Optimized PAH genes and expression cassettes and their uses

Abstract

The present disclosure relates to an optimized PAH gene and expression cassette and uses thereof, and discloses a polynucleotide molecule encoding a PAH protein. The polynucleotide molecule, expression cassette, expression vector, viral particle, and / or pharmaceutical composition disclosed herein can be used to effectively, sustainably, and stably express human PAH in the liver at a relatively low dose, thereby maintaining the phenylalanine concentration in a subject's blood at a low level for a long period of time and stably, and can be used to treat phenylketonuria.

Description

[Technical Field]

[0001] The present disclosure is in the field of gene therapy technology, and specifically relates to optimized PAH genes and expression cassettes and uses thereof. [Background technology]

[0002] Phenyleketonuria (PKU, OMIM 261600) is an autosomal recessive genetic disorder caused by a phenylalanine hydroxylase (PAH) gene mutation. The incidence in the United States is 1 / 13,500, and in China is approximately 1 / 15,924. In newborns and children, peripheral blood phenylalanine concentrations can be well controlled through low-phenylalanine formula and diet to promote nervous system development. Currently, oral tetrahydrobiopterin or sapropterin (trade name Kuvan) and subcutaneous prolonged phenylalanine aminolase (pegvaliase-pqpz, trade name Palynziq, PEGylated phenylalanine aminolase) are available for use in adults. However, both of these medications have drawbacks and limitations, are only applicable to a small proportion of patients, have immune-related side effects, and are slow to respond.

[0003] The current US classification criteria and treatment guidelines recommend lifelong dietary and drug treatment to maintain peripheral blood phenylalanine levels between 120 μmol / L and 360 μmol / L (Vockley, J., et al., Phenylalanine hydroxylase deficiency: diagnosis and management guideline. Genet Med, 2014.16(2):pp.188-200). The European Union treatment guidelines recommend a control level of less than 360 μmol / L for children under 12 years of age, pregnant women, and those planning to become pregnant, and less than 600 μmol / L for other groups (van Wegberg, A.M.J., et al., The complete European guidelines on phenylketonuria: diagnosis and treatment. Orphanet J Rare Dis, 2017.12(1):pp.162). However, adult patients have difficulty maintaining proper dietary control, and approximately 19% of patients do not progress beyond 9 months, resulting in a loss of progress and a state of hyperphenylalaninemia. Studies have shown that in adult PKU patients, 67% of patients have phenylalanine levels greater than 360 μmol / L, 45% greater than 600 μmol / L, and 18% greater than 1200 μmol / L, with only approximately 24% of adult patients able to control their phenylalanine levels below 360 μmol / L (Brown, C.S. and U. Lichter-Konecki, Phenylketonuria (PKU): A problem solved? Mol Genet Metab Rep, 2016.6:pp.8-12).

[0004] Even with early and effective treatment, adult patients with PKU who do not control peripheral blood phenylalanine levels are prone to neurological symptoms, such as tremors, brisk deep tendon reflexes, poor motor coordination, and white matter abnormalities. Adults with PKU also experience many quality-of-life problems, including reduced work capacity, lack of autonomy, increased despair, reduced motivation, depression and anxiety, difficulty maintaining long-term relationships with friends, and increased risk of running away from home in later life (Murphy, G.H., et al., Adults with untreated phenylketonuria: out of sight, out of mind. Br J Psychiatry, 2008, 193(6):501-2; Hoeks, M.P., M.den Heijer, and M.C.Janssen, Adult issues in phenylketonuria. Neth J Med, 2009, 67(1):2-7). In adolescents and adults who are diagnosed at a terminal stage and who do not receive early or adequate treatment, high peripheral blood phenylalanine levels can lead to epilepsy, convulsions, severe behavioral problems, such as aggression, self-injury, hyperactivity, hassle, anger, sleep disorders, anxiety, cutting, and neurological and cognitive problems (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).)。.

[0005] Therefore, there is still a great clinical need for the treatment of adult phenylketonuria. Research has shown that treating adult patients by controlling peripheral blood phenylalanine levels can reduce or stop epileptic seizures and improve behavioral and neurocognitive problems (van Spronsen, FJ, et al., Phenylketonuria. Nat Rev Dis Primers, 2021.7(1):p.36.).

[0006] Gene therapy has made remarkable progress over the past 60 years. With a single treatment, Zolgensma can enable children with spinal muscular atrophy (SMA) to grow up to be almost normal. With a single treatment, Luxturna can restore the vision of patients with Leber congenital amaurosis (LCA), enabling them to essentially live, learn, and work normally. Currently, two gene therapy programs for PKU are in Phase I clinical trials internationally. Both programs use high doses, and while no effective therapeutic effects have yet been reported, there are concerns about potential liver toxicity due to high doses. Summary of the Invention

[0007] The present disclosure aims to provide an optimized PAH gene, expression cassette, and viral vector that can effectively, sustainably, and stably express human PAH in the liver at relatively low doses and can be used to treat phenylketonuria.

[0008] In order to solve the above technical problems, the present disclosure provides the following inventions.

[0009] A first aspect of the present disclosure provides a polynucleotide molecule encoding a PAH protein, comprising a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth 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 comprising a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably comprising a nucleotide sequence having 98% or 99% or more identity.

[0010] A second aspect of the present disclosure provides an expression cassette comprising a polynucleotide molecule according to the first aspect of the disclosure and a promoter operably linked to said polynucleotide molecule.

[0011] In some embodiments, the expression cassette further comprises an expression control element operably linked to the polynucleotide molecule.

[0012] In some embodiments, the expression control element is at least one selected from transcriptional / translational control signals, enhancers, introns, polyA signals, ITRs, insulators, RNA processing signals, and elements that enhance mRNA and protein stability.

[0013] A third aspect of the present disclosure provides an expression vector comprising a polynucleotide molecule according to the first aspect of the disclosure or an expression cassette according to the second aspect of the disclosure.

[0014] In some embodiments, 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.

[0015] In some embodiments, the expression vector is an adeno-associated virus vector.

[0016] A fourth aspect of the present disclosure provides a viral particle comprising at least one of a polynucleotide molecule according to the first aspect of the present disclosure, an expression cassette according to the second aspect of the present disclosure, and an expression vector according to the third aspect of the present disclosure.

[0017] A fifth aspect of the present disclosure provides a pharmaceutical composition for treating phenylketonuria comprising at least one of a polynucleotide molecule according to the first aspect of the present disclosure, an expression cassette according to the second aspect of the present disclosure, an expression vector according to the third aspect of the present disclosure, and a viral particle according to the fourth aspect of the present disclosure.

[0018] A sixth aspect of the present disclosure provides the use of at least one of a polynucleotide molecule according to the first aspect of the present disclosure, an expression cassette according to the second aspect of the present disclosure, an expression vector according to the third aspect of the present disclosure, a viral particle according to the fourth aspect of the present disclosure, and a pharmaceutical composition according to the fifth aspect of the present disclosure in the preparation of a medicament for the treatment of phenylketonuria.

[0019] A seventh aspect of the present disclosure provides the use of at least one of a polynucleotide molecule according to the first aspect of the present disclosure, an expression cassette according to the second aspect of the present disclosure, an expression vector according to the third aspect of the present disclosure, a viral particle according to the fourth aspect of the present disclosure, and a pharmaceutical composition according to the fifth aspect of the present disclosure in the treatment of phenylketonuria.

[0020] 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 molecule, expression cassette, expression vector, viral particle, and pharmaceutical composition of the present disclosure.

[0021] By using the polynucleotide molecule, expression cassette, expression vector, viral particle, and / or pharmaceutical composition of the present disclosure, it is possible to more effectively express human PAH by optimizing the PAH-encoding gene, and further, it is possible to effectively, sustainably, and stably express human PAH in the liver at a relatively low dose, thereby maintaining the phenylalanine concentration in the subject's blood at a low level for a long period of time and stably, and it can be used to treat phenylketonuria. Furthermore, the polynucleotide molecule, expression cassette, expression vector, viral particle, and / or pharmaceutical composition of the present disclosure have a low dosage and lower potential liver toxicity. [Brief explanation of the drawings]

