Recombinant AAV vector for the treatment of type I glutaric aciduria

Abstract

Provided are codon-optimized sequences encoding hGCDH polypeptides and recombinant adeno-associated viral (rAAV) vectors comprising one of the sequences under the control of a promoter component. Also provided herein are viral particles comprising the rAAV vectors, pharmaceutical compositions comprising the rAAV vectors or the viral particles, and uses thereof in treating glutaric aciduria type I (GA-I).

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of PCT International Application No. PCT / CN2022 / 093084, filed on May 16, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure relates to the technical field of gene therapy. In particular, this disclosure provides a recombinant adeno-associated virus (rAAV) vector containing a nucleotide sequence encoding human glutaryl-CoA dehydrogenase (GCDH), said recombinant adeno-associated virus vector for treating disorders or conditions caused by glutaryl-CoA dehydrogenase (GCDH) deficiency, especially type I glutarateuria (GA-I).

[0004] sequence list

[0005] This disclosure includes a sequence list that is part of this disclosure. Background Technology

[0006] First described in 1974, glutarateuria is a hereditary neurometabolic disorder[1]. Patients suffer from neurodegenerative disorders as glutarate (GA) levels rise in plasma, urine, and cerebrospinal fluid (CSF)[1,2]. Type I glutarateuria (GA-I) is an autosomal recessive metabolic disorder caused by a deficiency of glutaryl-CoA dehydrogenase (GCDH; EC 1.3.99.7)[3]. GCDH, located in the mitochondria, is a key enzyme in the metabolism of L-lysine, L-hydroxylysine, and L-tryptophan. Glutamate-CoA is an intermediate in this pathway and is dehydrogenated and decarboxylated by GCDH to crotonyl-CoA. When GCDH is deficient, glutaryl-CoA cannot be properly catalyzed and the byproduct GA is produced. The accumulation of GA in the brain leads to neurotoxicity and neurodegenerative disorders[4]. Patients will develop malformed macrocephaly, hypotonia, and acute encephalopathy crisis. Without proper treatment, the life expectancy of patients with GA-I may be only 2-3 years. In mammals, GA can bind with carnitine and form glutarylcarnitine (C5DC), which can eliminate and detoxify GA to some extent. In clinical practice, carnitine supplementation is a widely used treatment for patients with GA-I. Carnitine supplementation can not only reduce GA levels, but also prevent secondary carnitine deficiency[5].

[0007] The global prevalence of GA-I is estimated at 1 in 100,000. Approximately 75,000 patients have this inherited metabolic disorder. Current treatments include dietary control and carnitine supplementation. However, dietary control involving protein restriction in daily life can be difficult to maintain, and carnitine supplementation does not eliminate 3-hydroxyglutaric acid (3-OH-GA) and pentenoic acid[6].

[0008] There remains an unmet need for effective treatment of disorders caused by GCDH deficiency, especially curative treatment of GA-I. Summary of the Invention

[0009] Gene therapy has proven effective in treating inherited metabolic disorders, as demonstrated in both animal models and clinical trials. Adeno-associated virus (AAV)-based gene substitution strategies have also proven effective in a variety of recessive genetic disorders.

[0010] GA-1 is a neurometabolic disorder, and GCDH deficiency primarily causes damage to the central nervous system (CNS). There is evidence that the blood-brain barrier has low permeability to dicarboxylic acids, and GCDH deficiency in CNS cells leads to in situ GA accumulation, thus resulting in neurotoxicity [7]. Patients with GA-1 can benefit from direct delivery of rAAV carrying a GCDH expression cassette to the CNS, thereby normalizing CNS amino acid metabolism and reducing CNS GA accumulation. - / - The life expectancy of the mice was similar to that of wild-type C57BL / 6 mice [8]. To simulate an acute encephalopathy crisis, GCDH knockout mice were challenged with a high-protein diet [9]. High-protein diet (HPD) challenge was performed on 4-week-old GCDH mice over 2–3 days. - / - In mice, it is fatal. Under HPD stimulation, Gcdh... - / - Mice developed GA accumulation, angioedema, neuronal loss, paralysis, and epilepsy [9]. Therefore, exposure to high-protein Gcdh... - / - Mice may be a useful model for human GA-1 (including developmental-dependent striatal vulnerability)[9].

[0011] The inventors have developed, for the first time, an rAAV vector containing an optimized GCDH coding sequence under the control of a specially designed promoter, and verified its performance under HPD excitation. - / - The invention was completed by demonstrating its effect in alleviating symptoms caused by GA accumulation in a mouse model.

[0012] Therefore, in a first aspect, this application provides an isolated nucleic acid molecule comprising a nucleotide sequence selected from a set of nucleotide sequences consisting of SEQ ID NO:11-18 (coding sequences C1-C8), wherein the nucleotide sequence encodes a human GCDH polypeptide having the amino acid sequence shown in SEQ ID NO:33. In a specific embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence shown in SEQ ID NO:12 (coding sequence C2). Compared to the wild-type coding sequence of hGCDH shown in SEQ ID NO:10, the coding sequence of this application has a reduced number of CpGs.

[0013] In a second aspect, this application provides a promoter component having a nucleotide sequence selected from a set of nucleotide sequences comprising SEQ ID NO:2-9 (promoter components P1-P8). In a preferred embodiment, the promoter component has a nucleotide sequence as shown in SEQ ID NO:5 or SEQ ID NO:9 (P4 or P8).

[0014] In a third aspect, this application provides an expression cassette comprising a coding sequence of a human GCDH polypeptide having the amino acid sequence shown in SEQ ID NO:33, operably linked to a promoter component, wherein the coding sequence has a nucleotide sequence selected from a set of nucleotide sequences consisting of SEQ ID NO:11-18, and the promoter component has a nucleotide sequence selected from a set of nucleotide sequences consisting of SEQ ID NO:2-9. In a preferred embodiment, the expression cassette comprises a coding sequence having the nucleotide sequence shown in SEQ ID NO:12, under the control of a promoter component having the nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:9. In a more specific embodiment, the expression cassette comprises a nucleotide sequence shown in SEQ ID NO:20 or SEQ ID NO:21 (V2 or V3).

[0015] In a fourth aspect, this application provides an rAAV vector comprising the isolated nucleic acid molecule of the first aspect or the expression cassette of the third aspect. In a preferred embodiment, the rAAV vector provides a desired expression level of human GCDH protein in a target tissue (e.g., disease-related tissue in the CNS).

[0016] In a fifth aspect, this application provides an AAV viral particle comprising an rAAV vector packaged into an AAV capsid. The AAV capsid can be derived from any AAV serotype, such as AAV1, AAV2, AAV3B, AAV5, AAV6, AAV7, AAV8, AAV9, AAVLK03, AAVS3, AAVKP1, AAVrh10, AAVNP40, AAVNP59, AAV-DJ, AAVANc80L65, AAVsL65, AAVHSC15, AAVC102, AAV204, and AAV214. In one embodiment, the AAV capsid is a CNS-tropy capsid (such as the AAV9 or AAV PHP.B capsid).

[0017] In a sixth aspect, this application provides a pharmaceutical composition comprising the rAAV carrier of the fourth aspect or the viral particles of the fifth aspect, and a pharmaceutically acceptable excipient.

[0018] In a seventh aspect, this application provides a method for treating a subject in need of GA-I, the method comprising administering to the subject a therapeutically effective amount of the rAAV carrier of the fourth aspect, the rAAV particles of the fifth aspect, or the pharmaceutical composition of the sixth aspect.

[0019] In an eighth aspect, this application provides the use of the rAAV carrier of the fourth aspect, the rAAV particles of the fifth aspect, or the pharmaceutical composition of the sixth aspect in the treatment of GA-I patients. Attached Figure Description

[0020] Figure 1 A diagram of the GCDH expression vector in Example 1 is shown.

[0021] Figure 2 The expression of GCDH protein at P0-P8 in U87 MG cells is compared.

[0022] Figure 3 The expression of GCDH protein at P0-P8 in HEK293 cells is shown as a comparison.

[0023] Figure 4 A diagram of the GCDH CDS evaluation carrier in Example 2 is shown.

[0024] Figure 5 The expression of GCDH protein in C0-C8 cells is evaluated in U87 MG cells.

[0025] Figure 6 The expression of GCDH protein in V1-V3 of U87 MG cells is evaluated.

[0026] Figure 7 The GCDH enzyme activity of V1-V3 in SH-sy5y cells is evaluated.

[0027] Figure 8 This demonstrates that Gcdh under high-protein diet stimulation following administration of rAAV9-V1. - / - Survival curves of mice.

[0028] Figure 9 This demonstrates that Gcdh under high-protein diet stimulation following rAAV9-V2 administration... - / - Survival curves of mice.

[0029] Figure 10 It is shown in Gcdh - / - Evaluation of GCDH protein expression in rAAV9-V1 and rAAV9-V2 in mice.

[0030] Figure 11 The study showed the survival and death of Gcdh after administration of rAAV9-V2. - / - LC-MS analysis of GA levels in the brain, liver, and plasma of mice.

