
本公开第五方面提供了一种治疗苯丙酮尿症的药物组合物，其包含本公开第一方面所提供的多核苷酸分子、本公开第二方面所提供的表达盒、本公开第三方面所提供的表达载体和本公开第四方面所提的病毒颗粒的至少一种。
本公开第六方面提供了本公开第一方面所提供的多核苷酸分子、本公开第二方面所提供的表达盒、本公开第三方面所提供的表达载体、本公开第四方面所提的病毒颗粒或本公开第五方面的药物组合物在制备治疗苯丙酮尿症的药物中的用途。
本公开第七方面提供了本公开第一方面所提供的多核苷酸分子、本公开第二方面所提供的表达盒、本公开第三方面所提供的表达载体、本公开第四方面所提的病毒颗粒或本公开第五方面的药物组合物在治疗苯丙酮尿症的中的用途。
本公开第八方面提供了一种治疗苯丙酮尿症的方法，其包括向有受试者施用有效量的本公开的多核苷酸分子、表达盒、表达载体、病毒颗粒和药物组合物的至少一种。
为了达到清楚和简洁描述的目的，本文中作为相同的或分开的一些实施方式的一部分来描述特征，然而，将要理解的是，本公开的范围可包括具有所描述的所有或一些特征的组合的一些实施方式。
下面，参考具体实施例更详细地描述本公开，然而，实施例仅用于说明目的，对于本公开不具有限制作用。
实验材料和方法：
PAH表达盒和AAV载体
在本研究中使用的所有PAH基因野生型和密码子优化的PAH序列由金斯瑞合成。密码子优化使用GenSmart codonoptimization tool加以改进进行。AAV载体介导的PAH表达盒如图1所示。
按SEQ ID NO.112序列由通用基因公司全基因合成初始AAV穿梭质粒载体，将野生型AAV2ITR序列重组至载体BamHI和AleI酶切位点间，以修复载体中突变的ITR序列；将初始穿梭质粒载体ApaLI酶切位点间的Amp抗性基因替换为Kan；接着将重组至载体的HindIII和NheI酶切位点之间，增加载体的长度以利于AAV病毒的包装；从pCAGGS载体(金唯智)中PCR扩增CAG启动子序列，重组至穿梭质粒载体的SpeI和KpnI酶切位点之间，得到最终的穿梭质粒载体(SEQ NO ID.113)。其中，CAG启动子包含CMV增强子(SEQ ID NO.40)、鸡β-肌动蛋白启动子(SEQ ID NO.41)和嵌合内含子(chimeric intron)(SEQ ID NO.42)。
所有的表达盒序列克隆至穿梭质粒载体的SalI和BamHI酶切位点之间，获得含AAV载体介导的表达PAH目的基因的穿梭质粒。
AAV载体生产及纯化方法
AAV载体的生产采用三质粒系统，即用含PAH目的基因的穿梭质粒、带AAV载体repcap基因的pRepCap质粒和辅助质粒Phelper(pRepCap质粒，按SEQ ID NO.110序列由金唯智合成和辅助质粒pHelper，按SEQ NO ID.111序列由金唯智合成)，以PEI为转染试剂，共同转染HEK293细胞，重组包装出AAV病毒载体。转染后48-72小时进行收获，收获液经纯化后获得一定纯度的重组AAV病毒载体。纯化方法如下：
首先对收获液进行预处理：充分裂解HEK293细胞，释放出细胞内的AAV病毒载体，同时加入核酸酶对游离的核酸进行消化，消化结束后，采用深层过滤除去大分子杂质和细胞碎片，深层过滤后滤液用二次过滤得到澄清液用于亲和上样。
亲和层析利用配基与蛋白的特异性吸附捕获收获液中的AAV病毒载体，并除去大部分工艺相关杂质，达到浓缩与除杂的效果。将收集的洗脱液混匀，并用中和缓冲液中和，保存在无菌储液瓶中作为阴离子层析上样液。
阴离子层析利用不同组分的等电点差异分离实心与空壳AAV病毒，同时继续去除残留杂质，洗脱液收集在新的无菌储液瓶中，再经超滤浓缩的方法置换缓冲液为制剂稳定的缓冲液，同时病毒滴度浓缩至约1×1013vg/mL，最后除菌过滤分装后备用。
AAV载体的滴度定量
在AAV病毒纯化完成之后，需要对病毒的含量进行测定，基因组滴度是表征AAV物理滴度最经典的检项。通过针对rAAV的基因组序列设计引物探针，然后进行Q-PCR检测是基因组滴度检测最通用的方法。
考虑到本公开中针对ORF编码框进行了密码子优化，涉及到多个载体结构的筛选，为了保证不同载体结构之间定量的稳定性和准确性，选择载体中的共有序列PolyA设计引物探针，F引物序列：5’-CAAGCCCATGTACACACCAG-3’(SEQ ID NO.114)，R引物序列：5’-GGGCAAAGCTTCTGTCTGAG-3’(SEQ ID NO.115)，探针序列：5’-CTGACATCTGCCACGAGCTGCTGGGCCA-3’(SEQ ID NO.116)。
在基因组滴度检测过程中，首先要进行标准曲线的建立，阳性标准质粒使用样品稀释液稀释至2×107、2×106、2×105、2×104、2×103、2×102拷贝/μl，作为标准曲线模板，标准曲线需要控制其线性和扩增效率，一般要求R2>0.99，扩增效率在90％-110％之间。之后采用预处理完成的rAAV样品稀释后进行QPCR检测，确保样品检测Ct值在标准曲线范围内，根据样品的Ct值代入到标准曲线中进行rAAV样品基因组滴度的计算，对产品的含量进行标识。
体外细胞质粒转染实验