[0022] [Figure 1] 1 shows a schematic diagram of the structure of the recombinant AAV (rAAV) vector PAH expression cassette of the present disclosure. [Figure 2] Figure 1 shows in vitro expression of the codon-optimized PAH opt gene in HepG2 cells transfected with the plasmid. A shows a representative image of a PAH protein Western blot, and B shows the quantitative results of the PAH protein Western blot to detect the expression levels of PAH and GAPDH (plasmid transfection control) in HepG2 cells, where the WT or opt gene was expressed with the pAAV8-ATT-PAH WT or pAAV8-ATT-PAH opt plasmid, respectively. Cell lysates were harvested two days after transfection with equal amounts of plasmid. Equal amounts of total protein were separated by SDS-PAGE and then subjected to Western blotting. PAH expression levels were normalized to GAPDH and calculated as a ratio to the WT expression level. [Figure 3A] The Phe-lowering effect of the PAH WT and optimized genes was demonstrated in PKU model mice after high-pressure injection into the tail vein. Blood samples were collected from the mice at 0 h, 6 h, 24 h, 3 days, 5 days, 10 days, 16 days, and 19 days after injection, and the Phe content in the blood was measured. [Figure 3B]The figure shows the Phe content in the blood of PKU model mice 10 days after injection. [Figure 4] This shows the Phe-lowering effect of AAV8-ATT-PAH WT and optimized gene viruses in PKU model mice. Blood samples were collected weekly for 4 weeks after injection, and the Phe content in the blood was measured. [Figure 5] Schematic diagram of the structure of recombinant AAV (rAAV) vector PAH expression cassettes containing different promoters. [Figure 6] The effect of PAH WT expression cassettes under different promoters was shown to be effective in lowering Phe in PKU model mice after high-pressure injection into the tail vein. Three days after injection, blood samples were taken and the Phe content in the blood was measured. [Figure 7] Schematic diagram of the structure of recombinant AAV (rAAV) vector PAH expression cassettes containing different expression regulatory elements. [Figure 8] We demonstrate the effect of high-pressure injection of a PAH expression cassette containing different expression regulatory elements into the tail vein of a PKU model mouse. Three days after injection, blood samples were collected and the Phe content in the blood was measured. [Figure 9] Schematic diagram of the structure of optimized recombinant AAV (rAAV) vector PAH expression cassettes containing different packaging sequences. [Figure 10A] To demonstrate the Phe-lowering effect of AAV8-ATT-PAH opt9 viruses with different loading sequences in PKU model mice, blood samples were taken weekly for 8 weeks after injection to measure the Phe content in the blood. [Figure 10B] To demonstrate the Phe-lowering effect of AAV8-ATT-PAH opt9 and AAV8-ATT-PAH opt-HPRT(4CpG) viruses in PKU model mice, blood samples were taken weekly for 4 weeks after injection and the Phe content in the blood was measured. [Figure 11]This shows the results of functional testing of the AAV8-ATT-PAH opt-HPRT(4CpG) virus to reduce the Phe concentration of the expressed PAH product in HepG2 cells. Transiently transfected AAVR HepG2 cells were infected with the AAV8-ATT-PAH opt-HPRT(4CpG) virus at an MOI of 0, 5e4, 1e5, or 2e5, and the change in Phe concentration was calculated using the uninfected HepG2 cell line as a reference. [Figure 12] We demonstrate the Phe-lowering effect of different doses of AAV8-ATT-PAH opt9-HPRT(4CpG) virus in male mice with a PKU model, where blood samples were taken weekly for 6 weeks after injection to measure the Phe content in the blood. [Figure 12B] The Phe-lowering effect of different doses of AAV8-ATT-PAH opt9-HPRT(4CpG) virus in female PKU mice was demonstrated. Six weeks after injection, blood samples were taken weekly and the Phe content in the blood was measured. [Figure 13A] PKU homozygous mice received 3.0e10 and 3.0e11 vg / mouse of AAV8-ATT-PAH opt9-HPRT(4CpG) virus, and the Tyr content in the brain tissue of the mice after 6 weeks of administration was shown. Untreated heterozygous mice served as normal control mice, and vehicle-treated homozygous mice served as untreated control mice. [Figure 13B] 1 shows the content of 5-hydroxyindoleacetic acid (5-HIAA) in mouse brain tissue. [Figure 13C] PKU homozygous mice were treated with a dose of 3.0e10vg / mouse for 3 weeks and then compared with the coat color of the vehicle group. DETAILED DESCRIPTION OF THE INVENTION

[0023] In order to more clearly explain the inventions in the embodiments of the present disclosure or the prior art, the following briefly introduces drawings required in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only one embodiment of the present disclosure, and those skilled in the art can also obtain other embodiments based on these drawings.

[0024] definition In this disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory operation steps used herein are terms and common steps widely used in the corresponding fields. At the same time, in order to better understand this disclosure, the following definitions and interpretations of related terms are provided.

[0025] As used herein, the terms "a," "an," "said," and similar referents refer to the singular and the plural unless the specification or context clearly contradicts otherwise.

[0026] As used herein, the terms "about," "essentially," and "similar" refer to a range of error for a particular value as determined by one of ordinary skill in the art, which may depend on how the value is measured or determined or on the limitations of the measurement system. As used herein, "about" a value or parameter includes embodiments relative to the value or parameter itself. For example, a description of "about X" includes a description of "X."

[0027] As used herein, "vector" refers to a recombinant plasmid or virus containing a nucleic acid to be delivered to a host cell (in vitro or in vivo).

[0028] As used herein, the term "polynucleotide molecule" or "nucleic acid" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, 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 non-natural or derivatized nucleotide bases, including purine and pyrimidine bases or other natural, chemical, or biochemical modifications. The backbone of a nucleic acid can contain sugars and phosphate groups (as typically found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of a nucleic acid can contain synthetic subunits such as aminophosphates, and thus can be oligodeoxynucleoside aminophosphate (P-NH) or mixed aminophosphate-phosphodiester oligomers. Double-stranded nucleic acids can also be obtained from single-stranded polynucleotide products by chemical synthesis (synthesizing the complementary strand under appropriate conditions and annealing the strands, or by using DNA polymerase to synthesize the complementary strand head-on with an appropriate primer).

[0029] "Recombinant viral vector" refers to a recombinant polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences of non-viral origin). In the case of a recombinant AAV vector, the side flaps of the recombinant nucleic acid are at least one, and preferably two, inverted terminal repeats (ITRs).

[0030] A "recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector containing one or more heterologous sequences (i.e., nucleic acid sequences of non-AAV origin), the side flaps of which are at least one, and preferably two, AAV inverted terminal repeats (ITRs). When present in a host cell that has been infected with and expressed the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins) using an appropriate accessory virus (or expressing appropriate accessory functions), the rAAV vector can replicate and package into infectious viral particles. When the rAAV vector is integrated into a larger polynucleotide (e.g., in a chromosome or in another vector, e.g., a plasmid, for cloning or transfer), the rAAV vector is referred to as a "pro-vector," which allows for "rescue" by replication and shelling in the presence of AAV packaging functions and appropriate accessory functions. rAAV vectors may be in any of several forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with or encapsulated within liposomes, and in embodiments, encapsulated within viral particles, particularly AAV particles. rAAV vectors can be packaged within the capsid of an AAV virus to produce "recombinant adeno-associated viral particles (rAAV particles)." AAV accessory functions (i.e., functions that allow AAV to be replicated and packaged by a host cell) may be provided in any of a variety of forms, including, but not limited to, accessory viruses or accessory viral genes that support AAV replication and packaging. Other AAV accessory functions are known in the art.

[0031] "rAAV virus" or "rAAV viral particle" refers to a viral particle consisting of at least one AAV capsid protein and a shelled rAAV vector genome.

[0032] "Heterologous" means a genotypically distinct entity compared to or derived from the entity into which it has been introduced or incorporated. For example, a nucleic acid introduced into a different cell type by genetic engineering techniques is a heterologous nucleic acid (and can encode a heterologous polypeptide when expressed). Similarly, a cellular sequence (e.g., a gene or portion thereof) incorporated into a viral vector is a nucleotide sequence heterologous to the vector.

[0033] The terms "genome particles (gp)," "genome equivalents," or "genome copies," as used to refer to viral titers, refer to the number of viral particles containing a recombinant AAV DNA genome, regardless of their infectiousness or functionality. The number of genome particles in a particular vector preparation can be determined by the methods described in the Examples herein or, for example, by Clark et al. (1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther., 6:272-278.

[0034] The terms "infectious unit (iu)," "infectious particle," or "replication unit," as used to refer to viral titer, refer to the number of infectious and replication-competent recombinant AAV vector particles as measured by the infectious center assay, also called the replication center assay, e.g., as described in McLaughlin et al. (1988) J. Virol., 62:1963-1973.

[0035] The term "transducing unit (tu)," as used to refer to viral titer, refers to the number of infectious recombinant AAV vector particles produced by a functional recombinant product, e.g., as measured in a functional assay, e.g., as measured in the embodiments herein or, e.g., in Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or Fisher et al. (1996) J. Virol., 70:520-532 (LFU assay).

[0036] "Inverted terminal repeat" or "ITR" sequences are a term well known in the art and refer to relatively truncated sequences found at the ends of viral genomes, in opposite orientations.

[0037] The term "AAV inverted terminal repeat (ITR)" is well known in the art and refers to a sequence of approximately 145 nucleotides present at both ends of a naturally occurring single-stranded AAV genome. The outermost 125 nucleotides of the ITR may be present in either of two interchangeable orientations, resulting in heterogeneity between different AAV genomes and between the ends of a single AAV genome. The outermost 125 nucleotides further contain multiple, relatively truncated regions (referred to as A, A', B, B', C, C', and D regions) that are complementary to each other, allowing interstrand base pairing to occur within the ITR portion.

[0038] The term "auxiliary virus" in relation to AAV refers to a virus that is replicated and packaged by AAV (a defective parvovirus) in a host cell. Examples of such auxiliary viruses include adenoviruses, herpes viruses, and smallpox viruses, such as cowpox. Adenoviruses include several different subtypes, with subtype C adenovirus type 5 (Ad5) being the most common. Numerous adenoviruses derived from humans, non-human mammals, and birds are known to be available from collection centers, such as the American College of Cardiology (ATCC). Examples of herpes viruses available from collection centers such as the ATCC include herpes simplex viruses (HSV), Epstein-Barr viruses (EBV), cytomegaloviruses (CMV), and pseudorabies viruses (PRV).