[0031] Figure 12 The results of LC-MS / MS analysis of GA and 3-OHGA levels in different tissues (cerebrospinal fluid (GA only), brain, liver, serum, and urine) are shown 4 weeks after AAV administration.

[0032] Figure 13 The results of LC-MS / MS analysis of GA and 3-OHGA levels in different tissues (cerebrospinal fluid (GA only), brain, liver, serum, and urine) are shown at 13 weeks after AAV administration.

[0033] Figure 14 The results showed that when a high dose (1.0 x 10) was administered... 10 GCDH protein levels in different tissues of mice 4 and 13 weeks after rAAV9-V2 administration (vg).

[0034] Figure 15 HE staining of mouse brains is shown 13 weeks after administration of AAV or the medium (200X). Detailed Implementation

[0035] Unless otherwise expressly defined elsewhere in this document, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0036] As used herein (including the appended claims), unless the context clearly indicates otherwise, singular terms such as “a”, “an”, and “the” include their corresponding plural indicators.

[0037] In the context of this disclosure, unless otherwise stated, the word “comprise” and its variations such as “comprises” and “comprising” will be understood to imply the inclusion of the stated elements (e.g., amino acid sequences, nucleotide sequences, properties, steps, or groups thereof), but not to exclude any other elements (e.g., amino acid sequences, nucleotide sequences, properties, and steps). When used herein, the term “comprise” or any variation thereof may be replaced by the terms “contain,” “include,” or sometimes “have,” or equivalent variations thereof. In some embodiments, the word “comprise” also includes the case of “consisting of.”

[0038] The encoding sequence of hGCDH

[0039] As an essential part of the expression cassette, this disclosure first provides a set of codon-optimized nucleotide sequences that encode an hGCDH polypeptide having the amino acid sequence shown in SEQ ID NO:33.

[0040] "Isolated nucleic acid" means DNA or RNA removed from all or part of a polynucleotide, wherein the isolated polynucleotide is found in nature or is associated with a polynucleotide not found in nature. Isolated nucleic acid molecules "containing" a specific nucleotide sequence may, in addition to the specified sequence, contain operatively linked regulatory sequences that control the expression of the coding regions of the enumerated nucleic acid sequences. Due to codon degeneracy, those skilled in the art will understand that any particular amino acid sequence can be encoded by several different nucleotide sequences.

[0041] In this article, "codon-optimized coding sequences" refer to nucleotide sequences encoding hGCDH proteins that have been modified from wild-type coding sequences adapted to codon bias. Optimization can be achieved by reducing sequence complexity, regulating GC content, modulating codon use, and / or avoiding rarely used codons. Codon-optimized coding sequences typically show increased translation efficiency of the target gene (GOI), leading to higher protein expression.

[0042] Compared to the wild-type coding sequence of hGCDH shown in SEQ ID NO:10, the codon-optimized coding sequence of hGCDH in this application has a reduced number of CpGs. "CpG content" or "CpG number" refers to the content or number of phosphate-linked cytosine (C)guanine (G) dinucleotides in a DNA sequence. A "CpG island" is a genomic region where CpG dinucleotides appear at a higher frequency. For example, the algorithm described by Gardiner-Garden and Frommer (1987) can be used to determine the presence of CpG islands. Specifically, a region containing at least 200 bp with a GC ratio exceeding 50% and an observed / predicted CpG ratio higher than 0.6 is called a "CpG island." The predicted CpG value can be calculated by multiplying the number of Cs in the observed window by the number of Gs in the window and then dividing by the window length. In mammals, unmethylated CpGs from exogenous genes are recognized by TLR9, leading to CD8... + T cell activation to eliminate infected cells is detrimental to the long-term expression of exogenous genes. Therefore, to more effectively express the GCDH-encoding gene, it is preferable to reduce the number of CpGs in the hGCDH coding sequence. The coding sequence of the present invention preferably has a low CpG content. When CpG content is a factor to be considered during codon optimization, it further increases the complexity of sequence design and validation.

[0043] The wild-type coding sequence of hGCDH shown in SEQ ID NO:10 has a CpG number of 73. Preferably, the CpG number of the hGCDH coding sequence of this application is less than 73. For example, the CpG number of the hGCDH coding sequence of this application does not exceed 65, 55, 50, 45, 40, 35, 30, 25, or 20. For example, the CpG number of the hGCDH coding sequence of this application is at least 10% less than the CpG number of the wild-type coding sequence of hGCDH (e.g., the nucleotide sequence shown in SEQ ID NO:10), and preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less than the CpG number of the wild-type coding sequence of hGCDH (e.g., the nucleotide sequence shown in SEQ ID NO:10). In a specific embodiment of this application, the number of CpGs in the hGCDH coding sequence of the nucleotide sequence having SEQ ID NO:12 (coding sequence C2) is as low as 16.

[0044] In a preferred embodiment, the codon-optimized coding sequence of the human GCDH protein comprises or consists of nucleotide sequences selected from or composed of SEQ ID NO:11-18. In a particularly preferred embodiment, the codon-optimized coding sequence of the human GCDH protein comprises or consists of nucleotide sequences as shown in SEQ ID NO:12.

[0045] Regulation sequence

[0046] In addition to the coding sequence, the expression cassette may contain one or more regulatory sequences. The regulatory sequences may be selected from one or more of promoters, enhancers, polyadenylation sequences, and translation termination signals. Certain combinations of the regulatory sequences in this disclosure can achieve unexpected effects in improving the expression efficiency of the coding sequence.

[0047] In one respect, this application provides a series of novel promoter components. A “promoter component” refers to a sequence component located upstream of the 5' of a coding sequence and consisting of a promoter and optionally one or more additional elements (such as enhancers and / or intron-derived segments), wherein the enhancer is typically located upstream of the promoter and the intron-derived segment is typically located downstream of the promoter.

[0048] The term "promoter" refers to a DNA sequence that can initiate the transcription of a downstream gene under the control of the promoter. Promoters include, but are not limited to, constitutive promoters, cell type-specific promoters, tissue-specific promoters, and developmental stage-specific promoters. Promoters can be naturally occurring promoters of genes, modified forms of naturally occurring promoters, or synthetic promoters.

[0049] In a preferred embodiment, the promoter of this disclosure may be a constitutive promoter. For example, the promoter may be a cytomegalovirus (CMV) promoter, a chicken β-actin (CBA) promoter, or a human elongation factor-1α (EF-1α) promoter. In one embodiment, the promoter is a CMV promoter having the nucleotide sequence shown in SEQ ID NO:25. In one embodiment, the promoter is a CBA promoter having the nucleotide sequence shown in SEQ ID NO:26. In one embodiment, the promoter is an EF-1α core promoter having the nucleotide sequence shown in SEQ ID NO:29.

[0050] An "enhancer" is a regulatory DNA sequence that, together with a promoter, can enhance the transcription of GOI in AAV. In some embodiments, the promoter component of this application contains an enhancer. More preferably, the enhancer may be a CMV enhancer. For example, a CMV enhancer may have a nucleotide sequence as shown in SEQ ID NO:24.

[0051] "Intron-derived fragments" are sequences derived from introns of genes. It has been reported that gene transcription can be enhanced by splicing-capable introns. In a preferred embodiment, the promoter component or expression cassette of this application contains intron-derived fragments.

[0052] In some embodiments, the intron-derived fragment is derived from the intron of SV40, for example, an intron-derived fragment having a nucleotide sequence as shown in SEQ ID NO:28.

[0053] In some embodiments, the intron-derived fragment is derived from any intron of human GCDH. For example, the intron sequence consists of one or more fragments derived from one or more intronic regions of the human GCDH gene. In a preferred embodiment, the promoter component or expression cassette of this application comprises an intron-derived fragment having a nucleotide sequence as shown in SEQ ID NO:27, 30, or 31 (hGCDH intron 1, hGCDH intron 2, or hGCDH intron 3, respectively). For example, the intron-derived fragment can be a combination of any two or three of hGCDH intron 1 (SEQ ID NO:27), hGCDH intron 2 (SEQ ID NO:30), and hGCDH intron 3 (SEQ ID NO:31).

[0054] In some embodiments, the intron-derived fragments are heterozygous introns. A "heterozygous intron" is an intron fragment containing at least two sequences from different origins or from the same origin but not contiguous in their natural state. For example, the promoter component or expression cassette of this application contains heterozygous introns as intron-derived fragments, wherein the heterozygous intron contains two intron-derived fragments derived from chicken β-actin (CBA) and mouse parvovirus (MMV) introns, as shown in SEQ ID NO:32.

[0055] Preferably, the total length of the intron-derived fragment is about or less than 200 bp, about or less than 250 bp, about or less than 300 bp, about or less than 350 bp, or about or less than 400 bp.

[0056] In some embodiments, the promoter component of this application comprises a CMV enhancer, a CBA promoter, and optionally an intron-derived fragment. In this case, the intron-derived fragment is preferably an intron derived from the hGCDH gene or SV40 (e.g., SEQ ID NO: 28). In specific embodiments, the promoter component comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NO: 2-4.