消化处理人肝癌细胞HepG2细胞，按2.5×104细胞/孔接种细胞至96孔板中。同时加入Lipo3K/DNA转染复合物进行质粒转染实验。首先将Lipofectamine 3000和P3000(Thermo，L3000015)转染试剂与质粒预混合，按照60ng/孔PAH-opt/WT表达质粒、90nL/孔P3000和90nL/孔Lipo 3000(Thermo，L3000015)，加至96孔板HepG2细胞中，置于CO2恒温培养箱中培养48h。
Western印迹分析
在接种培养细胞的96孔板的每个孔内加入60μL含1×SDS上样缓冲液的RIPA裂解液(Beyotime，P0013B)，振荡裂解10min，之后95℃变性10min。SDS-PAGE上样10μL蛋白电泳，之后转移到PVDF膜上，用Anti-PAH(SantaCruz，sc-271258)和Anti-GAPDH(TransGen，HC301)抗体孵育，用ChemiDoc Touch Imaging System(Bio-Rad)进行成像分析。
小鼠尾静脉高压注射质粒
将小鼠放入合适的容器中，放置红外灯下，打开开关，照射若干分钟，取出小鼠，用酒精棉球擦拭尾部，使尾部静脉充分扩张。将小鼠放入小鼠固定器中，露出尾部。使用合适的注射器及针头抽取待注射样本，注射使用1mL注射器的针头，匹配5mL注射器的针筒。抽取2mL(0.1ml/g×小鼠体重)的待注射样本，通过尾静脉进行注射，在5-8秒内推注完毕，注射需快速且速度均一。注射完毕后，用干棉球按压注射部位进行止血。
小鼠尾静脉注射病毒样品
将小鼠放入合适的容器中，放置红外灯下，打开开关，照射若干分钟，取出小鼠，用酒精棉球擦拭尾部，使尾部静脉充分扩张。将小鼠放入小鼠固定器中，露出尾部。使用合适的注射器及针头抽取待注射样本，注射使用1mL注射器的针头，匹配1mL注射器的针筒。按照病毒给药剂量将病毒样品稀释后，抽取200μL待注射样本的稀释液，通过尾静脉进行注射，在大于10秒时间推注完毕，注射需缓慢且速度均一。注射完毕后，用干棉球按压注射部位进行止血。
血液中Phe定量分析
小鼠眶周采血，取20μL滴加到采血卡上自然风干。使用苯丙氨酸测定试剂盒(FENGHUA，AN302)定量血液中苯丙氨酸浓度，用标准血卡进行校准。
体外PAH降Phe功能测试
待测细胞培养到24孔板中，测试时，收集待测细胞，用PBS漂洗一遍，每孔加入150μL反应液(其中含0.25％NP-40，50mM Hepes，150mM KCL，800mM L-Phe，100μg/mL Catalase，400μM FeNH4(SO4)2，400μM BH4，2mM DTT)37℃孵育3小时。取15μL反应液滴加到采血卡上自然风干。使用苯丙氨酸测定试剂盒(FENGHUA，AN302)定量血液中苯丙氨酸浓度，计算Phe浓度减少量。
5-HIAA检测
小鼠处死后取脑组织，委托苏州帕诺米克生物医药科技有限公司用质谱检测5-HIAA含量，具体方法如下：用5-羟吲哚乙酸标准品梯度稀释配制标准曲线溶液，将待测样品脑组织匀浆，按照1:1:10＝匀浆液:内标工作液:甲醇的体积比混匀，以12000rpm、4℃的条件离心10分钟，取上清待测；用XDB-C18analytical 4.6*150mm 5-Micron色谱柱和电喷雾电离源，采用多重反应监测(MRM)进行扫描检测；根据标准曲线定量法计算待测样品中5-HIAA的含量。
血液中Tyr定量分析
小鼠眶周采血，血清用3kDa超滤管离心，12000xg 20min，收集滤过液。用Tyr标准品梯度稀释配制标准曲线溶液，将血清样本用3kDa超滤管离心，12000xg 20min，收集滤过液待测；用Chromcore 120 C18色谱柱，检测波长210nm信号；根据标准曲线定量法计算待测样品中Tyr的含量。
实施例1：腺相关病毒载体的构建和分离纯化
1.1腺相关病毒重组载体的构建
PAH表达盒的结构如图1所示。所述PAH表达盒从5’端至3’端依次包含5’ITR、ApoE HCR增强子、SerpinA1启动子、截短的SerpinA1内含子、Kozak序列、目的基因：野生型人源PAH基因hPAH WT或优化的PAH基因hPAH opt、BGH polyA、HPRT(4CpG)填充序列和3’ITR。含有不同目的基因的PAH表达盒的核苷酸序列如SEQ ID NO.79-93所示。其中：
5’ITR的核苷酸序列如SEQ ID NO.1所示。
ApoE HCR增强子为人源载脂蛋白E的肝细胞控制区，其核苷酸序列如SEQ ID NO.2所示。
SerpinA1启动子为人α1抗胰蛋白酶启动子，其核苷酸序列如SEQ ID NO.3所示。
SerpinA1内含子为截短的α1抗胰蛋白酶内含子，长度为261bp，其核苷酸序列如SEQ ID NO.4所示。
Kozak序列被插入到PAH基因序列之前，其序列如SEQ ID NO.5所示。
hPAH WT基因源自野生型人源PAH基因(GeneID：5053)，其基因序列如SEQ ID NO.6所示，其编码的野 生型hPAH WT蛋白的NCBI登记号为NP_000268.1。
优化的PAH基因hPAH opt为编码野生型人源PAH蛋白的密码子优化的基因opt1～15，其核苷酸序列分别为SEQ ID NO.9～23。
BGH polyA为牛生长激素聚腺苷酸化信号，其核苷酸序列如SEQ ID NO.7所示。
HPRT(4CpG)填充序列为次黄嘌呤磷酸核糖基转移酶的部分内含子序列，其核苷酸序列如SEQ ID NO.43所示。