[0039] In this disclosure, the term "packing sequence" refers to a human non-coding sequence added to a recombinant AAV expression cassette to approximate the length of the nucleotide sequence of the wild-type AAV genome. In some embodiments of the present disclosure, adding a packing sequence results in higher viral packaging yields when the expression cassette or the polynucleotide sequence carrying it is expressed using an AAV viral vector.

[0040] "Percentage (%) sequence identity" 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 those in the reference polypeptide or nucleic acid sequence, after aligning the sequences and introducing deletions (if necessary to achieve the maximum percentage sequence identity and not considering any conservative substitutions as part of the sequence identity). Comparisons for the purpose of determining percentage amino acid or nucleic acid sequence identity can be performed using publicly available computer software programs, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. A preferred software is ALIGN Plus (Scientific and Educational Software, Pennsylvania). Those skilled in the art can determine appropriate parameters to use for measuring comparisons, including any algorithms necessary to achieve maximum comparison across the entire length of the sequences being compared. Herein, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (which can alternatively be referred to as amino acid sequence A having a certain % amino acid sequence identity to given amino acid sequence B) is calculated by multiplying 100 by the fraction X / Y, where X is the number of amino acid residues that have identical matches according to a sequence comparison program score in a program comparison of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the lengths of amino acid sequence A and amino acid sequence B are not equal, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Herein, the % nucleic acid sequence identity of a given nucleic acid sequence C to a given nucleic acid sequence D (which can alternatively be referred to as given nucleic acid sequence C having a certain % nucleic acid sequence identity to or containing a certain % nucleic acid sequence identity to given nucleic acid sequence D) is calculated by multiplying 100 by the fraction W / Z, where W is the number of nucleotides that have identical matches according to a sequence comparison program score in a program comparison of C and D, and Z is the total number of nucleotides in D.It is understood that if the lengths of nucleic acid sequence C and nucleic acid sequence D are not equal, then the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C.

[0041] An "effective amount" is an amount sufficient to provide a beneficial or expected result, including a clinical result (e.g., amelioration of symptoms, realization of a clinical endpoint, etc.). An effective amount can be administered one or more times. The disease state is an amount sufficient to ameliorate, stabilize, or slow the progression of the disease. For example, an effective amount of rAAV particles expresses a desired amount of a heterologous nucleic acid, such as a therapeutic polypeptide or therapeutic nucleic acid.

[0042] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is human.

[0043] As used herein, "treatment" is a method for obtaining beneficial or expected clinical results. In this disclosure, beneficial or expected clinical results include, but are not limited to, amelioration of symptoms, reduction in the extent of disease, stabilized (e.g., non-worsening) state of disease, prevention of disease spread (e.g., migration), slowing or alleviation of disease progression, and improvement or alleviation and remission (whether partial or complete) of the disease state, whether detectable or undetectable. "Treatment" means prolonging survival beyond the desired survival rate without treatment.

[0044] The unit vg (Vector Genomes) represents the number of copies of the viral genome.

[0045] The term multiplicity of infection (MOI) refers to the ratio of the number of viruses to the number of bacteria at the time of infection, i.e., the number of bacteriophages infecting each bacterium.

[0046] A first aspect of the present disclosure provides a polynucleotide molecule encoding a PAH protein, comprising a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth 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 comprising a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably comprising a nucleotide sequence having 98% or 99% or more identity.

[0047] In some embodiments, the polynucleotide molecule has a nucleotide sequence set forth 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.

[0048] In some embodiments, the codon-optimized human PAH protein-encoding gene has the nucleotide sequence set forth in SEQ ID NO. 17.

[0049] A second aspect of the present disclosure provides an expression cassette comprising a polynucleotide molecule according to the first aspect of the disclosure and a promoter operably linked to said polynucleotide molecule.

[0050] In some embodiments, the promoter is a specific or non-specific promoter.

[0051] In some embodiments, the promoter comprises a core promoter.

[0052] In some embodiments, the promoter may be a constitutive promoter, and preferably, the constitutive promoter is at least one selected from a CMV promoter, an EF1A promoter, an EFS promoter, a CAG promoter, a CBh promoter, an SFFV promoter, an MSCV promoter, an SV40 promoter, an mPGK promoter, an hPGK promoter, a UBC promoter, and the like.

[0053] In some embodiments, the promoter is an inducible promoter, preferably comprising 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. In some embodiments, the tetracycline-regulated promoter is selected from a Tet on promoter, a Tet off promoter, and a Tet Activator promoter. In some embodiments, the alcohol-regulated promoter is selected from an alcohol dehydrogenase I (alcA) gene promoter and a promoter responsive to alcohol transactivator protein (AlcR). In some embodiments, the steroid-regulated promoter is selected from a rat corticoid receptor promoter, a human estrogen receptor promoter, a minostrogen receptor promoter, a retinoid promoter, and a thyroid receptor superfamily promoter. In some embodiments, the metal-regulated promoter is selected from yeast, mouse, and human metallothioprotein promoters. In some embodiments, the pathogenicity-regulated promoter is selected from a salicylic acid-regulated promoter, an ethylene-regulated promoter, and a benzothiadiazole-regulated (BTH) promoter. In some embodiments of the present disclosure, the temperature / heat-inducible promoter is selected from an HSP-70 promoter, an HSP-90 promoter, and a soybean thermo-activated promoter. In some embodiments of the present disclosure, the light-regulated promoter is a light-responsive promoter in plant cells.

[0054] In some preferred embodiments, 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, thyroid hormone transport protein (transthyretin, TTR) promoter, alpha-antitrypsin (AAT or Serpin A1) promoter, TK (thymidine kinase) promoter, hemopexin promoter, alcohol dehydrogenase 6 promoter, cholesterol 7α-25-hydroxylase promoter, factor IX promoter, α-microglobulin promoter, SV40 promoter, CMV promoter, Rous sarcoma virus-LTR promoter, HBV promoter, ALB promoter, and TBG promoter. Needless to say, minimal promoters derived from these promoters may also be used. More preferably, the liver-specific promoter is the human alpha 1 antitrypsin promoter (hAAT or SERPINA1 promoter), and preferably the core promoter comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in SEQ ID NO. 3, 24, 25, 30, 33 or 38, preferably has a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably has a nucleotide sequence having 98% or more identity, and preferably the core promoter has the nucleotide sequence set forth in SEQ ID NO. 3, 24, 25, 30, 33 or 38, and more preferably the nucleotide sequence of the core promoter is set forth in SEQ ID NO. 3.

[0055] In some embodiments, the expression cassette further comprises an expression control element operably linked to the polynucleotide molecule.

[0056] In some embodiments, the expression control element is at least one selected from transcriptional / translational control signals, enhancers, introns, polyA signals, ITRs, insulators, RNA processing signals, and elements that enhance mRNA and protein stability.

[0057] In some embodiments, the expression cassette comprises a 5' ITR, preferably the 5' ITR comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in SEQ ID NO.1, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably 98% or 99% or more identity, and more preferably the 5' ITR has the nucleotide sequence set forth in SEQ ID NO.1.

[0058] In some embodiments, the expression cassette comprises a 3' ITR. In some preferred embodiments, the 3' ITR comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in SEQ ID NO. 8, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably 98% or 99% or more identity, and more preferably the 3' ITR has the nucleotide sequence set forth in SEQ ID NO. 8.

[0059] In some embodiments, the expression cassette further comprises an enhancer. In some preferred embodiments, the enhancer is selected from the group consisting of the ApoE HCR enhancer or an active fragment thereof, the CRMSBS2 enhancer or an active fragment thereof, the TTRm enhancer or an active fragment thereof, and the CMV enhancer or an active fragment thereof; more preferably, the enhancer comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth 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 99% or more identity; preferably, the enhancer has the nucleotide sequence set forth in SEQ ID NO. 2, 29, 32, 37, or 40, more preferably the nucleotide sequence of the enhancer is set forth in SEQ ID NO. 2.

[0060] In some embodiments, the expression cassette further comprises an intron, preferably the intron is a truncated alpha 1 antitrypsin intron or an active fragment thereof, the second intron of beta-globin or an active fragment thereof, an SV40 intron or an active fragment thereof, or a mouse parvovirus intron or an active fragment thereof; preferably the intron comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth 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 99% or more identity; preferably the intron has the nucleotide sequence set forth in SEQ ID NO. 4, 26, 27, 28, 31 or 34, more preferably the nucleotide sequence of the intron is set forth in SEQ ID NO. 4.

[0061] In some embodiments, the promoter of the expression cassette is a combinatorial promoter comprising an upstream regulatory element, a core promoter, and an intron.

[0062] In some embodiments, the upstream regulatory element is an enhancer or an active fragment thereof.

[0063] In some embodiments, the combined promoter comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in SEQ ID NO. 44, 45, 46, 47, 48 or 49, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably 98% or 99% or more identity, preferably the combined promoter has the nucleotide sequence set forth in SEQ ID NO. 44, 45, 46, 47, 48 or 49, more preferably the nucleotide sequence of the combined promoter is set forth in SEQ ID NO. 44.

[0064] In some embodiments, the expression cassette further comprises a polyA signal, preferably at least one of bovine growth hormone polyA (BGH polyA), short polyA, SV40 polyA, and human β-globin polyA. Preferably, the polyA signal comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in SEQ ID NO. 7, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably 98% or 99% or more identity, and more preferably the nucleotide sequence of the polyA signal is set forth in SEQ ID NO. 7.