[0057] In some embodiments, the promoter component of this application comprises an EF-1α promoter (e.g., an EF-1α core promoter (e.g., SEQ ID NO: 29)) and an intron-derived fragment. In this case, the intron-derived fragment is preferably an intron or a heterozygous intron derived from the hGCDH gene (e.g., SEQ ID NO: 32). In specific embodiments, the promoter component comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NO: 5-9. In a more preferred embodiment, the promoter component comprises or consists of a nucleotide sequence as shown in SEQ ID NO: 5 or 9.

[0058] In a preferred embodiment, due to the limited packaging capacity of AAV, the length of the promoter component does not exceed 1,000 bp, 900 bp, 850 bp, 800 bp, 700 bp, 600 bp, 500 bp, or 400 bp.

[0059] Expression Box

[0060] The term "expression cassette" as used herein refers to a DNA component contained in a vector (e.g., an rAAV vector) and consisting of a gene (e.g., the human GCDH gene) to be expressed in a host cell transfected with the vector and one or more regulatory sequences.

[0061] By optimizing the cDNA sequence (codons) and regulatory sequences of the human GCDH gene, particularly the promoter component, the hGCDH expression cassette inserted into the AAV vector can provide the desired expression level and reduced immunogenicity after rAAV is delivered to the subject.

[0062] In one specific embodiment, the expression cassette comprises any coding sequence having a nucleotide sequence as shown in any one of SEQ ID NO:11-18, preferably as shown in SEQ ID NO:12, operably linked to a promoter component having a nucleotide sequence as shown in SEQ ID NO:2-9, preferably as shown in SEQ ID NO:5 or 9. "operably linked" means that the promoter component is in a functionally appropriate position and / or orientation relative to the coding sequence to control transcription of the coding sequence.

[0063] In a specific implementation, the expression cassette comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NO:19-21, preferably as shown in SEQ ID NO:20 or SEQ ID NO:21.

[0064] Recombinant AAV vector and viral particles

[0065] The nucleic acid molecules or expression cassettes of this disclosure can be constructed into recombinant AAV (rAAV) vectors to obtain rAAV particles for delivery to subjects in need.

[0066] In addition to the inserted nucleotide sequence as described above, the rAAV vector is self-complementary. The rAAV vector consists of two inverted terminal repeat (ITR) sequences at both ends of the inserted nucleotide sequence. The ITRs in this disclosure can be ITRs derived from any AAV serotype. When referring to an AAV ITR serotype, the phrase "derived from" means that the ITR can be an ITR of a certain serotype or a variant derived therefrom with one or more modifications. In a preferred embodiment of this disclosure, the rAAV vector comprises two ITRs derived from AAV2. For example, the rAAV vector comprises two AAV2 ITRs, or comprises a wild-type AAV2 ITR and a truncated form of the AAV2 ITR lacking the C or C' region. For example, the wild-type AAV2 ITR is located at the 5' end of the inserted nucleotide sequence, while the AAV2 ITR variant is located at the 3' end of the inserted nucleotide sequence; or vice versa. In one embodiment, the ITR contained in the rAAV of this application has the nucleotide sequence shown in SEQ ID NO:22 (5'ITR) and SEQ ID NO:23 (3'ITR).

[0067] The rAAV genome is packaged into an AAV capsid. The capsid can be derived from any AAV serotype known in the art or to be characterized in the future. The capsid and ITR can be derived from the same AAV serotype or different AAV serotypes. For example, the capsid can be suitable for intravenous (IV) delivery (e.g., IV injection) to peripheral tissues. In the context of this application, the term "peripheral tissues" refers to any tissue that is not part of the brain or spinal cord. For example, the capsid can be suitable for delivery through the nervous system (e.g., intrathecal, intracerebellomedullary cistern, or intraventricular delivery (e.g., by injection)). In some embodiments, the AAV vector comprises a capsid of serotypes AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV PHP.B, AAV2.7m8, or AAVAnc80L65, or a variant thereof. In a preferred embodiment, the capsid is an AAV9 capsid.

[0068] Pharmaceutical Composition

[0069] The term "pharmaceutical composition" refers to a composition suitable for delivery to a subject. The pharmaceutical compositions of this disclosure comprise isolated nucleic acids, rAAV vectors, or viral particles of this disclosure and pharmaceutically acceptable excipients. Conventional pharmaceutically acceptable excipients are known in the art and can be solid or liquid excipients. In one embodiment, the pharmaceutical composition may be a liquid for injection.

[0070] deliver

[0071] When applied to a subject (e.g., an animal, including a human) or to cells, tissues, organs, or biological fluids, the terms “administration,” “administering,” “treating,” and “treatment” as used herein mean contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with a subject, cells, tissues, organs, or biological fluid. Cellular treatment encompasses contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid contacts the cells. The terms “administration” and “treatment” also include in vitro and ex vivo treatment of, for example, cells by means of a reagent, diagnostic agent, conjugated compound, or another cell.

[0072] In some embodiments, the rAAV carrier of this application can be administered to a subject via systemic or local delivery. In some embodiments, the rAAV carrier of this application can be delivered to peripheral tissues or organs (e.g., to peripheral blood) via any parenteral or enteral route, rather than to the nervous system. For example, the rAAV carrier of this application can be administered via intravenous (IV), intramuscular (IM), subcutaneous (SC), intraarterial, intraperitoneal (IP), intradermal, transdermal, oral, nasal, or rectal routes. In some embodiments, the rAAV carrier of this application can be delivered to the nervous system, for example, to cerebrospinal fluid (CSF). For example, the rAAV carrier of this application can be administered via intraventricular (ICV), intrathecal, or intracerebellomedullary cistern (ICM) routes. For example, rAAV can be delivered by injection. In some embodiments, the rAAV carrier can be delivered via a combination of administrations via more than one delivery route. For example, the rAAV carrier can be delivered sequentially or simultaneously to both peripheral tissues and the nervous system.

[0073] The rAAV vector can be administered via single or multiple doses. In a specific implementation, the rAAV vector is administered via a single injection.

[0074] Therapeutic uses

[0075] The terms “treat,” “treating,” or “treatment” include curing or at least alleviating the symptoms of a disorder or condition caused by GCDH deficiency (such as the symptoms of GA-I).

[0076] Example

[0077] To facilitate understanding and utilization of the invention, its advantages will be described in more detail with reference to embodiments and accompanying drawings. However, it should be understood that the following embodiments are intended to illustrate the invention only and are not intended to limit the scope of the invention. The scope of the invention should be defined by the claims.

[0078] Example 1. Constitutive promoter with minimum CpG number for GCDH protein expression

[0079] A GCDH expression AAV vector V1 was constructed, consisting of an ITR, a CMV enhancer / promoter (P0, SEQ ID NO:1), wild-type GCDH CDS, and SV40 polyA. However, due to the silencing risk of the CMV enhancer / promoter, it was replaced with artificially synthesized promoter components P1-P8 (SEQ ID NO:2-9, respectively) to achieve more robust GCDH expression. In addition to the constitutive promoter, each tested promoter component (P1-P8) also included an enhancer and / or intron. The specific structures of promoter components P1-P8 are provided in Table 1. Illustrations of the vector structures are shown in... Figure 1 As shown in the image.

[0080] Table 1. Information on promoter components P0-P8

[0081] serial number P0 CMV enhancer (SEQ ID NO:24) - CMV promoter (SEQ ID NO:25) P1 CMV enhancer-chicken β-actin promoter (SEQ ID NO:26) P2 CMV enhancer-chicken β-actin promoter-hGCDH intron 1 (SEQ ID NO:27) P3 CMV enhancer-chicken β-actin promoter-SV40 intron (SEQ ID NO:28) P4 EF-1α core promoter (SEQ ID NO:29)-hGCDH intron 1 P5 EF-1α core promoter - hGCDH intron 2 (SEQ ID NO:30) P6 EF-1α core promoter - hGCDH intron 3 (SEQ ID NO:31) P7 EF-1α core promoter - hGCDH intron 1 - hGCDH intron 2 P8 EF-1α core promoter-hybrid intron (SEQ ID NO:32)

[0082] U87 MG or HEK293 cells were maintained in DMEM + 10% FBS and passaged every 3 days via TrypLE. One day before transfection, cells were cultured at 1 × 10⁻⁶ cells / day. 5 / cm 2The cells were seeded into 24-well plates. Following the user guide, plasmids were transfected into U87 MG or HEK293 cells using Lipofectamine 3000 transfection reagent (Invitrogen, L3000008). 72 hours post-transfection, cells were collected in RIPA lysis buffer (Beyotime, P0013C) containing a protease inhibitor mixture (Roche, 04693159001) and SDS-PAGE loading buffer (Cowin Bio, CW0027), denatured at 95°C for 10 min, and centrifuged at 12,000 rpm for 10 min. The supernatant was separated on a 4%–10% SDS-PAGE gel (Cowin Bio, CW0022M) and blotted onto a 0.2 μm PVDF transfer membrane (Merck, ISEQ00010). Protein levels of GCDH (Abcam, ab232774) and the housekeeping gene GAPDH (Cell Signaling Technology, 2118S) were detected using antibodies targeting GCDH (Abcam, ab232774) and GAPDH (Cell Signaling Technology, 2118S), respectively. Western blot images were obtained in... Figure 2 and Figure 3 As shown in the image.