3’ITR的核苷酸序列如SEQ ID NO.8所示。
包含野生型人源PAH基因hPAH WT的表达盒构建为穿梭质粒pAAV8-ATT-PAH-WT-HPRT。其中，SEQ ID NO.51示出了包含具有野生型人源PAH基因hPAH WT的表达盒的穿梭质粒序列。
包含优化的PAH基因opt 1～15的表达盒构建为穿梭质粒pAAV8-ATT-PAH-opt1～15-HPRT。其中，SEQ ID NO.52～66分别示出了包含具有密码子优化基因opt1～15的表达盒的穿梭质粒序列。
1.2腺病毒载体的分离和纯化
AAV载体的生产采用三质粒系统，即用含PAH目的基因的穿梭质粒pAAV8-ATT-PAH-WT-HPRT或pAAV8-ATT-PAH-opt1～15-HPRT、带AAV载体repcap基因的pRepCap质粒和辅助质粒pHelper，以PEI为转染试剂，共同转染HEK293细胞，重组包装出AAV病毒载体，分别命名为AAV8-ATT-PAH-WT-HPRT、AAV8-ATT-PAH-opt1～15-HPRT。转染后48-72小时进行收获，收获液经亲和层析纯化后，通过阴离子层析进一步纯化，超滤浓缩，置换缓冲液。纯化后的重组AAV病毒载体测定基因组滴度，除菌过滤分装后备用。
实施例2：密码子优化的PAH opt的体外表达检测
本实施例在HepG2细胞系(购自中国科学院典型培养物保藏委员会细胞库，货号：TCHu72)中评估了密码子优化的PAH opt的体外表达水平。构建了包含ATT启动子驱动的野生型人源PAH基因hPAH WT的穿梭质粒pAAV8-ATT-PAH WT，其核苷酸序列如SEQ ID NO.94；以及包含优化的PAH基因hPAH opt1-15的穿梭质粒pAAV8-ATT-PAH opt 1～15，其核苷酸序列如SEQ ID NO.95～109，采用前述“体外细胞质粒转染实验”的方法，将目的基因导入细胞。
在HepG2中，瞬时转染了表达PAH WT的质粒pAAV8-ATT-PAH WT或表达PAH opt的质粒pAAV8-ATT-PAH opt1～15，并通过Western印迹分析评估了细胞裂解物中的蛋白质表达，结果如图2所示。其中，A图示出了PAH蛋白质Western印迹的代表性图像；B图示出了定量结果，密码子优化后PAH的基因opt2～9以及opt11-15在HepG2细胞中的表达水平都明显高于野生型人源PAH基因(WT)，说明本公开的密码子优化后的PAH基因具有更高的表达水平。
实施例3：密码子优化的PAH opt在PKU小鼠模型中的体内药效评价
3.1密码子优化序列的质粒降低PKU小鼠Phe功能活性比较
采用尾静脉高压注射方式，向PKU模型小鼠(购自北京澄天生物科技有限公司，品系为BTBR-Pah<enu2>/J)注射40μg的pAAV8-ATT-PAH WT或pAAV8-ATT-PAH opt2、opt9、opt14质粒。在注射后0h、6h、24h、3天、5天、10天、16天、19天的时间点，采集小鼠血液，测定血液中Phe的含量，比较分析PAH opt质粒降低Phe的效果，结果如图3A和图3B所示。由图3A可见，在D3～D16期间，注射PAH opt9质粒的小鼠血液Phe浓度低于PAH WT，而注射PAH opt2和PAH opt14质粒的小鼠Phe都高于PAH WT。由于质粒在细胞内会被代谢降解，因此随着时间的增加降低Phe效果会逐渐减弱。由图3B可见，在注射后第10天，注射PAH opt9质粒的小鼠血液Phe浓度低于PAH WT、PAH opt2和PAH opt14。因此表明，密码子优化的PAH opt9具有更好的降低Phe效果。
3.2密码子优化序列的AAV病毒降低PKU小鼠Phe功能活性比较
采用实施例2的穿梭质粒，通过与实施例1的1.2相同的方法，重组包装出AAV病毒载体，分别命名为AAV8-ATT-PAH WT、以及AAV8-ATT-PAH opt2、9、11、14。
采用静脉注射方式，以1e10vg/只小鼠的剂量向PKU模型小鼠注射AAV8-ATT-PAH WT或AAV8-ATT-PAH opt2、opt9、opt11、opt14病毒。注射后每周采集小鼠血液，测定血液中Phe的含量，直至4周，比较分析AAV8-ATT-PAH opt病毒降低Phe的效果，结果如图4所示。由图4可见，注射了AAV8-ATT-PAH opt2、9、14病毒的模型小鼠血液Phe浓度都低于AAV8-ATT-PAH WT，因此表明，携带密码子优化的AAV8-ATT-PAH opt2、9、14病毒具有更好的降低Phe效果。
实施例4：肝脏特异性启动子组合在PKU小鼠模型中的筛选