[0065] In some embodiments, the expression cassette comprises an optimized filler sequence, preferably the filler 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 a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), preferably the filler sequence is a partial intron sequence of hypoxanthine phosphoribosyltransferase (HPRT), preferably the filler sequence is selected from a partial intron sequence of SEQ ID NO. 39 or SEQ ID NO. The nucleotide sequence of the filler sequence may be set forth in SEQ ID NO. 39 or SEQ ID NO. 43, and may have 90% or more identity to the nucleotide sequence set forth in SEQ ID NO. 39 or SEQ ID NO. 43, and preferably has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the nucleotide sequence set forth in SEQ ID NO. 39 or SEQ ID NO. 43, and more preferably has 98% or 99% or more identity to the nucleotide sequence set forth in SEQ ID NO. 39 or SEQ ID NO. 43.

[0066] In some embodiments, the expression cassette comprises a Kozak initiation sequence, wherein the Kozak initiation sequence comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in SEQ ID NO. 5, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably 98% or 99% or more identity, and more preferably the Kozak initiation sequence has the nucleotide sequence set forth in SEQ ID NO. 5.

[0067] In some embodiments, the expression cassette comprises a 5' ITR, an ApoE HCR enhancer, a human alpha 1 antitrypsin promoter (SERPINA1 promoter), a truncated alpha 1 antitrypsin intron (SerpinA1 intron), a Kozak initiation sequence (GCCACC, SEQ ID NO. 5), the polynucleotide molecule, BGH poly A, a partial intron sequence of hypoxanthine phosphoribosyltransferase (HPRT) (HPRT(4CpG)), and a 3' ITR, and preferably the expression cassette is comprised of 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, or SEQ ID NO. and more preferably, the expression cassette comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in 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 or SEQ ID NO.93.

[0068] A third aspect of the present disclosure provides an expression vector comprising a polynucleotide molecule according to the first aspect of the disclosure or an expression cassette according to the second aspect of the disclosure.

[0069] In some embodiments, the expression vector further comprises a gene encoding a marker, and preferably the marker is at least one selected from an antibiotic resistance protein, a toxin resistance protein, a colored, fluorescent, or luminescent protein, and a protein that mediates enhanced cell growth and / or gene amplification.

[0070] In some embodiments, the antibiotic is selected from at least one of ampicillin, neomycin, G418, puromycin, and blasticidin.

[0071] In some embodiments, the toxin is at least one selected from anthrax toxin and diphtheria toxin.

[0072] In some embodiments, the colored, fluorescent, or luminescent protein is selected from at least one of green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, and luciferase.

[0073] In some embodiments, the protein that mediates enhanced cell growth and / or gene amplification is dihydrofolate reductase (DHFR).

[0074] In some embodiments, the expression vector comprises an origin of replication, and preferably, the origin of replication sequence is selected from at least one of f1 bacteriophage ori, RK2oriV, pUC ori, and pSC101ori.

[0075] In some embodiments, 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.

[0076] In some embodiments, the plasmid is selected from pCI, puc57, pcDNA3, pSG5, pJ603, or pCMV.

[0077] In some embodiments, the viral vector is selected from a retrovirus, adenovirus, parvovirus (e.g., adeno-associated virus), coronavirus, negative-strand RNA virus such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles virus and Sendai virus), positive-strand RNA virus (e.g., picornavirus and alphavirus), or double-stranded DNA virus, wherein the double-stranded DNA virus is selected from adenovirus, herpes virus (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), pox virus (e.g., cowpox virus, fowlpox virus, and canarypox virus), norovirus, togavirus, flavivirus, reovirus, polyomavirus, hepatotropic DNA virus, baculovirus, or hepatitis virus.

[0078] In some embodiments, the retrovirus is selected from avian leukocytoproliferative sarcoma, mammalian C virus, mammalian B virus, mammalian D virus, HTLV-BLV assemblage, rentovirus, or foam virus.

[0079] In some embodiments, the lentiviral vector is selected from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or ovine demyelinating encephalitis lentivirus.

[0080] In some embodiments, the expression vector is an adeno-associated virus (AAV) vector.

[0081] In some embodiments, the adeno-associated virus is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV.

[0082] In some embodiments, the expression cassette may be packaged in a vector having a shell derived from any AAV serotype or hybrid or variant thereof in an rAAV.

[0083] In some embodiments, 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. The expression vector comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in 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. 79, 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. 88, SEQ ID NO. 89, SEQ ID NO. 90, SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97, SEQ ID NO. 98, SEQ ID NO. 99, SEQ ID NO. 99, SEQ ID NO. 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. 109, and more preferably, the expression vector has the nucleotide sequence set forth in SEQ ID NO. 76, SEQ ID NO. 73, SEQ ID NO. 60 or SEQ ID NO. 78.

[0084] In some embodiments, the expression vector has the nucleotide sequence shown in SEQ ID NO.73.

[0085] A fourth aspect of the present disclosure provides a viral particle comprising at least one of a polynucleotide molecule according to the first aspect of the present disclosure, an expression cassette according to the second aspect of the present disclosure and an expression vector according to the third aspect of the present disclosure.

[0086] A fifth aspect of the present disclosure provides a pharmaceutical composition for treating phenylketonuria comprising at least one of a polynucleotide molecule according to the first aspect of the present disclosure, an expression cassette according to the second aspect of the present disclosure, an expression vector according to the third aspect of the present disclosure, and a viral particle according to the fourth aspect of the present disclosure.

[0087] A sixth aspect of the present disclosure provides the use of at least one of a polynucleotide molecule according to the first aspect of the present disclosure, an expression cassette according to the second aspect of the present disclosure, an expression vector according to the third aspect of the present disclosure, a viral particle according to the fourth aspect of the present disclosure and a pharmaceutical composition according to the fifth aspect of the present disclosure in the preparation of a medicament for treating phenylketonuria.

[0088] A seventh aspect of the present disclosure provides the use of at least one of the polynucleotide molecule described in the first aspect of the present disclosure, the expression cassette described in the second aspect of the present disclosure, the expression vector described in the third aspect of the present disclosure, the viral particle described in the fourth aspect of the present disclosure and the pharmaceutical composition described in the fifth aspect of the present disclosure in the treatment of phenylketonuria.

[0089] An eighth aspect of the present disclosure provides a method for treating phenylketonuria, comprising the step of administering to a subject an effective amount of at least one of the polynucleotide molecule, expression cassette, expression vector, viral particle, and pharmaceutical composition of the present disclosure.

[0090] Although features are described herein as part of several embodiments, either the same or different, in order to achieve clear and concise stated objectives, the scope of the disclosure may include several embodiments having combinations of all or some of the described features.

[0091] The present disclosure will be described in more detail below with reference to specific examples. However, the examples are for illustrative purposes only and do not have any limiting effect on the present disclosure.

[0092] Materials and Methods: PAH expression cassette and AAV vector The wild-type and codon-optimized PAH sequences of all PAH genes used in this study were synthesized using GenScript. Codon optimization was performed using the GenSmart codon optimization tool. The PAH expression cassette mediated by the AAV vector is shown in Figure 1.

[0093] An initial AAV shuttle plasmid vector was synthesized from the entire gene of GeneWiz using SEQ ID NO. 112. The wild-type AAV2 ITR sequences were recombined between the BamHI and AleI enzyme sites of the vector to repair the mutated ITR sequences in the vector. The Amp resistance gene between the ApaLI enzyme sites of the initial shuttle plasmid vector was replaced with Kan, which was then recombined between the HindIII and NheI enzyme sites of the vector to increase the length of the vector and facilitate AAV viral packaging. The CAG promoter sequence was amplified by PCR from the pCAGGS vector (GENEWIZ) and recombined between the SpeI and KpnI enzyme sites of the shuttle plasmid vector to obtain the final shuttle plasmid vector (SEQ ID NO. 113). The CAG promoter contained a CMV enhancer (SEQ ID NO. 40), a chicken β-actin promoter (SEQ ID NO. 41), and a chimeric intron (SEQ ID NO. 42).

[0094] All expression cassette sequences are cloned between the SalI and BamHI enzyme cleavage sites of the shuttle plasmid vector, resulting in a shuttle plasmid that expresses the PAH target gene mediated by the AAV vector.

[0095] AAV vector production and purification methods AAV vectors were produced using a three-plasmid system: a shuttle plasmid containing the PAH target gene, the pRepCap plasmid containing the AAV vector repcap gene, and the helper plasmid Phelper (pRepCap plasmid, synthesized by GENEWIZ according to sequence SEQ ID NO. 110, and the helper plasmid pHelper, synthesized by GENEWIZ according to sequence SEQ ID NO. 111). HEK293 cells were co-transfected with PEI as a transfection reagent to recombinantly package the AAV viral vector. The cells were harvested 48-72 hours after transfection, and the harvest solution was purified to obtain recombinant AAV viral vectors of a certain purity. The purification method was as follows.

[0096] First, the harvested solution is pretreated to thoroughly lyse the HEK293 cells and release the intracellular AAV viral vector. At the same time, nuclease is added to digest the free nucleic acids. After digestion is complete, large molecular impurities and cell debris are removed by deep filtration. After deep filtration, the filtrate is subjected to a second filtration and the supernatant is loaded onto affinity chromatography.

[0097] Affinity chromatography captures AAV viral vectors in the harvested solution through specific adsorption between ligands and proteins, removing most process-related impurities, achieving the effects of concentration and impurity removal. The collected eluate is mixed homogeneously, neutralized with a neutralization buffer, and stored in a sterile bottle as the loading solution for anion chromatography.