[0083] like Figure 2 and Figure 3 The results show that P4 and P8 mediated the highest GCDH protein expression in both U87 MG and HEK293 cell lines.

[0084] Example 2. Codon optimization to minimize immunogenicity risk and enhance expression

[0085] It has been reported that CpGs in AAV vectors induce immune responses and silencing of exogenous genes [10,11]. In this embodiment, the coding sequence of GCDH was optimized to enhance expression and reduce the number of CpGs, and the expression efficiency of the codon-optimized sequence was tested using the CMV promoter. The vector structure is as follows: Figure 4 As shown in Table 2, a total of eight different optimized coding sequences, namely C1-C8, were synthesized, having nucleotide sequences as shown in SEQ ID NO:11-18. The CpG numbers of the modified coding sequences C1-C8 together with the wild-type coding sequence C0 are summarized in Table 2.

[0086] Table 2. Number of CpGs in codon-optimized sequences C0-C8

[0087] serial number CpG number serial number CpG number serial number CpG number C0 73 C3 30 C6 46 C1 51 C4 70 C7 33 C2 16 C5 65 C8 46

[0088] To evaluate the expression of the codon-optimized sequences C1-C8, U87 MG cells were maintained in DMEM + 10% FBS and passaged every 3 days via TrypLE. One day before transfection, cells were cultured at 1 × 10⁻⁶ cells / day. 5 pcs / cm 2 Cells were seeded into 24-well plates. Following the user guide, plasmids were transfected into U87 MG cells using Lipofectamine 3000 transfection reagent (Invitrogen, L3000008). 72 hours post-transfection, cells were collected in RIPA lysis buffer (Beyotime, P0013C) containing a protease inhibitor mixture (Roche, 04693159001) and SDS-PAGE loading buffer (Cowin Bio, CW0027), denatured at 95°C for 10 min, and centrifuged at 12,000 rpm for 10 min. The supernatant was separated on a 4%–10% SDS-PAGE gel (Cowin Bio, CW0022M) and blotted onto a 0.2 μm PVDF transfer membrane (Merck, ISEQ00010). Protein levels of GCDH and the housekeeping gene GAPDH were detected using antibodies targeting GCDH (Abcam, ab232774) and β-tubulin (Proteintech, 66240-1), respectively. Western blot images were obtained in... Figure 5 As shown in the image.

[0089] like Figure 5 The results show that the codon-optimized sequence C2 mediates the highest GCDH protein expression in U87 MG cells.

[0090] Example 3. GCDH construct containing optimized promoter components and coding sequences

[0091] In Example 2, the codon-optimized sequence C2 was identified as containing the optimal GCDH protein coding sequence. Therefore, C2 was combined with either of the first two promoter components (P4 and P8) identified in Example 1 to generate two plasmid constructs, V2 (P4-C2) and V3 (P8-C2), for further evaluation of GCDH protein expression efficiency.

[0092] U87 MG cells were maintained in DMEM + 10% FBS and passaged every 3 days via TrypLE. One day before transfection, cells were cultured at 1 × 10⁻⁶ cells / day. 5 pcs / cm 2Cells were seeded into 24-well plates. Following user instructions, plasmids containing constructs V2 (P4-C2), V3 (P8-C2), and control V1 (P0-C0) were transfected into U87 MG cells using Lipofectamine 3000 transfection reagent (Invitrogen, L3000008). 72 hours post-transfection, cells were collected in RIPA lysis buffer (Beyotime, P0013C) containing a protease inhibitor mixture (Roche, 04693159001) and SDS-PAGE loading buffer (Cowin Bio, CW0027), denatured at 95°C for 10 min, and centrifuged at 12,000 rpm for 10 min. The supernatant was separated on a 4%–10% SDS-PAGE gel (Cowin Bio, CW0022M) and blotted onto a 0.2 μm PVDF transfer membrane (Merck, ISEQ00010). Protein levels of GCDH and the housekeeping gene GAPDH were detected using antibodies targeting GCDH (Abcam, ab232774) and β-tubulin (Proteintech, 66240-1), respectively. Western blot images were obtained in... Figure 6 As shown in the image.

[0093] like Figure 6 The results show that V1, V2, and V3 mediate similar GCDH protein expression levels in U87 MG cells, indicating that the V2 and V3 constructs achieve comparable GCDH protein expression compared to V1, and have the advantage of a reduced number of CpGs in the coding sequence.

[0094] Constructor V2 was chosen for further study because it has a lower CpG number than V3.

[0095] The enzyme activity of GCDH protein expressed by constructs V1 and V2 in SH-sy5y cells was evaluated by incubating cell lysates with pentenyl-CoA and then measuring the catalytic product crotonyl-CoA by LC-MS / MS analysis.

[0096] SH-sy5y cells were maintained in DMEM + 10% FBS and passaged every 3 days via TrypLE. One day before transfection, cells were cultured at 3 × 10⁻⁶ cells / day. 6 Cells were seeded per 100 mm culture dish. Following the user guide, the plasmid was transfected into SH-sy5y cells using jetOptimus reagent (Polyplus, 117-15). Cells were collected 48 hours after transfection. The cell volume was adjusted to 3 × 10⁶ cells / dish. 7Total protein was measured in the cell lysis buffer (0.2 mM flavin adenine dinucleotide disodium hydrate (Sigma, F6625-25MG), 1 mM L-cysteine ​​(Sangon Biotech, A600132-0100)) by sonication in 1×PBS (Sangon Biotech, B540626-0500). Total protein in the cell lysis buffer was measured using a BCA protein quantification kit (YEASEN, 20201ES76). GCDH activity was measured by mixing 0.5 g of total protein with GCDH reaction buffer [0.15 mM lithium glutaryl-CoA (Sigma G9510-5MG), 0.5 mM L-cysteine, 0.1 mM flavin adenine dinucleotide disodium hydrate, 1 mM methyl phenazine sulfate (Sigma, P9625-1G)] to a final volume of 500 μL. The reaction mixture was incubated at 37°C for different time points: 0 min, 5 min, 10 min, and 15 min, and the incubation was terminated with 500 μL of 7M trichloroacetic acid (Sigma, T9159-100G). GCDH activity was measured by increasing crotonyl-CoA production. The production of crotonyl-CoA in the reaction buffer was monitored and determined by LC-MS / MS. The time course of crotonyl-CoA production was... Figure 7 As shown in the image.

[0097] like Figure 7 As shown, the GCDH protein expressed by V2 surprisingly mediates a significantly faster catalytic reaction compared to the protein expressed by V1, suggesting that the GCDH protein expressed by the optimized coding sequence of C2 has increased enzymatic activity compared to the protein expressed by the construct V1 containing a wild-type coding sequence under the control of the CMV enhancer / promoter.

[0098] Example 4. In vivo proof-of-concept efficacy study

[0099] Both V1 and V2 constructs were introduced into the AAV9 vector to obtain rAAV9-V1 and rAAV9-V2, which were then used to evaluate their performance in Gcdh. - / - In vivo efficacy in mouse models.

[0100] Gcdh under normal diet - / - The mice exhibited a similar life expectancy to wild-type C57BL / 6 mice. However, after a 2-day high-protein diet (HPD) challenge, half of the 4-week-old Gcdh mice showed a significantly shorter life expectancy. - / - The mice will die within 3 days.

[0101] Within 24 hours of birth, the dose was 4.38 × 10⁻⁶. 8 4.38×109 4.38×10 10 The dose of vg given to Gcdh - / - Young mice were administered a single intracerebroventricular injection of rAAV9-V1 or rAAV9-V2. Young mice injected with PBS served as a control group. Four weeks after administration, HPD was administered for two consecutive days, and survival rates were evaluated in each group.

[0102] Following administration of PBS to the ventricles, Gcdh under HPD stimulation - / - The survival rate of the mice was 46%. Figure 8 and Figure 9 ). At 4.38×10 8 4.38×10 9 4.38×10 10 Following intraventricular administration of rAAV9-V1 at a dose of vg, Gcdh under HPD stimulation - / - The survival rates of the mice were 46%, 83%, and 83%, respectively. Figure 8 ). At 4.38×10 8 4.38×10 9 4.38×10 10 Following intraventricular administration of rAAV9-V2 at a dose of vg, Gcdh was stimulated under HPD. - / - The survival rates of the mice were 83%, 81%, and 100%, respectively. Figure 9 Treatment with rAAV9-V2 resulted in HPD excitation of Gcdh compared to treatment with rAAV9-V1. - / - The survival rate of mice was significantly higher.