本实施例在PKU模型小鼠中，评估不同启动子驱动PAH的降低Phe的效果，启动子的组合元件如表1所示。ApoE HCR增强子为人源载脂蛋白E的肝细胞控制区，其核苷酸序列如SEQ ID NO.2所示；Core ApoE HCR增强子为人源载脂蛋白E的肝细胞控制区，其核苷酸序列如SEQ ID NO.37所示；CRMSBS2增强子为修饰的Serpinl增强子，其核苷酸序列如SEQ ID NO.29所示；TTRm增强子为突变的转甲状腺素增强子区，其核苷酸序列如SEQ ID NO.32所示；SerpinA1启动子为人α1抗胰蛋白酶启动子，其核苷酸序列如SEQ ID NO.3所示；Core SerpinA1启动子(218bp)为人α1抗胰蛋白酶启动子核心区，其核苷酸序列由SEQ ID NO.24、25组成；Core SerpinA1启动子(254bp)为人α1抗胰蛋白酶启动子核心区，其核苷酸序列如SEQ ID NO.38所示；TTRm启动子(223bp)为突变的转甲状腺素启动子，其核苷酸序列如SEQ ID NO.30所示；TTRm启动子(228bp)为突变的转甲状腺素启动子， 其核苷酸序列如SEQ ID NO.33所示；truncated SerpinA1内含子(261bp)为截短的α1抗胰蛋白酶内含子，其核苷酸序列如SEQ ID NO.4所示；Truncated SerpinA1内含子(206bp)为截短的α1抗胰蛋白酶内含子，其核苷酸序列如SEQ ID NO.26所示；Modified humanβ-globin 2nd内含子为修饰的人β-珠蛋白2号内含子部分序列，其核苷酸序列如SEQ ID NO.27所示；Modified SV40内含子为猴空泡病毒40内含子部分序列，其核苷酸序列如SEQ ID NO.28所示；SBR内含子3为修饰的小鼠微小病毒内含子，其核苷酸序列如SEQ ID NO.31所示；MVM内含子为小鼠微小病毒内含子部分序列，其核苷酸序列如SEQ ID NO.34所示。
采用所述组合启动子构建表达盒，表达盒结构如图5所示，将表达盒构建为穿梭质粒，获得质粒载体ATT-PAH-WT(SEQ ID NO.94)、100-AT-PAH-WT(SEQ ID NO.67)、ATG-PAH-WT(SEQ ID NO.68)、ATS-PAH-WT(SEQ ID NO.69)、CTS-PAH-WT(SEQ ID NO.70)和TTM-PAH-WT(SEQ ID NO.71)。
采用尾静脉高压注射方式，向PKU模型小鼠注射40μg的本实施例的上述质粒载体，在注射后3天，采集小鼠血液，测定血液中Phe的含量，比较分析不同启动子驱动PAH的降低Phe的效果，结果如图6所示，结果显示ATT启动子驱动的表达质粒降低PKU模型小鼠Phe的效果最明显。
表1.不同启动子的构成元件
实施例5：其他表达调控元件对PKU小鼠模型的体内药效的影响
本实施例在PKU模型小鼠中，评估不同表达调控元件(U6启动子(SEQ ID NO.35)、CAG启动子(SEQ ID NO.50)、填充序列HPRT(47CpG)(SEQ ID NO.39)和填充序列WPRE(SEQ ID NO.36))对AAV8-ATT-PAH病毒降低Phe效果的影响，不同表达盒的构建如图7所示。
将图7所示的表达盒分别构建为穿梭质粒，获得质粒载体ATT-PAH-opt9-HPRT(47CpG)(SEQ ID NO.73)、U6-ATT-PAH-opt9-HPRT(47CpG)(SEQ ID NO.75)、ATT-PAH-opt9-WPRE(SEQ ID NO.74)、U6-ATT-PAH-opt9-WPRE(SEQ ID NO.72)、CAG-PAH-opt9(SEQ ID NO.77)、ATT-PAH-opt9(SEQ ID NO.76)，采用尾静脉高压注射方式，向PKU模型小鼠注射40μg的上述质粒。在注射后第3天采集小鼠血液，测定血液中Phe的含量，比较分析不同构建的PAH表达盒降低Phe的效果，如图8所示，结果显示，添加填充序列HPRT的载体降低Phe效果(ATT-PAH-opt9-HPRT(47CpG))要好于不添加的(ATT-PAH-opt9)。
实施例6：填充序列的优化对PKU小鼠模型的体内药效的影响
有研究表面先天免疫刺激可能由CpG富集驱动，从而导致AAV载体介导的基因表达在体内失效(Faust,Susan M et al.CpG-depleted adeno-associated virus vectors evade immune detection.The Journal of clinical investigation vol.123,7(2013):2994-3001.；Konkle,Barbara A et al.BAX 335 hemophilia B gene therapy clinical trial results:potential impact of CpG sequences on gene expression.Blood vol.137,6(2021):763-774.)。为了研究CpG数目对AAV8-ATT-PAH病毒降低Phe效果的影响，本实施例在PKU模型小鼠中，采用具有较多CpG数目的填充序列(HPRT(47CpG))以及具有较少CpG数目的填充序列HPRT(4CpG))进行了实验。含有不同填充序列的优化重组AAV(rAAV)载体PAH表达盒的结构示意如图9所示。其中，HPRT(47CpG)核苷酸序列如SEQ ID NO.39所示，HPRT(4CpG)核苷酸序列如SEQ ID NO.43所示。
将包含不同填充序列的表达盒分别构建为穿梭质粒，采用实施例1的三质粒系统获得重组病毒载体 AAV8-ATT-PAH-opt9(其中，穿梭质粒序列如SEQ ID NO.76所示)、AAV8-ATT-PAH-opt9-HPRT(47CpG)(其中，穿梭质粒序列如SEQ ID NO.73所示)、AAV8-ATT-PAH-opt9-HPRT(4CpG)(其中，穿梭质粒序列如SEQ ID NO.60所示)、AAV8-HPRT(4CpG)-ATT-PAH-opt9(其中，穿梭质粒序列如SEQ ID NO.78所示)。