[0098] Anion chromatography utilizes the difference in isoelectric point between different compositions to separate the core and empty-shell AAV viruses while continuing to remove residual impurities. The eluate is collected in a new sterile bottle, and the buffer is replaced with a formulation-stable buffer by ultrafiltration concentration. At the same time, the virus titer is concentrated to approximately 1 x 1013 vg / mL, and finally, it is dispensed for reserve use by sterilization filtration.

[0099] AAV vector titer quantification After AAV virus purification is complete, the viral content must be measured. The most common method for measuring the viral genome titer is to measure the AAV physical titer. The most commonly used method for measuring the viral genome titer is to design primer probes against the rAAV genome sequence and then perform Q-PCR detection.

[0100] In this disclosure, considering that codon optimization was performed on the ORF reading frame, in order to ensure quantitative stability and accuracy between different vector structures when screening multiple vector structures, primer probes were designed for the shared PolyA sequence in the vector. The F primer sequence: 5'-CAAGCCCATGTACACACCAG-3' (SEQ ID NO. 114), the R primer sequence: 5'-GGGCAAAGCTTCTGTCTGAG-3' (SEQ ID NO. 115), and the probe sequence: 5'-CTGACATCTGCCACGAGCTGCTGGGCCA-3' (SEQ ID NO. 116) were selected.

[0101] For genome titration testing, a standard curve was first constructed. The positive standard plasmid was diluted with sample diluent to 2 x 107, 2 x 106, 2 x 105, 2 x 104, 2 x 103, and 2 x 102 copies / µL. This was used as the standard curve template. The standard curve's linearity and amplification efficiency must be controlled; an R2 of >0.99 is generally required, with an amplification efficiency between 90% and 110%. The pretreated rAAV sample was then diluted and subjected to QPCR detection. The sample's Ct value was then measured within the range of the standard curve. The sample's Ct value was then substituted into the standard curve to calculate the rAAV sample genome titer and label the product.

[0102] In vitro cell plasmid transfer experiment Human hepatoma HepG2 cells were digested and seeded at 2.5 x 10 cells / well in a 96-well plate. Simultaneously, Lipo3K / DNA transfer complex was added to perform plasmid transfer experiments. Lipofectamine 3000 and P3000 (Thermo, L3000015) transfer reagents were premixed with the plasmid. The plasmid was then expressed using 60 ng / well PAH-opt / WT, 90 nL / well P3000, and 90 nL / well Lipo3000 (Thermo, L3000015). The resulting mixture was added to the HepG2 cells in a 96-well plate and incubated for 48 h in a CO2-controlled incubator.

[0103] Western blot analysis 60 μL of RIPA lysis solution (Beyotime, P0013B) containing 1× SDS loading buffer was added to each well of a 96-well plate inoculated with cultured cells, and the wells were lysed by shaking for 10 minutes, followed by denaturation at 95°C for 10 minutes. 10 μL of protein was loaded onto SDS-PAGE and electrophoresed. Subsequently, the protein was transferred to a PVDF membrane and incubated with anti-PAH (SantaCruz, sc-271258) and anti-GAPDH (TransGen, HC301) antibodies. Imaging analysis was performed using a ChemiDoc Touch Imaging System (Bio-Rad).

[0104] High-pressure injection of plasmid into mouse tail vein The mouse was placed in a suitable container and placed under an infrared lamp. The lamp was turned on and irradiated for several minutes. The mouse was then removed and its tail was wiped with an alcohol cotton ball to allow the tail vein to fully dilate. The mouse was then placed in a mouse holder and its tail was exposed. A sample was extracted using an appropriate syringe and needle, and injected with the needle of a 1 mL syringe, matching the syringe of a 5 mL syringe. A 2 mL sample (0.1 mL / g × mouse body weight) was extracted and injected via the tail vein. The injection was completed within 5-8 seconds, requiring rapid and uniform injection. After the injection was completed, the injection site was pressed with a dry cotton ball to stop bleeding.

[0105] Inject virus samples into the mouse tail vein The mouse was placed in a suitable container and placed under an infrared lamp. The lamp was turned on and irradiated for several minutes. The mouse was then removed and its tail was wiped with an alcohol cotton ball to fully dilate the tail vein. The mouse was placed in a mouse holder and its tail was exposed. A sample was extracted using an appropriate syringe and needle, matching the needle of a 1 mL syringe to the syringe of a 1 mL syringe. After diluting the virus sample according to the virus dose, 200 μL of the diluted sample was extracted and injected via the tail vein. The injection took more than 10 seconds to complete, and the injection speed should be slow and uniform. After the injection was completed, the injection site was pressed with a dry cotton ball to stop bleeding.

[0106] Quantitative analysis of Phe in blood Blood was collected from the eye margins of the mice, and 20 μL of the blood was dropped onto a blood collection card and allowed to dry naturally. The phenylalanine concentration in the blood was quantified using a phenylalanine measurement kit (FENGHUA, AN302) and calibrated with a standard blood card.

[0107] Testing the Phe-lowering function of PAH in vitro Test cells were cultured in 24-well plates. At the time of testing, the test cells were harvested and rinsed once with PBS. 150 μL of reaction solution (containing 0.25% NP-40, 50 mM Hepes, 150 mM KCl, 800 mM L-Phe, 100 μg / mL catalase, 400 μM FeNH4(SO4)2, 400 μM BH4, and 2 mM DTT) was added to each well and incubated at 37°C for 3 hours. 15 μL of the reaction solution was dropped onto a blood collection card and allowed to air dry. Phenylalanine concentrations in the blood were quantified using a phenylalanine assay kit (FENGHUA, AN302), and the decrease in Phe concentration was calculated.

[0108] 5-HIAA detection After sacrificing the mice, brain tissues were collected and the content of 5-HIAA was detected by mass spectrometry at Suzhou PANOMIX Biomedical Tech. The specific method was to prepare a standard curve solution by diluting with a 5-hydroxyindoleacetic acid standard gradient. The brain tissues of the test samples were homogenized and mixed uniformly at a volume ratio of 1:1:10 (homogenate solution: internal standard working solution: methanol). The mixture was centrifuged at 12,000 rpm and 4°C for 10 minutes to obtain the supernatant. Scan detection was performed using an XDB-C18 analytical 4.6 x 150 mm 5-Micron chromatography column and an electrospray ionization source with multiple reaction monitoring (MRM). The content of 5-HIAA in the test samples was calculated using the standard curve quantification method.

[0109] Quantitative analysis of blood Tyr Blood was collected from the eye margins of mice, and the serum was centrifuged in a 3 kDa ultrafiltration tube and filtered at 12,000 × g for 20 min. The filtrate was collected. A standard curve solution was prepared by gradient dilution of the Tyr standard. Serum samples were centrifuged in a 3 kDa ultrafiltration tube and filtered at 12,000 × g for 20 min. The filtrate was collected and analyzed using a Chromcore 120 C18 chromatography column. The signal at 210 nm was detected, and the Tyr content in the test samples was calculated using the standard curve method.

[0110] Example 1: Construction and isolation / purification of adeno-associated virus vector 1.1 Construction of adeno-associated virus recombinant vectors The structure of a PAH expression cassette is shown in Figure 1. From the 5' to 3' end, the PAH expression cassette contains the 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 (4 CpG) filling sequence, and 3' ITR. The nucleotide sequences of PAH expression cassettes containing different target genes are shown in SEQ ID NOs. 79 to 93.

[0111] The nucleotide sequence of the 5'ITR is shown in SEQ ID NO.1.

[0112] The ApoE HCR enhancer is the hepatocyte regulatory region of apolipoprotein E from humans, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0113] The SerpinA1 promoter is the human α1 antitrypsin promoter, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0114] The Serpin1 intron is a truncated α1 antitrypsin intron, 261 bp in length, and its nucleotide sequence is shown in SEQ ID NO.4.

[0115] The Kozak sequence, before it was inserted into the PAH gene sequence, is shown in SEQ ID NO. 5.

[0116] The hPAH WT gene is derived from the wild-type human PAH gene (Gene ID: 5053), the gene sequence of which is shown in SEQ ID NO. 6, and the NCBI accession number of the encoded wild-type hPAH WT protein is NP_000268.1.

[0117] The optimized PAH genes hPAH opt are codon-optimized genes opt1 to opt15 encoding wild-type human PAH proteins, and their nucleotide sequences are SEQ ID NOs. 9 to 23, respectively.

[0118] BGH polyA is the polyadenylation signal of bovine growth hormone, the nucleotide sequence of which is shown in SEQ ID NO.7.

[0119] The HPRT (4 CpG) filling sequence is a partial intron sequence of hypoxanthine phosphoribosyltransferase, the nucleotide sequence of which is shown in SEQ ID NO.43.

[0120] The nucleotide sequence of the 3'ITR is shown in SEQ ID NO.8.

[0121] An expression cassette containing the wild-type human PAH gene, hPAH WT, was constructed as the shuttle plasmid pAAV8-ATT-PAH-WT-HPRT, where SEQ ID NO. 51 represents the shuttle plasmid sequence containing the expression cassette with the wild-type human PAH gene, hPAH WT.

[0122] An expression cassette containing the optimized PAH genes opt1-15 was constructed as the shuttle plasmid pAAV8-ATT-PAH-opt1-15-HPRT, where SEQ ID NOS. 52-66 represent the shuttle plasmid sequences containing the expression cassettes with the codon-optimized genes opt1-15, respectively.