[0103] Eight weeks after AAV administration, surviving mice were sacrificed to collect brain, liver, and plasma. Brain and liver tissues were homogenized, and mitochondria were isolated (QIAGEN, 37612). Mitochondria were collected in RIPA lysis buffer (Beyotime, P0013C) containing a protease inhibitor mixture (Roche, 04693159001) and SDS-PAGE loading buffer (Cowin Bio, CW0027), denatured at 95°C for 10 min, and centrifuged at 12,000 rpm for 10 min. The supernatant was separated on a 4%–10% SDS-PAGE gel (Cowin Bio, CW0022M) and blotted onto a 0.2 μm PVDF transfer membrane (Merck, ISEQ00010). Protein levels of GCDH and the housekeeping gene COXIV were detected using antibodies against GCDH (Abcam, ab232774) and COXIV (Abcam, ab16056), respectively. Protein blot images in Figure 10As shown in the figure. Brain GCDH expression was detected in a dose-dependent manner. Figure 10 Because the blood-brain barrier (BBB) ​​is immature in mice when AAV is administered, hepatic GCDH expression was detected in the highest dose group treated with rAAV9-V2, indicating that AAV translocates from the CNS across the BBB to peripheral tissues and organs. Figure 10 rAAV9-V2 mediates higher levels of GCDH protein expression than rAAV9-V1, suggesting that higher levels of GCDH protein expression protect more GCDH proteins. - / - Mice were spared from HPD-induced death.

[0104] For Gcdh administered rAAV9-V2 - / - In mice, liquid chromatography-mass spectrometry (LC-MS) analysis for measuring GA levels showed a dose-dependent significant decrease in GA levels in the brain (analysis of variance was performed using a two-way ANOVA test). Figure 11 There were no significant differences in liver and plasma GA levels between the different treatment groups. Figure 11 The brain and liver GA levels of the dead mice were also examined, and the results showed a sharp increase in GA levels compared to the surviving mice. Figure 11 Considering the mortality of mice, ICV administration of rAAV9-V2 effectively reduced GA accumulation and improved survival rate after HPD challenge. It was also observed that 4.38 × 10⁻⁶ GA was significantly reduced. 8 There was no significant difference in brain GA levels among the vg-treated groups, but the survival rate was significantly improved, indicating that even a moderate reduction in brain GA levels can protect against Gcdh. - / - Mice were spared from acute encephalopathy crisis induced by HPD provocation, suggesting that reducing GA levels in the CNS is more important than reducing GA levels in peripheral tissues.

[0105] Example 5. Long-term efficacy study

[0106] Long-term studies were conducted to test the durability of rAAV9-V2 efficacy. AAV9-V2 was applied at concentrations of 0 and 5 × 10⁻⁶. 8 2.5×10 9 1×10 10 VG dose administered intraventricularly to neonatal Gcdh - / - In mice. Unlike the study described in Example 4, HPD stimulation was not used in this study. Animals were sacrificed at 4 and 13 weeks after AAV administration for biochemical and histopathological analysis.

[0107] The levels of glutaric acid (GA) and 3-hydroxyglutaric acid (3-OHGA) were determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Figure 12The results, measured 4 weeks after AAV administration, are shown. Figure 12 As shown, brain GA and 3-OHGA levels decreased significantly in a dose-dependent manner. CSF GA, serum GA, and serum 3-OHGA levels showed a dose-dependent decreasing trend, but this was not significant. No significant changes were observed in GA and 3-OHGA levels in the liver and urine. Figure 13 The LC-MS / MS results measured 13 weeks after AAV administration are shown. Figure 13 As shown, brain GA and 3-OHGA levels decreased significantly in a dose-dependent manner. Serum GA and 3-OHGA levels showed a dose-dependent decreasing trend, with a more significant decrease in 3-OHGA levels. No significant changes were observed in GA and 3-OHGA in the liver and urine. CSF GA showed a dose-dependent decreasing trend. Figure 13 Compared to the results at 4 weeks, at 13 weeks after AAV administration, GA and 3-OHGA levels in the brain and serum, as well as GA levels in the CSF, showed even greater reductions.

[0108] GCDH expression was measured by ELISA. After AAV administration, GCDH expression was most abundant in the brain, followed by the heart, spinal cord, and liver. Figure 14 Four weeks after AAV administration, the mean brain GCDH concentration was 6,178.5 ng / mg total protein, and 13 weeks after AAV administration, the mean brain GCDH concentration was 12,143.5 ng / mg total protein. Four weeks after AAV administration, the mean liver GCDH concentration was 227.0 ng / mg total protein, and 13 weeks after AAV administration, the mean liver GCDH concentration was 92.2 ng / mg total protein.

[0109] Brain tissue pathology was evaluated using hematoxylin-eosin (HE) staining. Compared to wild-type mice, it was observed in Gcdh... - / - Void formation was observed in the cortex and striatum of mice. Void formation decreased in a dose-dependent manner after administration of AAV. Figure 15 ).

[0110] The results of this long-term study validated the role of AAV9-V2 in enhancing GCDH expression (particularly in the brain) and in reducing GA and 3-OHGA levels in the CNS and peripheral tissues. Furthermore, the results showed that long-term GCDH expression achieved through AAV9-V2 therapy protected CNS tissues from GCDH deficiency-induced vacuolation. Therefore, this 13-week study provides strong evidence that AAV9-V2 can be an effective therapy for extended periods.

[0111] References

[0112] 1.Goodman SI,Moe P,Markey SP,O'brien D.Glutaric acidemia:A newdisorder of amino acidmetabolism.Pediatric Research.1974;8:389-389.

[0113] 2.Goodman SI,Markey SP,Moe PG,Miles BS,Teng CC.Glutaric aciduria;a“new”disorder ofamino acid metabolism.Biochemical medicine.1975;12:12-21.

[0114] 3.Goodman SI,Stein DE,Schlesinger S,Christensen E,Schwartz M,Greenberg CR et al.Glutaryl-coa dehydrogenase mutations in glutaric acidemia(type i):Review and report of thirty novelmutations.Human mutation.1998;12:141-144.

[0115] 4.Besrat A,Polan CE,Henderson L.Mammalian metabolism of glutaricacid.Journal of BiologicalChemistry.1969;244:1461-1467.

[0116] 5. S,Christensen E,Leonard J,Greenberg C,Burlina A,Burlina A etal.Guideline for thediagnosis and management of glutaryl-coa dehydrogenasedeficiency(glutaric aciduria type i).

[0117] Journal of Inherited Metabolic Disease:Official Journal of theSociety for the Study of InbornErrors of Metabolism.2007;30:5-22.

[0118] 6.Ullrich K,Flott-Rahmel B,Schluff P,Musshoff U,Das A,Lücke T etal.Glutaric aciduria type i:

[0119] Pathomechanisms of neurodegeneration.Journal of inherited metabolicdisease.1999;22:392-403.

[0120] 7.Sauer SW,Okun JG,Fricker G,Mahringer A,Muller I,Crnic LR etal.Intracerebral accumulationof glutaric and 3-hydroxyglutaric acidssecondary to limited flux across the blood-brain barrierconstitute abiochemical risk factor for neurodegeneration in glutaryl-coadehydrogenasedeficiency.J Neurochem.2006;97:899-910.

[0121] 8.Koeller DM,Woontner M,Crnic LS,Kleinschmidt-Demasters B,Stephens J,Hunt EL et al.

[0122] Biochemical,pathologic and behavioral analysis of a mouse model ofglutaric acidemia type i.

[0123] Human molecular genetics.2002;11:347-357.

[0124] 9.Zinnanti WJ,Lazovic J,Wolpert EB,Antonetti DA,Smith MB,Connor JR etal.A diet-inducedmouse model of glutaric aciduria type i.Brain.2006:129:899-910.

[0125] [ PubMed ] 10.Bertolini TB,Shirley JL,Zolotukhin I,Li X,Kaisho T,Xiao W etal.Effect of cpg depletion ofvector genome on cd8+t cell responses in aavgene therapy NC, Young G, Von Drygalski A et al.Bax 335

[0126] Results of a clinical trial of hemophilia b gene therapy:Potential impact ofcpg sequences on geneexpression.Blood,The Journal of the American Society ofHematology.2021:137:763-774. Sequence information

[0127] >P0(SEQ ID NO:1)

[0128] GACATTGATTATTGACTAGTTATTAATGTAATCAATTACGGGGTCATTAGTTCATAGCCCA

[0129] FATHERGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAAC

[0130] GACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACT

[0131] TTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAA

[0132] GTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGG

[0133] CATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAG

[0134] TCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGT

[0135] TTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGC

[0136] ACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATG

[0137] GGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT

[0138] >P1(SEQ ID NO:2)

[0139] CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT

[0140] GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCA

[0141] ATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCC

[0142] AAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA

[0143] CATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACC

[0144] ATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCC

[0145] CCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGG

[0146] GGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGG

[0147] AGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGA

[0148] GGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG

[0149] >P2(SEQ ID NO:3)

[0150] CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT

[0151] GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCA

[0152] ATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCC

[0153] AAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA

[0154] CATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACC

[0155] ATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCC

[0156] CCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGG

[0157] GGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGG

[0158] AGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGA

[0159] GGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGTAAGTTTAGT

[0160] CTTTTTGTCTTTTATTTCAGGTCCCGGATCCGGTGGTGGTGCAAATCAAAGAACTGCTCC

[0161] TCAGTGGATGTTGCCTTTACTTCTAG

[0162] >P3(SEQ ID NO:4)