采用静脉注射方式，以2e11vg/只小鼠的剂量向PKU模型小鼠注射病毒载体。注射后每周采集小鼠血液，测定血液中Phe的含量，直至8周，比较分析不同填充序列的优化对降低Phe效果的影响。由图10A可见，向PKU模型小鼠分别注射上述4种表达盒的病毒后，小鼠血液Phe含量均达到了最佳治疗效果(<120μM)。
为了避免CpG富集引起的免疫刺激，选择了CpG数目较少的填充序列的表达盒在1e10vg/只小鼠的较低剂量下进一步研究AAV8-ATT-PAH-opt9-HPRT(4CpG)对PKU模型小鼠的治疗效果，注射后每周采集小鼠血液，测定血液中Phe的含量，直至4周，结果如图10B所示，由图10B可见，低剂量下，AAV8-ATT-PAH-opt9-HPRT(4CpG)的降低Phe的效果优于AAV8-ATT-PAH-opt9。
实施例7：优化的表达盒AAV8-ATT-PAH-opt9-HPRT(4CpG)病毒表达产物的体外PAH降Phe功能测试
本实施例在HepG2细胞系中评估了密码子优化的AAV8-ATT-PAH-opt9-HPRT(4CpG)病毒表达产物降Phe的功能。首先在HepG2中，瞬时转染了表达腺相关病毒受体AAVR的质粒(AAVR基因按SEQ ID NO.117序列由金唯智合成，然后插入到pcDNA3.1(+)质粒中NheI和XbaI酶切位点之间)，AAVR的过表达会提高AAV8侵染HepG2的效率。24h后，用实施例1的AAV8-ATT-PAH-opt9-HPRT(4CpG)病毒分别以0、5e4、1e5、2e5的MOI侵染细胞，培养48小时后，进行Phe含量检测，以未感染病毒的HepG2细胞系为参照，通过计算Phe浓度的变化量，来评估PAH降Phe的活性，结果如图11所示，表明AAV8-ATT-PAH-opt9-HPRT(4CpG)病毒表达产物具有降Phe的功能，病毒的侵染MOI具有剂量效应。
实施例8：优化的表达盒在PKU小鼠模型的体内药效：低剂量，高活性
本实施例分别在PKU模型雄性和雌雄小鼠中，评估不同剂量AAV8-ATT-PAH-opt9-HPRT(4CpG)病毒对降低Phe效果的影响。
采用静脉注射方式，分别以3.0e11、1.0e11、3.3e10、1.5e10、1.1e10、3.0e9vg/只小鼠的剂量向PKU模型雄性小鼠注射实施例1的AAV8-ATT-PAH-opt9-HPRT(4CpG)病毒。注射后每周采集小鼠血液，测定血液中Phe的含量，直至6周，比较分析不同病毒施用剂量对降低Phe效果的影响，结果如图12A所示。由图12A可见，病毒注射后2～6周的时间内，在3.0e11、1.0e11、3.3e10vg/只小鼠的剂量下小鼠血液Phe浓度低于120μM，达到了治疗效果。因此认为AAV8-ATT-PAH-opt9-HPRT(4CpG)病毒在雄性小鼠中治疗PKU的最低有效剂量约为3.3e10vg/只小鼠。
采用静脉注射方式，分别以4.0e11、2.0e11、1.0e11、5.0e10、2.5e10vg/只小鼠的剂量向PKU模型雌性小鼠注射实施例1的AAV8-ATT-PAH-opt9-HPRT(4CpG)病毒。注射后每周采集小鼠血液，测定血液中Phe的含量，直至6周，比较分析不同病毒施用剂量对降低Phe效果的影响，结果如图12B所示。由图12B可见，病毒注射后2～6周的时间内，在4.0e11、2.0e11、1.0e11、5.0e10vg/只小鼠的剂量下小鼠血液Phe浓度低于120μM，达到了治疗效果。因此认为AAV8-ATT-PAH-opt9-HPRT(4CpG)病毒在雌性小鼠中治疗PKU的最低有效剂量约为5.0e10vg/只小鼠。
实施例9：优化的表达盒在PKU小鼠模型的其他药效
本实施例在PKU模型雄性小鼠中，评估了3.0e10和3.0e11vg/只小鼠剂量下AAV8-ATT-PAH-opt9-HPRT(4CpG)病毒对降低PKU模型小鼠的其他治疗效果。在接受病毒给药6周后，检测了血液中Tyr的含量，结果如图13A所示，结果显示相较于无疾病表型的PKU杂合子小鼠(Jackson Lab)，有疾病表型的PKU纯合子小鼠(即PKU模型小鼠)血液中的Tyr含量显著降低，在接受3.0e10和3.0e11vg/只小鼠剂量的AAV8-ATT-PAH-opt9-HPRT(4CpG)病毒后，PKU小鼠血液中Tyr都恢复到与正常杂合子小鼠相当的水平。图13B显示了PKU纯合子小鼠脑组织中的5-羟基吲哚乙酸(5-HIAA，一种PKU患者的药效学标志物)的含量，同时将未给药的杂合子小鼠作为正常小鼠对照。结果显示相较于无疾病表型的PKU杂合子小鼠，有疾病表型的PKU纯合子小鼠脑组织中的5-HIAA含量显著降低，在接受3.0e10和3.0e11vg/只小鼠剂量的AAV8-ATT-PAH-opt9-HPRT(4CpG)病毒后，PKU小鼠脑组织中5-HIAA都恢复到与正常杂合子小鼠相当的水平。并且经过治疗的PKU小鼠，毛色均发黑变亮，图13C展示了PKU纯合子小鼠，在3.0e10vg/只小鼠剂量下给药3周后与溶媒组的毛色对比。