[0123] 1.2 Isolation and purification of adenoviral vectors AAV vectors were produced using a three-plasmid system: the shuttle plasmids pAAV8-ATT-PAH-WT-HPRT or pAAV8-ATT-PAH-opt1-15-HPRT containing the PAH target gene, the pRepCap plasmid containing the AAV vector repcap gene, and the auxiliary plasmid pHelper. HEK293 cells were co-transfected with PEI as the transfection reagent, and recombinantly packaged AAV viral vectors, designated AAV8-ATT-PAH-WT-HPRT and AAV8-ATT-PAH-opt1-15-HPRT, respectively. The harvested solution was purified by affinity chromatography, further purified by anion chromatography, concentrated by ultrafiltration, and buffer replacement was performed. The purified recombinant AAV viral vectors were used to measure genome titers, and a reserve aliquot was prepared by sterile filtration.

[0124] Example 2: In vitro expression detection of codon-optimized PAH opt In this example, the in vitro expression level of codon-optimized PAH opt was evaluated in the HepG2 cell line (purchased from the Cell Archive of the Committee for Standard Culture Collection, Chinese Academy of Sciences, catalog number: TCHu72). A shuttle plasmid, pAAV8-ATT-PAH WT, containing the wild-type human PAH gene hPAH WT driven by the ATT promoter (nucleotide sequence: SEQ ID NO. 94), and a shuttle plasmid, pAAV8-ATT-PAH opt 1-15, containing the optimized PAH gene hPAH opt 1-15 (nucleotide sequences: SEQ ID NOs. 95-109), were constructed. The target gene was introduced into the cells using the method described in the "In vitro cell plasmid transfer experiment" above.

[0125] HepG2 cells were instantly transfected with the PAH WT-expressing plasmid pAAV8-ATT-PAH WT or the PAH opt-expressing plasmid pAAV8-ATT-PAH opt 1-15, and protein expression in cell lysates was evaluated by Western blot analysis. The results are shown in Figure 2. Here, A shows a representative image of the PAH protein Western blot, and B shows the quantification results. The expression levels of the codon-optimized PAH genes opt2-9 and opt11-15 in HepG2 cells were all significantly higher than that of the wild-type human PAH gene (WT), indicating that the codon-optimized PAH genes of the present disclosure have higher expression levels.

[0126] Example 3: In vivo efficacy evaluation of codon-optimized PAH opt in a PKU mouse model 3.1 Comparison of codon-optimized plasmid sequences for reduced Phe functional activity in PKU mice PKU model mice (purchased from iBio Logistics, product name: BTBR-Pah) were injected using the high-pressure tail vein injection method. <enu2>Mice (J / J) were injected with 40 μg of pAAV8-ATT-PAH WT or pAAV8-ATT-PAH opt2, opt9, or opt14 plasmid. Blood samples were collected at 0 h, 6 h, 24 h, and 3, 5, 10, 16, and 19 days post-injection. Blood Phe levels were measured, and the Phe-lowering effects of the PAH opt plasmids were analyzed. The results are shown in Figures 3A and 3B. As can be seen from Figure 3A, from D3 to D16, blood Phe levels in mice injected with the PAH opt9 plasmid were lower than those in PAH WT, while those injected with the PAH opt2 and PAH opt14 plasmids both had higher Phe levels than those in PAH WT. The Phe-lowering effect gradually weakened with time due to intracellular metabolic degradation of the plasmids. As can be seen from Figure 3B, at 10 days after injection, the blood Phe concentrations of mice injected with the PAH opt9 plasmid were lower than those of PAH WT, PAH opt2, and PAH opt14, indicating that the codon-optimized PAH opt9 had a better Phe-lowering effect.

[0127] 3.2 Comparison of codon-optimized AAV virus sequences to reduce Phe functional activity in PKU mice Using the shuttle plasmid of Example 2, AAV viral vectors were recombinantly packaged in the same manner as in 1.2 of Example 1, and named AAV8-ATT-PAH WT, and AAV8-ATT-PAH opt2, 9, 11, and 14, respectively.

[0128] PKU model mice were injected intravenously with AAV8-ATT-PAH WT or AAV8-ATT-PAH opt2, opt9, opt11, or opt14 viruses at a dose of 1e10vg / mouse. Blood samples were collected weekly for up to 4 weeks after injection, and the Phe levels in the blood were measured. The Phe-lowering effects of the AAV8-ATT-PAH opt viruses were compared and analyzed. The results are shown in Figure 4. As can be seen from Figure 4, the blood Phe levels in the model mice injected with AAV8-ATT-PAH opt2, 9, and 14 viruses were all lower than those in AAV8-ATT-PAH WT, indicating that the codon-optimized AAV8-ATT-PAH opt2, 9, and 14 viruses had better Phe-lowering effects.

[0129] Example 4: Screening of liver-specific promoter combinations in a PKU mouse model This example evaluates the Phe-lowering effects of PAH driven by different promoters in PKU model mice, and the promoter combination elements are as shown in Table 1. The ApoE HCR enhancer is the hepatocyte regulatory region of apolipoprotein E from human origin, and its nucleotide sequence is set forth in SEQ ID NO. 2. The Core ApoE HCR enhancer is the hepatocyte regulatory region of apolipoprotein E from human origin, and its nucleotide sequence is set forth in SEQ ID NO. 37. The CRMSBS2 enhancer is a modified Serpin1 enhancer, and its nucleotide sequence is set forth in SEQ ID NO. 29. The TTRm enhancer is a mutated transthyretin promoter region, and its nucleotide sequence is set forth in SEQ ID NO. 32. The SerpinA1 promoter is the human alpha1 antitrypsin promoter, and its nucleotide sequence is set forth in SEQ ID NO. 3. The core SerpinA1 promoter (218 bp) is the human alpha 1 antitrypsin promoter core region, the nucleotide sequence of which consists of SEQ ID NOs. 24 and 25. The core SerpinA1 promoter (254 bp) is the human alpha 1 antitrypsin promoter core region, the nucleotide sequence of which is set forth in SEQ ID NO. 38. The TTRm promoter (223 bp) is a mutated transthyretin promoter, the nucleotide sequence of which is set forth in SEQ ID NO. 30. The TTRm promoter (228 bp) is a mutated transthyretin promoter, the nucleotide sequence of which is set forth in SEQ ID NO. 33. The truncated SerpinA1 intron (261 bp) is a truncated alpha 1 antitrypsin intron, the nucleotide sequence of which is set forth in SEQ ID NO. 4. The truncated SerpinA1 intron (206 bp) is a truncated alpha 1 antitrypsin intron, the nucleotide sequence of which is set forth in SEQ ID NO. 26.Modified human β-globin 2 intron is a modified human β-globin 2 intron subsequence, the nucleotide sequence of which is set forth in SEQ ID NO. 27. Modified SV40 intron is a monkeypox virus 40 intron subsequence, the nucleotide sequence of which is set forth in SEQ ID NO. 28. SBR intron 3 is a modified mouse parvovirus intron, the nucleotide sequence of which is set forth in SEQ ID NO. 31. MVM intron is a mouse parvovirus intron subsequence, the nucleotide sequence of which is set forth in SEQ ID NO. 34.

[0130] The combined promoters were used to construct expression cassettes, the structure of which is shown in Figure 5. The expression cassettes were constructed as shuttle plasmids, resulting in the 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).

[0131] The plasmid vector of this example was injected into PKU model mice at a rate of 40 μg via high pressure tail vein injection. Three days after injection, blood samples were collected and the Phe content in the blood was measured. The Phe-lowering effects of PAHs driven by different promoters were compared and analyzed. The results are shown in Figure 6, which showed that the expression plasmid driven by the ATT promoter had the most significant Phe-lowering effect in PKU model mice.

[0132] [Table 1]

[0133] Example 5: Effects of other expression regulatory elements on in vivo drug efficacy in PKU mouse models This example evaluated the effect of different expression regulatory elements (U6 promoter (SEQ ID NO. 35), CAG promoter (SEQ ID NO. 50), filling sequence HPRT (47 CpG) (SEQ ID NO. 39), and filling sequence WPRE (SEQ ID NO. 36)) on the Phe-lowering effect of the AAV8-ATT-PAH virus in PKU model mice. The construction of different expression cassettes is shown in Figure 7.

[0134] The expression cassettes shown in Figure 7 were constructed as shuttle plasmids to obtain the plasmid vectors ATT-PAH-opt9-HPRT(47 CpG) (SEQ ID NO. 73), U6-ATT-PAH-opt9-HPRT(47 CpG) (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). 40 μg of the plasmids were injected into PKU model mice via high pressure tail vein injection. Three days after injection, blood samples were collected from the mice to measure the Phe content in the blood. The Phe-lowering effects of the different PAH expression cassettes were compared and analyzed. As shown in Figure 8, the Phe-lowering effect of the vector containing the HPRT filling sequence (ATT-PAH-opt9-HPRT(47CpG)) was better than that of the vector without the HPRT filling sequence (ATT-PAH-opt9).