[0163] CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT

[0164] GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCA

[0165] ATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCC

[0166] AAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA

[0167] CATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACC

[0168] ATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCC

[0169] CCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGG

[0170] GGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGG

[0171] AGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGA

[0172] GGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGTCAGTGTGG

[0173] GGTCGGGAGTGTGGAGGGAAGGAGGGAGGAACTGGGGGTTTAGGGACTTTCCGGGGT

[0174] GACTTTCCCGTTCTGTGCTTGCAG

[0175] >P4(SEQ ID NO:5)

[0176] GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGG

[0177] GGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGG

[0178] AAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATA

[0179] AGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGT

[0180] CAGTGTGGGGTCGGGAGTGTGGAGGGAAGGAGGGAGGAACTGGGGGTTTAGGGACTT

[0181] TCCGGGGTGACTTTCCCGTTCTGTGCTTGCAG

[0182] >P5(SEQ ID NO:6)

[0183] GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGG

[0184] GGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGG

[0185] AAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATA

[0186] AGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGT

[0187] AAGGACCTCTGGTCGCACCGTGTGTCTGCTGCCCCTGTTCAGCTGTCTGTCTGCCGCAG

[0188] GTGGACTCTGTCCCAGAATCCGAGAGCTGCCCGAGCGGGGTGGCAGGGTCGTGGCCAG

[0189] GGTCAGAGGCACTAAGGCAGTGAGTGCGCTGTGCCTGCGGGGCCGGAGAAAAGTCACC

[0190] TGATCAGTCTCGCTTGCAGCTCGCACTAGCCGGGGGGCGACATGGGTGTTGGGGGGTAG

[0191] GGCTGATGAGGGTCCGAGAAGGGAGGGCACAGTGATCTTGCGGACTGGACCGAGGCG

[0192] AATTCCCCTTCCCAG

[0193] >P6(SEQ ID NO:7)

[0194] GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGG

[0195] GGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGG

[0196] AAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATA

[0197] AGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGT

[0198] GGGCGGGCTGGTGGGTGCCCTGAGACTGCTCCTCCGCCTGGAGCCATAGCCACCCCACC

[0199] TCAAGGCCCCTCTGTCCTTGGGGCTGGGGCTTCCTGTGGCCTAGGCCTGGGCCTGAATT

[0200] TGGGCACTGGTCCCTTTGCAG

[0201] >P7(SEQ ID NO:8)

[0202] GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGG

[0203] GGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGG

[0204] AAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATA

[0205] AGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGT

[0206] CAGTGTGGGGTCGGGAGTGTGGAGGGAAGGAGGGAGGAACTGGGGGTTTAGGGACTT

[0207] TCCGGGGTGACTTTCCCGTTCTGTGCTTGCAGGTAAGGACCTCTGGTCGCACCGTGTGT

[0208] CTGCTGCCCCTGTTCAGCTGTCTGTCTGCCGCAGGTGGACTCTGTCCCAGAATCCGAGA

[0209] GCTGCCCGAGCGGGGTGGCAGGGTCGTGGCCAGGGTCAGAGGCACTAAGGCAGTGAG

[0210] TGCGCTGTGCCTGCGGGGCCGGAGAAAAGTCACCTGATCAGTCTCGCTTGCAGCTCGC

[0211] ACTAGCCGGGGGGCGACATGGGTGTTGGGGGGTAGGGCTGATGAGGGTCCGAGAAGGG

[0212] AGGGCACAGTGATCTTGCGGACTGGACCGAGGCGAATTCCCCTTCCCAG

[0213] >P8(SEQ ID NO:9)

[0214] GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGG

[0215] GGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGG

[0216] AAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATA

[0217] AGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGG

[0218] AGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCG

[0219] CCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCC

[0220] TCCGGGCTGTAATTAGCTGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGG

[0221] TATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAG

[0222] >C0(SEQ ID NO:10)

[0223] ATGGCCCTGAGAGGCGTCTCCGTGCGGCTGCTGAGCCGCGGACCCGGCCTGCACGTCC

[0224] TTCGCACGTGGGTCTCGTCGGCGGCGCAGACCGAGAAAGGCGGGAGAACACAGAGCC

[0225] AACTGGCTAAGTCCTCGCGTCCCGAGTTTGACTGGCAGGACCCGCTGGTGCTGGAGGA

[0226] GCAGCTGACCACAGATGAGATCCTCATCAGGGACACCTTCCGCACCTACTGCCAGGAGA

[0227] GACTCATGCCTCGCATCCTGTTGGCCAATCGCAACGAAGTTTTTCATCGGGAGATCATTT

[0228] CGGAGATGGGGGAGTTGGGTGTGCTGGGCCCCACCATCAAAGGATATGGCTGTGCTGGG

[0229] GTTTCGTCTGTGGCCTATGGGCTCCTGGCCCGAGAGCTGGAGCGGGTGGACAGTGGCTA

[0230] CAGGTCGGCGATGAGTGTCCAGTCCTCCCTCGTCATGCACCCTATCTATGCCTATGGCAG

[0231] CGAGGAACAGCGGCAGAAGTACCTGCCCCAGCTGGCCAAGGGGGAGCTCCTGGGCTGC

[0232] TTCGGGCTCACAGAGCCCAACAGCGGAAGTGACCCCAGCAGCATGGAGACCAGAGCCC

[0233] ACTACAACTCATCCAACAAGAGCTACACCCTCAATGGGACCAAGACCTGGATCACGAAC

[0234] TCGCCTATGGCCGATCTGTTTGTAGTGTGGGCTCGGTGTGAAGATGGCTGCATTCGGGGC

[0235] TTCCTGCTGGAGAAGGGGATGCGGGGTCTCTCGGCCCCCAGGATCCAGGGCAAGTTCTC

[0236] GCTGCGGGCCTCAGCCACAGGCATGATCATCATGGACGGTGTGGAGGTGCCAGAGGAG

[0237] AATGTGCTCCCTGGTGCATCCAGCCTGGGGGGTCCCTTCGGCTGCCTGAACAACGCCCG

[0238] GTACGGCATCGCGTGGGGCGTGCTTGGAGCTTCGGAGTTCTGCTTGCACACAGCCCGGC

[0239] AGTACGCCCTCGACAGGATGCAGTTTGGTGTCCCACTGGCCAGGAACCAGCTGATTCAG

[0240] AAGAAGCTGGCAGACATGCTCACTGAGATTACCCTGGGCCTTCACGCCTGCCTGCAGCT

[0241] CGGCCGCTTGAAGGACCAGGACAAGGCTGCCCCCGAGATGGTTTCTCTGCTGAAGAGG

[0242] AATAACTGTGGGAAAGCCCTGGACATCGCCCGCCAGGCCCGAGACATGCTGGGGGGGA

[0243] ATGGGATTTCTGACGAGTATCACGTGATCCGGCACGCCATGAACCTGGAGGCCGTGAAC

[0244] ACCTACGAAGGTACACATGACATTCACGCCCTGATCCTTGGGAGAGCTATCACGGGAAT

[0245] CCAGGCGTTCACGGCCAGCAAGTAA

[0246] >C1(SEQ ID NO:11)

[0247] ATGGCCCTGAGAGGCGTGTCCGTCAGACTGCTGAGCAGAGGCCCTGGCCTGCATGTGCT

[0248] CAGAACCTGGGTCAGCAGCGCTGCTCAAACAGAAAAGGGGGGCAGAACACAAAGCCA

[0249] ACTGGCTAAGAGCAGCAGACCTGAATTCGATTGGCAAGACCCCCTGGTCCTGGAAGAA

[0250] CAGCTGACAACAGACGAGATTCTGATTAGAGACACATTCAGAACATATTGCCAAGAAAG

[0251] ACTGATGCCTAGAATCCTGCTGGCCAACAGAAATGAAGTGTTTCATCGGGAAATCATTA

[0252] GCGAGATGGGCGAGCTGGGCGTGCTGGGCCCCACCATTAAGGGCTACGGCTGTGCTGG

[0253] GGTGTCCTCCGTGGCCTATGGCCTCCTGGCTAGAGAACTCGAAAGAGTCGATAGCGGCT

[0254] ACAGAAGCGCTATGAGCGTGCAGAGCAGCCTGGTGATGCATCCTATCTATGCTTATGGCA

[0255] GCGAAGAGCAGAGACAAAAGTATCTGCCTCAGCTGGCTAAGGGCGAGCTGCTCGGCTG

[0256] CTTCGGGCTGACAGAACCCAATAGCGGGTCCGATCCTAGCAGCATGGAGACAAGAGCT

[0257] CATTATAATAGCAGCAACAAGAGCTATACCCTGAACGGGACAAAAACATGGATCACAAA

[0258] TAGCCCTATGGCTGACCTGTTTGTGGTGTGGGCCAGATGTGAGGATGGCTGTATCAGAG

[0259] GCTTTCTGCTGGAGAAGGGCATGCGGGGGCTGTCCGCTCCTAGAATCCAAGGCAAATTT

[0260] AGCCTGAGAGCTAGCGCTACAGGCATGATTATTATGGACGGCGTCGAGGTGCCTGAGGA

[0261] AAATGTGCTGCCTGGCGCTAGCAGCCTGGGCGGGCCTTTCGGCTGCCTGAATAACGCTA

[0262] GATATGGCATCGCCTGGGGGGTGCTGGGCGCCTCCGAGTTTTGTCTGCACACAGCTAGA

[0263] CAGTATGCCCTGGACAGAATGCAATTCGGGGTGCCCCTGGCTAGAAATCAGCTGATTCA

[0264] AAAGAAACTGGCTGACATGCTGACAGAAATTACACTCGGCCTCCATGCCTGTCTGCAGC

[0265] TGGGCAGACTCAAAGATCAAGATAAGGCTGCCCCTGAAATGGTCAGCCTGCTCAAAAG

[0266] AAACAATTGCGGCAAAGCTCTGGATATCGCTAGACAAGCTAGAGATATGCTCGGCGGCA

[0267] ACGGGATTAGCGACGAGTATCATGTGATCAGACACGCTATGAATCTGGAAGCCGTGAAC

[0268] ACCTATGAAGGCACACACGACATCCACGCTCTGATCCTCGGGAGAGCTATCACCGGCAT

[0269] TCAAGCCTTCACAGCTAGCAAGTAA

[0270] >C2(SEQ ID NO:12)