本公开通过对基因和表达调控元件的优化和筛选，实现了用低剂量在PKU小鼠中有效持久且稳定地抑制外周血苯丙氨酸的浓度，改善PKU其他表型。此外，本公开能有效地降低用于治疗PKU的基因治疗药物的剂量和可能的副作用，提高治疗效果。
以上所述仅为本公开的较佳实施例，并不用以限制本公开，凡在本公开的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本公开保护的范围之内。

Claims (10)

  1. 一种编码PAH蛋白的多核苷酸分子，其包含与SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22或SEQ ID NO.23所示核苷酸序列具有90％或以上同一性的核苷酸序列；优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸列；更优选具有98％或99％以上同一性的核苷酸序列；
    更优选地，所述多核苷酸分子具有SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22或SEQ ID NO.23所示的核苷酸序列。
  2. 一种表达盒，其包含权利要求1所述的多核苷酸分子，以及与所述多核苷酸分子可操作连接的启动子；
    优选地，所述启动子为特异性或非特异性启动子；
    优选地，所述启动子是组成型启动子或可诱导启动子；优选地，所述组成型启动子选自CMV启动子、EF1A启动子、EFS启动子、CAG启动子、CBh启动子、SFFV启动子、MSCV启动子、SV40启动子、mPGK启动子、hPGK启动子和UBC启动子；优选地，所述可诱导启动子包含四环素调控启动子、醇调控启动子、类固醇调控启动子、金属调控启动子、致病调控启动子、温度/热诱导型启动子和光调控启动子、IPTG诱导型启动子；
    优选地，所述启动子包含核心启动子；
    优选地，所述核心启动子包含肝脏特异性启动子或其活性片段；优选地，所述肝脏特异性启动子选自ApoA-I启动子、ApoA-II启动子、ApoA-IV启动子、ApoB启动子、ApoC-1启动子、ApoC-II启动子、ApoC-III启动子、ApoE启动子、白蛋白启动子、甲胎蛋白启动子、磷酸烯醇丙酮酸羧化激酶(PCK1)启动子、磷酸烯醇丙酮酸羧化激酶2(PCK2)启动子、甲状腺素转运蛋白(transthyretin，TTR)启动子、α-抗胰蛋白酶(AAT或SerpinA1)启动子、TK(胸苷激酶)启动子、血色素结合蛋白启动子、醇脱氢酶6启动子、胆固醇7α-25羟化酶启动子、IX因子启动子、a-微球蛋白启动子、SV40启动子、CMV启动子、劳斯肉瘤病毒-LTR启动子、HBV启动子、ALB启动子和TBG启动子；更优选地，所述肝脏特异性启动子为人α1抗胰蛋白酶启动子，优选地，所述核心启动子包含与SEQ ID NO.3、24、25、30、33或38所示核苷酸序列具有90％或以上同一性的核苷酸序列，优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列；优选地，所述核心启动子具有如.SEQ ID NO.3、24、25、30、33或38所示的核苷酸序列；更优选地，所述核心启动子的核苷酸序列如SEQ ID NO.3所示。
  3. 根据权利要求2所述的表达盒，其还包含表达控制元件，所述表达控制元件与所述多核苷酸分子可操作地连接；
    优选地，所述表达控制元件选自转录/翻译控制信号、增强子、内含子、polyA信号、ITR、绝缘子、RNA加工信号、增强mRNA和蛋白质的稳定性的元件中的至少一种；
    优选地，所述表达盒包含5’ITR；优选地，所述5’ITR包含与SEQ ID NO.1所示核苷酸序列具有90％或以上同一性的核苷酸序列，优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列；更优选地，所述5’ITR具有如SEQ ID NO.1所示的核苷酸序列；
    优选地，所述表达盒包含3’ITR；优选地，所述3’ITR包含与SEQ ID NO.8所示核苷酸序列具有90％或以上同一性的核苷酸序列，优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列；更优选地，所述3’ITR具有如SEQ ID NO.8所示的核苷酸序列；
    优选地，所述表达盒还包含增强子；优选地，所述增强子选自ApoE HCR增强子或其活性片段、CRMSBS2增强子或其活性片段、TTRm增强子或其活性片段以及CMV增强子或其活性片段；更优选地，所述增强子包含与SEQ ID NO.2、29、32、37或40所示核苷酸序列具有90％或以上同一性的核苷酸序列，优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列；优选地，所述增强子具有如SEQ ID NO.2、29、32、37或40所示的核苷酸序列；更优选地，所述增强子的核苷酸序列如SEQ ID NO.2所示；