[0135] Example 6: Effect of optimizing the packing sequence on in vivo drug efficacy in a PKU mouse model Research has shown that innate immune stimulation may be driven by CpG-rich sequences, which may neutralize AAV vector-mediated gene expression in vivo (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). To investigate the influence of CpG number on the Phe-lowering effect of the AAV8-ATT-PAH virus, this example conducted experiments in PKU model mice using a CpG-rich filler sequence (HPRT (47 CpGs)) and a CpG-lowering filler sequence (HPRT (4 CpGs)). The structures of optimized recombinant AAV (rAAV) vector PAH expression cassettes containing different filling sequences are shown in Figure 9, where the HPRT(47 CpG) nucleotide sequence is shown in SEQ ID NO. 39 and the HPRT(4 CpG) nucleotide sequence is shown in SEQ ID NO. 43.

[0136] Expression cassettes containing different filling sequences were each constructed as shuttle plasmids, and the three-plasmid system of Example 1 was used to obtain the recombinant viral vectors AAV8-ATT-PAH-opt9 (where the shuttle plasmid sequence is shown in SEQ ID NO. 76), AAV8-ATT-PAH-opt9-HPRT(47 CpG) (where the shuttle plasmid sequence is shown in SEQ ID NO. 73), AAV8-ATT-PAH-opt9-HPRT(4 CpG) (where the shuttle plasmid sequence is shown in SEQ ID NO. 60), and AAV8-HPRT(4 CpG)-ATT-PAH-opt9 (where the shuttle plasmid sequence is shown in SEQ ID NO. 78).

[0137] Viral vectors were injected intravenously into PKU model mice at a dose of 2e11vg / mouse. Blood samples were taken weekly for up to 8 weeks after injection to measure the Phe content in the blood. The effects of optimizing different loading sequences on the Phe-lowering effect were compared and analyzed. As can be seen in Figure 10A, after PKU model mice were injected with the viruses containing the four expression cassettes, the Phe content in the blood of the mice all reached the optimal therapeutic effect (<120 μM).

[0138] To avoid immune stimulation due to CpG-rich genes, we selected an expression cassette with a low CpG-containing sequence. The therapeutic effect of AAV8-ATT-PAH-opt9-HPRT(4CpG) on PKU model mice was further investigated at a low dose of 1e10vg / mouse. Blood samples were collected weekly for up to four weeks after injection, and the Phe content in the blood was measured. The results are shown in Figure 10B. As can be seen from Figure 10B, at low doses, the Phe-lowering effect of AAV8-ATT-PAH-opt9-HPRT(4CpG) was superior to that of AAV8-ATT-PAH-opt9.

[0139] Example 7: Testing the in vitro PAH Phe-lowering function of the optimized expression cassette AAV8-ATT-PAH-opt9-HPRT(4CpG) viral expression product In this example, the Phe-reducing function of the codon-optimized AAV8-ATT-PAH-opt9-HPRT (4CpG) viral expression product was evaluated in HepG2 cells. First, a plasmid expressing the adeno-associated virus receptor AAVR (the AAVR gene was synthesized by GENEWIZ according to SEQ ID NO. 117 and then inserted between the NheI and XbaI enzyme cleavage sites in the pcDNA3.1(+) plasmid) was rapidly transfected into HepG2 cells. Overexpression of AAVR improved the efficiency of AAV8 infection in HepG2 cells. After 24 hours, the cells were infected with the AAV8-ATT-PAH-opt9-HPRT(4CpG) virus from Example 1 at MOIs of 0, 5e4, 1e5, and 2e5, respectively, and cultured for 48 hours. Phe content was detected, and the Phe concentration change was calculated using the uninfected HepG2 cell line as a reference to evaluate the Phe-lowering activity of PAH. The results are shown in Figure 11, demonstrating that the AAV8-ATT-PAH-opt9-HPRT(4CpG) virus expression product has Phe-lowering function and that the viral infection MOI has a dose effect.

[0140] Example 8: In vivo efficacy of optimized expression cassettes in PKU mouse models: low dose, high activity This example evaluated the effect of different doses of the AAV8-ATT-PAH-opt9-HPRT(4CpG) virus on the Phe-lowering effect in male and female mice of a PKU model.

[0141] Male PKU mice were intravenously injected with the AAV8-ATT-PAH-opt9-HPRT(4CpG) virus of Example 1 at doses of 3.0e11, 1.0e11, 3.3e10, 1.5e10, 1.1e10, and 3.0e9 vg / mouse. Blood samples were collected weekly for up to six weeks after injection, and the Phe levels in the blood were measured. The effects of different viral doses on Phe-lowering efficacy were compared and analyzed. The results are shown in Figure 12A. As can be seen from Figure 12A, at doses of 3.0e11, 1.0e11, and 3.3e10 vg / mouse, blood Phe levels in mice were below 120 μM, achieving therapeutic efficacy, from two to six weeks after viral injection. Therefore, the AAV8-ATT-PAH-opt9-HPRT(4CpG) virus is thought to have a minimum effective dose of approximately 3.3e10vg / mouse for treating PKU in male mice.

[0142] The AAV8-ATT-PAH-opt9-HPRT(4CpG) virus of Example 1 was injected intravenously into female PKU mice at doses of 4.0e11, 2.0e11, 1.0e11, 5.0e10, and 2.5e10 vg / mouse. Blood samples were collected weekly for up to six weeks after injection, and the Phe content in the blood was measured. The effects of different viral doses on Phe-lowering efficacy were compared and analyzed. The results are shown in Figure 12B. As can be seen from Figure 12B, at doses of 4.0e11, 2.0e11, 1.0e11, and 5.0e10 vg / mouse, blood Phe concentrations in mice were below 120 μM, demonstrating therapeutic efficacy, from two to six weeks after viral injection. Therefore, the AAV8-ATT-PAH-opt9-HPRT(4CpG) virus appears to have a minimum effective dose of approximately 5.0e10vg / mouse for treating PKU in female mice.

[0143] Example 9: Further efficacy of optimized expression cassettes in a PKU mouse model In this example, the therapeutic effect of AAV8-ATT-PAH-opt9-HPRT(4CpG) virus on male PKU mice was evaluated at doses of 3.0e10 and 3.0e11 vg / mouse. Six weeks after virus administration, the blood Tyr levels were measured. The results are shown in Figure 13A. Compared to the disease-free PKU heterozygous mice (Jackson Lab), the blood Tyr levels of PKU homozygous mice (i.e., PKU model mice) were significantly lower. After receiving the AAV8-ATT-PAH-opt9-HPRT(4CpG) virus at doses of 3.0e10 and 3.0e11 vg / mouse, the blood Tyr levels of PKU mice returned to levels comparable to those of normal heterozygous mice. Figure 13B shows the levels of 5-hydroxyindoleacetic acid (5-HIAA, a pharmacological marker for PKU patients) in the brain tissue of PKU homozygous mice, with untreated heterozygous mice serving as normal controls. Results indicated that the 5-HIAA levels in the brain tissue of PKU homozygous mice with a diseased phenotype were significantly lower than those of PKU heterozygous mice without a diseased phenotype. After receiving the AAV8-ATT-PAH-opt9-HPRT(4CpG) virus at doses of 3.0e10 and 3.0e11 vg / mouse, the 5-HIAA levels in the brain tissue of PKU mice returned to levels comparable to those of normal heterozygous mice. Furthermore, the treated PKU mice developed a dark coat color. Figure 13C shows the coat color of PKU homozygous mice treated with 3.0e10 vg / mouse for 3 weeks, compared to the vehicle group.

[0144] The present disclosure has achieved effective, sustained, and stable suppression of peripheral blood phenylalanine levels in PKU mice at low doses through optimization and screening of genes and expression regulatory elements, and has also improved other PKU phenotypes. Furthermore, the present disclosure can effectively reduce the dosage and potential side effects of gene therapy drugs for treating PKU, thereby improving therapeutic efficacy.

[0145] The above are merely preferred embodiments of the present disclosure and are not intended to limit the present disclosure. Any amendments, equivalent replacements, improvements, etc. made within the spirit and principles of the present disclosure should be included within the scope of protection of the present disclosure.

Claims

1. 1. A polynucleotide molecule encoding a PAH protein, comprising: The nucleotide sequence has 90% or more identity to the nucleotide sequence set forth 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 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity, and more preferably 98% or 99% or more identity; More preferably, the polynucleotide molecule is a polynucleotide molecule encoding a PAH protein having a nucleotide sequence set forth 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.

2. An expression cassette comprising:

10. A method for producing a gene encoding a gene encoding a gene for a human genomic DNA comprising the steps of: Preferably, the promoter is a specific or non-specific promoter, Preferably, the promoter is a constitutive promoter or an inducible promoter, preferably, the constitutive promoter is selected from CMV promoter, EF1A promoter, EFS promoter, CAG promoter, CBh promoter, SFFV promoter, MSCV promoter, SV40 promoter, mPGK promoter, hPGK promoter and UBC promoter, preferably, the inducible promoter comprises 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 and a light-regulated promoter, an IPTG-inducible promoter, Preferably, the promoter comprises a core promoter, Preferably, the core promoter comprises a liver-specific promoter or an active fragment thereof, and preferably, the liver-specific promoter is an ApoA-I promoter, an ApoA-II promoter, an ApoA-IV promoter, an ApoB promoter, an ApoC-1 promoter, an ApoC-II promoter, an ApoC-III promoter, an ApoE promoter, an albumin promoter, an alpha-fetoprotein promoter, a phosphoenolpyruvate carboxykinase (PCK1) promoter, a phosphoenolpyruvate carboxykinase 2 (PCK2) promoter, a thyroid hormone transport protein (transthyre) promoter, a thyroid hormone transport protein (THM ... Preferably, the liver-specific promoter is selected from the group consisting of human α1-antitrypsin (AAT or Serpin A1) promoter, α-antitrypsin (AAT or Serpin A1) promoter, TK (thymidine kinase) promoter, hemopexin promoter, alcohol dehydrogenase 6 promoter, cholesterol 7α-25 hydroxylase promoter, factor IX promoter, α-microglobulin promoter, SV40 promoter, CMV promoter, Rous sarcoma virus-LTR promoter, HBV promoter, ALB promoter and TBG promoter, more preferably, the liver-specific promoter is human α1-antitrypsin promoter, and preferably, the core promoter is selected from the group consisting of human α1-antitrypsin (AAT or Serpin A1) promoter, α-antitrypsin (TTR ... and a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in SEQ ID NO. 3, 24, 25, 30, 33 or 38, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably 98% or 99% or more identity, and preferably the core promoter has the nucleotide sequence set forth in SEQ ID NO. 3, 24, 25, 30, 33 or 38, more preferably the nucleotide sequence of the core promoter is set forth in SEQ ID NO.