[0271] ATGGCTCTGAGAGGGGTGAGCGTCAGACTGCTGAGCAGAGGCCCTGGCCTGCATGTGC

[0272] TGAGAACATGGGTGTCCAGCGCTGCTCAGACAGAGAAGGGGGGCAGAACACAGAGCC

[0273] AACTGGCCAAGAGCAGCAGACCTGAATTTGACTGGCAAGACCCCCTGGTCCTGGAGGA

[0274] GCAGCTGACCACAGATGAGATCCTGATCAGAGACACCTTCAGAACCTACTGCCAAGAG

[0275] AGACTGATGCCTAGAATCCTGCTGGCCAACAGAAATGAGGTCTTCCACAGAGAAATCAT

[0276] TAGCGAGATGGGGGAGCTGGGGGTGCTGGGCCCTACAATCAAGGGCTATGGCTGTGCTG

[0277] GGGTGAGCAGCGTGGCCTATGGCCTGCTGGCTAGAGAGCTGGAGAGAGTGGACAGCGG

[0278] GTACAGAAGCGCTATGAGCGTGCAGAGCAGCCTGGTCATGCACCCCATCTATGCCTATG

[0279] GCAGCGAGGAGCAGAGACAGAAATATCTCCCTCAGCTGGCCAAGGGGGAGCTGCTGGGG

[0280] CTGCTTTGGCCTCACAGAGCCCAATAGCGGCAGCGACCCTAGCAGCATGGAGACAAGA

[0281] GCCCACTACAACAGCAGCAACAAGAGCTACACCCTGAATGGCACCAAGACATGGATCA

[0282] CAAACAGCCCCATGGCTGATCTCTTGTGGTCTGGGCTAGATGTGAGGATGGCTGTATCA

[0283] GAGGCTTTCTCCTGGAGAAGGGCATGAGAGGCCTGAGCGCTCCTAGAATCCAAGGCAA

[0284] ATTCAGCCTCAGAGCTTCCGCCACCGGGATGATCATCATGGATGGGGTGGAGGTCCCTG

[0285] AGGAGAATGTGCTGCCTGGGGCTAGCTCCCTGGGGGGCCCCTTTGGCTGTCTCAATAAT

[0286] GCTAGATATGGCATTGCCTGGGGGGTGCTGGGGGCCAGCGAGTTCTGCCTGCATACAGC

[0287] TAGACAATATGCCCTGGACAGAATGCAGTTTGGGGTGCCCCTGGCTAGAAATCAGCTGA

[0288] TTCAGAAGAGCTGGCTGACATGCTGACAGAGATCACACTGGGCCTGCATGCCTGTCTG

[0289] CAGCTGGGGAGACTGAAGGACCAAGATAAGGCTGCCCCTGAGATGGTGAGCCTGCTGA

[0290] AGAGAAATAACTGTGGGAAAGCTCTGGACATTGCTAGACAAGCTAGAGACATGCTGGG

[0291] GGGCAATGGCATCTCCGATGAGTACCATGTCATCAGACATGCCATGAACCTGGAGGCTG

[0292] TGAACACCTATGAGGGCACACATGACATCCATGCCCTGATCCTGGGCAGAGCCATCACC

[0293] GGCATCCAAGCCTTCACAGCTAGCAAGTGA

[0294] >C3(SEQ ID NO:13)

[0295] ATGGCCCTGAGAGGCGTCTCCGTGAGGCTGCTGAGCAGAGGACCTGGCCTGCATGTCCT

[0296] TAGAACGTGGGTCTCGTCGGCGGCGCAGACCGAGAAAGGCGGGAGAACACAGAGCCA

[0297] ACTGGCTAAGTCCTCGCGTCCTGAGTTTGACTGGCAGGACCCGCTGGTGCTGGAGGAG

[0298] CAGCTGACCACAGATGAGATCCTCATCAGGGACACCTTCAGAACCTACTGCCAGGAGA

[0299] GACTCATGCCTAGAATCCTGTTGGCCAATAGAAATGAAGTTTTTCATAGGGAGATCATTT

[0300] CGGAGATGGGGGAGTTGGGTGTGCTGGGCCCCACCATCAAAGGATATGGCTGTGCTGGG

[0301] GTTTCGTCTGTGGCCTATGGGCTCCTGGCCCGAGAGCTGGAGAGGGTGGACAGTGGCTA

[0302] CAGGTCGGCGATGAGTGTCCAGTCCTCCCTTGTCATGCACCCTATCTATGCCTATGGCAG

[0303] TGAGGAACAGAGGCAGAAGTACCTGCCCCAGCTGGCCAAGGGGAGCTCCTGGGCTGC

[0304] TTTGGGCTCACAGAGCCCAACTCTGGAAGTGACCCCAGCAGCATGGAGACCAGAGCCC

[0305] ACTACAACTCATCCAACAAGAGCTACACCCTCAATGGGACCAAGACCTGGATCACGAAC

[0306] TCGCCTATGGCCGATCTGTTTGTAGTGTGGGCTAGGTGTGAAGATGGCTGCATTAGGGGC

[0307] TTCCTGCTGGAGAAGGGGATGAGGGGTCTTCTCGGCCCCCAGGATCCAGGGCAAGTTCT

[0308] CGCTGAGGGCCTCAGCCACAGGCATGATCATCATGGATGGTGTGGAGGTGCCAGAGGA

[0309] GAATGTGCTCCCTGGTGCATCCAGCCTGGGGGGTCCCTTTGGCTGCCTGAACAATGCCA

[0310] GGTATGGCATTGCGTGGGGCGTGCTTGGAGCTTCGGAGTTCTGCTTGCACACAGCCAGG

[0311] CAGTATGCCCTTGACAGGATGCAGTTTGGTGTCCCACTGGCCAGGAACCAGCTGATTCA

[0312] GAAGAAGCTGGCAGACATGCTCACTGAGATTACCCTGGGCCTTCATGCCTGCCTGCAGC

[0313] TTGGCAGATTGAAGGACCAGGACAAGGCTGCCCCTGAGATGGTTTTCTCTGCTGAAGAG

[0314] GAATAACTGTGGGAAAGCCCTGGACATTGCCAGACAGGCCCGAGACATGCTGGGGGGG

[0315] AATGGGATTTCTGATGAGTATCATGTGATCAGGCATGCCATGAACCTGGAGGCCGTGAAC

[0316] ACCTATGAAGGTACACATGACATTCATGCCCTGATCCTTGGGAGAGCTATCACGGGAATC

[0317] CAGGCGTTCACGGCCAGCAAGTAA

[0318] >C4(SEQ ID NO:14)