    优选地，所述表达盒还包含内含子；优选地，所述内含子选自α1抗胰蛋白酶内含子或其活性片段、β-珠蛋白2号内含子或其活性片段、SV40内含子或其活性片段、以及小鼠微小病毒内含子或其活性片段；优选地，所述内含子包含与SEQ ID NO.4、26、27、28、31或34所示核苷酸序列具有90％或以上同一性的核苷酸序列，优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列；优选地，所述内含子具有如SEQ ID NO.4、26、27、28、31或34所示的核苷酸序列；更优选地，所述内含子的核苷酸序列如SEQ ID NO.4所示；
    优选地，所述表达盒的启动子为包含上游调控元件、核心启动子和内含子的组合启动子；优选地，所述上游调控元件为增强子或其活性片段；
    优选地；所述组合启动子包含与SEQ ID NO.44、45、46、47、48或49所示核苷酸序列具有90％或以上同一 性的核苷酸序列，优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列；优选地，所述组合启动子具有如SEQ ID NO.44、45、46、47、48或49所示的核苷酸序列；更优选地，所述组合启动子的核苷酸序列如SEQ ID NO.44所示；
    优选地，所述表达盒还包含polyA信号；优选地，所述polyA信号为牛生长激素poly A(BGH poly A)、短poly A、SV40 polyA、和人β珠蛋白poly A中的至少一种；优选地，所述polyA信号包含与SEQ ID NO.7所示核苷酸序列具有90％或以上同一性的核苷酸序列，优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列；更优选地，所述polyA信号的核苷酸序列如SEQ ID NO.7所示；
    优选地，所述表达盒包含优化的填充序列；优选地，填充序列选自次黄嘌呤磷酸核糖基转移酶(HPRT)的部分内含子序列和土拨鼠肝炎病毒转录后调控元件(WPRE)；优选地，所述部分内含子序列中含有的CpG序列个数不超过100、80、60、50、40、30、20、15、10、9、8、7、6、5、4、3、2或1；优选地，所述部分内含子序列或土拨鼠肝炎病毒转录后调控元件(WPRE)中不含CpG序列；优选地，所述填充序列为次黄嘌呤磷酸核糖基转移酶(HPRT)的部分内含子序列；优选地，所述填充序列包含与SEQ ID NO.39或SEQ ID NO.43所示核苷酸序列具有90％或以上同一性的核苷酸序列，优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸序列，更优选具有98％或99％以上同一性的核苷酸序列；更优选地，所述填充序列的核苷酸序列如SEQ ID NO.39或SEQ ID NO.43所示；
    优选地，所述表达盒包含Kozak起始序列；所述Kozak起始序列包含与SEQ ID NO.5所示核苷酸序列具有90％或以上同一性的核苷酸序列，优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸序列，更优选具有98％或99％以上同一性的核苷酸序列；更优选地，所述Kozak起始序列具有如SEQ ID NO.5所示的核苷酸序列。
  4. 根据权利要求2或3所述的表达盒，其包含5’ITR、ApoE HCR增强子、人α1抗胰蛋白酶启动子、截短的α1抗胰蛋白酶内含子、Kozak起始序列、所述多核苷酸分子、BGH poly A、次黄嘌呤磷酸核糖基转移酶(HPRT)的部分内含子序列和3’ITR；优选地，所述表达盒包含与SEQ ID NO.80、SEQ ID NO.81、SEQ ID NO.82、SEQ ID NO.83、SEQ ID NO.84、SEQ ID NO.85、SEQ ID NO.86、SEQ ID NO.87、SEQ ID NO.89、SEQ ID NO.90、SEQ ID NO.91、SEQ ID NO.92或SEQ ID NO.93所示核苷酸序列具有90％或以上同一性的核苷酸序列，优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列；更优选地，所述表达盒具有如SEQ ID NO.80、SEQ ID NO.81、SEQ ID NO.82、SEQ ID NO.83、SEQ ID NO.84、SEQ ID NO.85、SEQ ID NO.86、SEQ ID NO.87、SEQ ID NO.89、SEQ ID NO.90、SEQ ID NO.91、SEQ ID NO.92或SEQ ID NO.93所示的核苷酸序列。
  5. 一种表达载体，其包含权利要求1所述的多核苷酸分子或权利要求2-4中任一项所述的表达盒；优选地，所述表达载体还包含编码标志物的基因，优选地，所述标志物选自抗生素抗性蛋白质、毒素抗性蛋白质、有色的或荧光的或发光的蛋白质和介导增强的细胞生长和/或基因扩增的蛋白质中的至少一种；
    优选地，所述抗生素选自氨苄青霉素、新霉素、G418、嘌呤霉素和杀稻瘟素的至少一种；
    优选地，所述毒素选自炭疽毒素和白喉毒素的至少一种；
    优选地，所述有色的或荧光的或发光的蛋白质选自绿色荧光蛋白、增强型绿色荧光蛋白、红色荧光蛋白和萤光素酶的至少一种；
    优选地，所述介导增强的细胞生长和/或基因扩增的蛋白质为二氢叶酸还原酶(DHFR)；