3.

3. further comprising an expression control element operably linked to the polynucleotide molecule; Preferably, the expression control element is at least one selected from 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; Preferably, the expression cassette comprises a 5' ITR, preferably the 5' ITR comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth 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 99% or more identity, more preferably the 5' ITR has the nucleotide sequence set forth in SEQ ID NO. 1; Preferably, the expression cassette comprises a 3' ITR, and preferably the 3' ITR comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in SEQ ID NO. 8, 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 99% or more identity, and more preferably the 3' ITR has the nucleotide sequence set forth in SEQ ID NO. 8; Preferably, the expression cassette further comprises an enhancer, preferably the enhancer is selected from the group consisting of an ApoE HCR enhancer or an active fragment thereof, a CRMSBS2 enhancer or an active fragment thereof, a TTRm enhancer or an active fragment thereof, and a CMV enhancer or an active fragment thereof, more preferably the enhancer comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth 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 99% or more identity, preferably the enhancer has the nucleotide sequence set forth in SEQ ID NO. 2, 29, 32, 37 or 40, more preferably the nucleotide sequence of the enhancer is a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in SEQ ID NO. 2, Preferably, the expression cassette further comprises an intron, preferably the intron is selected from the group consisting of an α1 antitrypsin intron or an active fragment thereof, a β-globin second intron or an active fragment thereof, an SV40 intron or an active fragment thereof, and a mouse parvovirus intron or an active fragment thereof, preferably the intron comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth 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 99% or more identity, preferably the intron has the nucleotide sequence set forth in SEQ ID NO. 4, 26, 27, 28, 31 or 34, more preferably the nucleotide sequence of the intron is a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in SEQ ID NO. 4, Preferably, the promoter of the expression cassette is a combined promoter comprising an upstream regulatory element, a core promoter and an intron, and preferably, the upstream regulatory element is an enhancer or an active fragment thereof; Preferably, the combined promoter comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth 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, more preferably a nucleotide sequence having 98% or 99% or more identity, preferably the combined promoter has the nucleotide sequence set forth in SEQ ID NO. 44, 45, 46, 47, 48 or 49, more preferably the nucleotide sequence of the combined promoter is set forth in SEQ ID NO. 44, Preferably, the expression cassette further comprises a polyA signal, and preferably the polyA signal is at least one of bovine growth hormone polyA (BGH polyA), short polyA, SV40 polyA, and human β-globin polyA. Preferably, the polyA signal comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth 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 99% or more identity, and more preferably the nucleotide sequence of the polyA signal is set forth in SEQ ID NO. 7, Preferably, the expression cassette comprises an optimized filler sequence, preferably the filler sequence is selected from the partial intron sequence of hypoxanthine phosphoribosyltransferase (HPRT) and the woodchuck hepatitis virus post-transcriptional 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 or the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) does not contain any CpG sequences, preferably the filler sequence is the partial intron sequence of hypoxanthine phosphoribosyltransferase (HPRT), preferably the filler sequence is set forth in SEQ ID NO. 39 or SEQ ID NO. The nucleotide sequence of the filler sequence may be a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in SEQ ID NO. 39 or SEQ ID NO. 43, 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 99% or more identity, more preferably the nucleotide sequence of the filler sequence is set forth in SEQ ID NO. 39 or SEQ ID NO. 43, 3. The expression cassette of claim 2, wherein the Kozak initiation sequence comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in SEQ ID NO. 5, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably 98% or 99% or more identity, and more preferably the Kozak initiation sequence has the nucleotide sequence set forth in SEQ ID NO.

5.

4. The expression cassette comprises a 5' ITR, an ApoE HCR enhancer, a human alpha 1 antitrypsin promoter, a truncated alpha 1 antitrypsin intron, a Kozak initiation sequence, the polynucleotide molecule, BGH poly A, a partial intron sequence of hypoxanthine phosphoribosyltransferase (HPRT), and a 3' ITR, and preferably comprises the sequences of 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. The expression cassette preferably comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in 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. 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. 93, or SEQ ID NO.

94.

4. The expression cassette of claim 2 or 3, having the nucleotide sequence shown in SEQ ID NO. 92 or SEQ ID NO.

93.

5. An expression vector comprising: The present invention relates to a method for producing a recombinant vector comprising the polynucleotide molecule of claim 1 or the expression cassette of any one of claims 2 to 4, wherein the expression vector further comprises a gene encoding a marker, and the marker is preferably at least one selected from the group consisting of an antibiotic resistance protein, a toxin resistance protein, a colored, fluorescent or luminescent protein and a protein mediating enhanced cell growth and / or gene amplification; Preferably, the antibiotic is at least one selected from ampicillin, neomycin, G418, puromycin, and blasticidin; Preferably, the toxin is at least one selected from anthrax toxin and diphtheria toxin; Preferably, the colored, fluorescent, or luminescent protein is at least one selected from green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, and luciferase; Preferably, the protein that mediates enhanced cell growth and / or gene amplification is dihydrofolate reductase (DHFR); Preferably, the expression vector comprises an origin of replication, and preferably, the sequence of the origin of replication is at least one selected from the group consisting of f1 bacteriophage ori, RK2 oriV, pUC ori, and pSC101 ori.

6. selected from a plasmid, a cosmid, a viral vector, an RNA vector or a linear or circular DNA or RNA molecule, Preferably, the plasmid is selected from pCI, puc57, pcDNA3, pSG5, pJ603 or pCMV; Preferably, the viral vector is selected from retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-stranded RNA viruses, such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles viruses and Sendai viruses), positive-stranded RNA viruses (e.g., picornaviruses and alphaviruses), or double-stranded DNA viruses, and the double-stranded DNA viruses are selected from adenoviruses, herpes viruses (e.g., herpes simplex viruses types 1 and 2, Epstein-Barr virus, cytomegalovirus), pox viruses (e.g., cowpox virus, fowlpox virus, and canarypox virus), noroviruses, togaviruses, flaviviruses, reoviruses, polyomaviruses, hepatotropic DNA viruses, baculoviruses, or hepatitis viruses; Preferably, the retrovirus is selected from avian leukocytoproliferative sarcoma, mammalian C virus, mammalian B virus, mammalian D virus, HTLV-BLV aggregate, rentovirus or foam virus; 6. The expression vector of claim 5, wherein the lentovirus vector is selected from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or ovine demyelinating encephalitis lentovirus.

7. an adeno-associated virus vector, Preferably, 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; Preferably, the expression vector is selected from the group consisting of 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 expression vector comprises a nucleotide sequence having 90% or more identity to the nucleotide sequence set forth in 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. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, 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. 79, 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. 88, SEQ ID NO. 89, SEQ ID NO. 90, SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97, SEQ ID NO. 98, SEQ ID NO. 99, SEQ ID NO. 99, SEQ ID NO. 100, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103, SEQ ID NO. 104, SEQ ID NO. 105, 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 ID7. The expression vector of claim 5 or 6, wherein the expression vector has the nucleotide sequence set forth in SEQ ID NO. 106, SEQ ID NO. 107, SEQ ID NO. 108 or SEQ ID NO. 109, more preferably the expression vector has the nucleotide sequence set forth in SEQ ID NO. 76, SEQ ID NO. 73, SEQ ID NO. 60 or SEQ ID NO. 78, more preferably the expression vector has the nucleotide sequence set forth in SEQ ID NO.

73.

8. A virus particle, A viral particle comprising at least one of the polynucleotide molecule according to claim 1, the expression cassette according to any one of claims 2 to 4, and the expression vector according to any one of claims 5 to 7.

9. A pharmaceutical composition for treating phenylketonuria, comprising:

10. A pharmaceutical composition for treating phenylketonuria, comprising at least one of the polynucleotide molecule of claim 1, the expression cassette of any one of claims 2 to 4, the expression vector of any one of claims 5 to 7, and the viral particle of claim 8, and optionally further comprising a pharmaceutically acceptable carrier, wherein the pharmaceutical composition expresses a wild-type or codon-optimized PAH protein.

10. Use of at least one of the polynucleotide molecule of claim 1, the expression cassette of any one of claims 2 to 4, the expression vector of any one of claims 5 to 7, the viral particle of claim 8, and the pharmaceutical composition of claim 9 in the preparation of a drug for treating phenylketonuria.

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