    优选地，所述表达载体包含复制起点；优选地，所述复制起点序列选自f1噬菌体ori、RK2oriV、pUC ori以及pSC101ori的至少一种。
  6. 根据权利要求5所述的表达载体，其选自质粒、粘粒、病毒载体、RNA载体或线性或圆形DNA或RNA分子；
    优选地，所述质粒选自pCI、puc57、pcDNA3、pSG5、pJ603或pCMV；
    优选地，所述病毒载体选自逆转录病毒、腺病毒、细小病毒(例如，腺伴随病毒)、冠状病毒、负链RNA病毒诸如正粘病毒(例如，流感病毒)、弹状病毒(例如，狂犬病和水疱性口炎病毒)、副粘病毒(例如，麻疼和仙台)、正链RNA病毒(诸如小RNA病毒和甲病毒)，或双链DNA病毒，所述双链DNA病毒选自腺病毒、疱疹病毒(例如，单纯疱疹病毒1和2型、愛泼斯坦-巴尔病毒、巨细胞病毒)、痘病毒(例如，牛痘病毒、鸡痘病毒和金丝雀痘病毒)、诺沃克病毒、披膜病毒、黄病毒、呼肠孤病毒、乳多泡病毒、嗜肝DNA病毒、杆状病毒或肝炎病毒；
    优选地，所述逆转录病毒选自禽造白细胞组织增生-肉瘤、哺乳动物C-型、B-型病毒、D-型病毒、HTLV-BLV集合、慢病毒或泡沫病毒；
    优选地，所述慢病毒载体选自HIV-1、HIV-2、SIV、FIV、BIV、EIAV、CAEV或绵羊脱髓鞘性脑白质炎慢病毒。
  7. 根据权利要求5或6所述的表达载体，其为腺相关病毒载体；
    优选地，所述腺相关病毒选自AAV 1型、AAV 2型、AAV 3型、AAV 4型、AAV 5型、AAV 6型、AAV 7型、AAV 8型、AAV 9型、AAV 10型、禽AAV、牛AAV、犬AAV、马AAV或绵羊AAV；
    优选地，所述表达载体包含与SEQ ID NO.53、SEQ ID NO.54、SEQ ID NO.55、SEQ ID NO.56、SEQ ID NO.57、SEQ ID NO.58、SEQ ID NO.59、SEQ ID NO.60、SEQ ID NO.62、SEQ ID NO.63、SEQ ID NO.64、SEQ ID NO.65、SEQ ID NO.66、SEQ ID NO.72、SEQ ID NO.73、SEQ ID NO.74、SEQ ID NO.75、SEQ ID NO.76、SEQ ID NO.77、SEQ ID NO.78、SEQ ID NO.96、SEQ ID NO.97、SEQ ID NO.98、SEQ ID NO.99、SEQ ID NO.100、SEQ ID NO.101、SEQ ID NO.102、SEQ ID NO.103、SEQ ID NO.105、SEQ ID NO.106、SEQ ID NO.107、SEQ ID NO.108或SEQ ID NO.109所示核苷酸序列具有90％或以上同一性的核苷酸序列，优选具有91％、92％、93％、94％、95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列；更优选地，所述表达载体具有如SEQ ID NO.53、SEQ ID NO.54、SEQ ID NO.55、SEQ ID NO.56、SEQ ID NO.57、SEQ ID NO.58、SEQ ID NO.59、SEQ ID NO.60、SEQ ID NO.62、SEQ ID NO.63、SEQ ID NO.64、SEQ ID NO.65、SEQ ID NO.66、SEQ ID NO.72、SEQ ID NO.73、SEQ ID NO.74、SEQ ID NO.75、SEQ ID NO.76、SEQ ID NO.77、SEQ ID NO.78、SEQ ID NO.96、SEQ ID NO.97、SEQ ID NO.98、SEQ ID NO.99、SEQ ID NO.100、SEQ ID NO.101、SEQ ID NO.102、SEQ ID NO.103、SEQ ID NO.105、SEQ ID NO.106、SEQ ID NO.107、SEQ ID NO.108或SEQ ID NO.109所示的核苷酸序列；更优选地，表达载体具有如SEQ ID NO.76、SEQ ID NO.73、SEQ ID NO.60或SEQ ID NO.78所示的核苷酸序列；更优选地，表达载体具有如SEQ ID NO.73所示的核苷酸序列。
  8. 一种病毒颗粒，其包含权利要求1所述的多核苷酸分子、权利要求2-4中任一项所述的表达盒和权利要求5-7中任一项所述的表达载体中的至少一种。
  9. 一种治疗苯丙酮尿症的药物组合物，其包含权利要求1所述的多核苷酸分子、权利要求2-4中任一项所述的表达盒、权利要求5-7任一所述的表达载体和权利要求8所述的病毒颗粒中的至少一种；任选地，其还包括药学上可接受的载体；其中，所述的药物组合物表达野生型或密码子优化的PAH蛋白。
  10. 权利要求1所述的多核苷酸分子、权利要求2-4中任一项所述的表达盒、权利要求5-7任一所述的表达载体、权利要求8所述的病毒颗粒和权利要求9所述的药物组合物中的至少一种在制备治疗苯丙酮尿症的药物中的用途。
PCT/CN2023/128981 2022-11-02 2023-11-01 优化的pah基因和表达盒及其用途 WO2024094044A1 (zh)

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