[0319] ATGGCCCTGAGAGGCGTCTCCGTGCGGCTGCTGAGCCGCGGACCCGGCCTGCACGTCC

[0320] TTCGCACGTGGGTCTCGTCGGCGGCGCAGACCGAGAAAGGCGGGAGAACACAGAGCC

[0321] AACTGGCTAAGTCCTCGAGGCCCGAGTTTGACTGGCAGGACCCCCTGGTGCTGGAGGA

[0322] GCAGCTGACAACCGATGAGATCCTGATCAGGGATACCTTCAGAACCTACTGTCAGGAGA

[0323] GGCTGATGCCCAGGATCCTGCTGGCCAACAGAAACGAGGTGTTCCACAGAGAGATCAT

[0324] CAGCGAGATGGGCGAGCTGGGCGTGCTGGGCCCTACAATCAAGGGCTACGGCTGCGCC

[0325] GGCGTGAGCAGCGTTGCCTACGGCCTGCTGGCCAGGGAGCTGGAGAGAGTGGATTCCG

[0326] GCTACAGAAGCGCCATGAGCGTGCAGAGCTCCCTGGTCATGCACCCTATCTACGCCTAC

[0327] GGCAGCGAGGAGCAGAGACAGAAGTACCTGCCCCAGCTGGCCAAAGGCGAGCTGCTG

[0328] GGCTGCTTCGGCCTGACAGAGCCTAATTCCGGCTCCGACCCCAGCTCCATGGAGACCAG

[0329] AGCCCACTACAATAGCTCCAATAAGAGCTACACACTGAACGGCACAAAGACCTGGATCA

[0330] CAAACAGCCCCATGGCCGACCTGTTTGTGGTGTGGGCCAGGTGTGAGGATGGCTGTATC

[0331] AGGGGCTTTCTGCTGGAGAAGGGCATGAGAGGCCTGTCCGCCCCCAGGATCCAGGGCA

[0332] AGTTTAGCCTGAGAGCCAGCGCCACCGGCATGATCATCATGGATGGCGTGGAGGTGCCC

[0333] GAGGAGAACGTGCTGCCTGGCGCCAGCAGCCTGGGCGGACCTTTTGGCTGCCTGAACA

[0334] ATGCCAGATACGGCATCGCCTGGGGCGTGCTGGGAGCCTCTGAGTTCTGCCTGCACACC

[0335] GCCAGGCAGTACGCCCTGGATAGGATGCAGTTTGGCGTGCCCCTGGCCAGAAACCAGCT

[0336] GATCCAGAAGAAGCTGGCCGACATGCTGACCGAGATCACACTGGGCCTGCACGCCTGC

[0337] CTGCAGCTGGGAAGGCTGAAGGATCAGGACAAGGCCGCCCCCGAGATGGTGTCCCTGC

[0338] TGAAGAGAAATAATTGTGGCAAGGCCCTGGACATCGCCAGACAGGCCAGAGATATGCTG

[0339] GGCGGCAATGGCATCAGCGATGAGTACCACGTGATCAGGCACGCCATGAACCTGGAGG

[0340] CCGTGAACACCTACGAGGGCACCCACGACATCCACGCCCTGATCCTGGGCAGGGCCATC

[0341] ACCGGCATCCAGGCCTTTACCGCCAGCAAGTAA

[0342] >C5(SEQ ID NO:15)

[0343] ATGGCCCTGAGAGGCGTCTCCGTGCGGCTGCTGAGCCGCGGACCCGGCCTGCACGTCC

[0344] TTCGCACGTGGGTCTCGTCGGCGGCGCAGACCGAGAAAGGCGGGAGAACACAGAGCC

[0345] AACTGGCTAAGTCCTCGAGACCCGAGTTCGACTGGCAGGACCCTCTTGTGCTGGAAGA

[0346] GCAACTGACAACAGATGAGATCCTGATCAGAGACACCTTCAGAACCTACTGCCAGGAG

[0347] AGACTGATGCCCAGAATCCTGCTGGCCAACAGAAACGAGGTGTTCCACAGAGAGATCA

[0348] TCAGCGAGATGGGCGAGCTGGGCGTGCTGGGCCCTACAATTAAAGGATATGGCTGCGCC

[0349] GGAGTGAGCAGCGTGGCTTATGGACTTCTGGCTAGAGAGCTGGAGAGAGTGGACAGCG

[0350] GCTATAGAAGCGCCATGAGCGTGCAGAGCAGCCTGGTGATGCATCCCATTTATGCCTACG

[0351] GCAGCGAGGAGCAAAGACAGAAGTACCTGCCCCAGCTGGCCAAGGGCGAGCTGCTGG

[0352] GATGTTTTGGACTTACAGAACCCAACAGCGGAAGCGACCCCAGCAGCATGGAAACCAG

[0353] AGCTCATTATAACAGCAGCAACAAGAGCTACACCCTGAACGGCACCAAGACCTGGATCA

[0354] CCAACAGCCCCATGGCCGACCTTTTTGTGGTGTGGGCTAGATGCGAGGACGGCTGTATT

[0355] AGAGGCTTTCTGCTGGAAAAGGGCATGAGAGGCCTGAGCGCCCCTAGAATTCAAGGCA

[0356] AATTTAGCCTGAGAGCCAGCGCCACCGGAATGATTATCATGGACGGCGTGGAGGTGCCC

[0357] GAGGAGAATGTGCTGCCTGGAGCTAGCAGCCTGGGAGGCCCTTTTGGATGTCTGAATAA

[0358] TGCCAGATACGGCATCGCCTGGGGCGTGCTGGGAGCTAGCGAGTTTTGTCTGCATACAG

[0359] CCAGACAGTACGCCCTGGACAGAATGCAGTTCGGCGTGCCCCTTGCTAGAAATCAGCTG

[0360] ATCCAGAAGAAGCTGGCCGACATGCTGACCGAGATCACCCTGGGACTTCACGCCTGTCT

[0361] GCAACTGGGAAGACTGAAAGATCAGGACAAGGCCGCCCCCGAAATGGTGTCTCTGCTT

[0362] AAAAGAAACAACTGCGGCAAGGCCCTGGACATCGCCAGACAAGCTAGAGATATGCTGG

[0363] GCGGCAATGGCATTAGCGATGAATATCACGTGATTAGACACGCCATGAACCTGGAGGCC

[0364] GTGAACACCTATGAGGGCACACATGACATCCACGCCCTGATTCTGGGAAGAGCCATTAC

[0365] CGGCATCCAGGCCTTTACCGCCAGCAAGTAA

[0366] >C6(SEQ ID NO:16)

[0367] ATGGCCCTGAGAGGCGTCTCCGTGCGGCTGCTGAGCCGCGGACCCGGCCTGCACGTCC

[0368] TTCGCACGTGGGTCTCGTCGGCGGCGCAGACCGAGAAAGGCGGGAGAACACAGAGCC

[0369] AACTGGCTAAGTCCTCGCGTCCCGAGTTTGACTGGCAGGACCCGCTGGTGCTGGAGGA

[0370] GCAGCTGACCACAGATGAGATCCTCATCAGGGACACCTTCCGCACCTACTGCCAGGAGA

[0371] GACTCATGCCTCGCATCCTGTTGGCCAATCGCAACGAAGTTTTTCATCGGGAGATCATTT

[0372] CGGAGATGGGGGAGTTGGGTGTGCTGGGCCCCACCATCAAAGGATATGGCTGTGCTGGG

[0373] GTTTCGTCTGTGGCCTATGGGCTCCTGGCCCGAGAGCTGGAGCGGGTGGACAGTGGCTA

[0374] CAGGTCGGCGATGAGTGTCCAGTCCTCCCTCGTCATGCACCCTATCTATGCCTATGGCAG

[0375] CGAGGAACAGCGGCAGAAGTACCTGCCCCAGCTGGCCAAGGGGAGCTCCTGGGCTGC

[0376] TTTGGGCTCACAGAGCCCAACTCTGGAAGTGACCCCAGCAGCATGGAGACCAGAGCCC

[0377] ACTACAACTCATCCAACAAGAGCTACACCCTCAATGGGACCAAGACCTGGATCACGAAC

[0378] TCGCCTATGGCCGATCTGTTTGTAGTGTGGGCTAGGTGTGAAGATGGCTGCATTAGGGGC

[0379] TTCCTGCTGGAGAAGGGGATGAGGGGTCTTCTCGGCCCCCAGGATCCAGGGCAAGTTCT

[0380] CGCTGAGGGCCTCAGCCACAGGCATGATCATCATGGATGGTGTGGAGGTGCCAGAGGA

[0381] GAATGTGCTCCCTGGTGCATCCAGCCTGGGGGGTCCCTTTGGCTGCCTGAACAATGCCA

[0382] GGTATGGCATTGCGTGGGGCGTGCTTGGAGCTTCGGAGTTCTGCTTGCACACAGCCAGG

[0383] CAGTATGCCCTTGACAGGATGCAGTTTGGTGTCCCACTGGCCAGGAACCAGCTGATTCA

[0384] GAAGAAGCTGGCAGACATGCTCACTGAGATTACCCTGGGCCTTCATGCCTGCCTGCAGC

[0385] TTGGCAGATTGAAGGACCAGGACAAGGCTGCCCCTGAGATGGTTTTCTCTGCTGAAGAG

[0386] GAATAACTGTGGGAAAGCCCTGGACATTGCCAGACAGGCCCGAGACATGCTGGGGGGG

[0387] AATGGGATTTCTGATGAGTATCATGTGATCAGGCATGCCATGAACCTGGAGGCCGTGAAC

[0388] ACCTATGAAGGTACACATGACATTCATGCCCTGATCCTTGGGAGAGCTATCACGGGAATC

[0389] CAGGCGTTCACGGCCAGCAAGTAA

[0390] >C7(SEQ ID NO:17)

[0391] ATGGCCCTGAGAGGCGTCTCCGTGCGGCTGCTGAGCCGCGGACCCGGCCTGCACGTCC

[0392] TTCGCACGTGGGTCTCGTCGGCGGCGCAGACCGAGAAAGGCGGGAGAACACAGAGCC

[0393] AACTGGCTAAGTCCTCGCGTCCCGAGTTTGACTGGCAGGACCCGCTGGTGCTGGAGGA

[0394] GCAGCTGACCACAGATGAGATCCTCATCAGGGACACCTTCCGCACCTACTGCCAGGAGA

[0395] GACTCATGCCTCGCATCCTGTTGGCCAATCGCAACGAAGTTTTTCATCGGGAGATCATTT

[0396] CGGAGATGGGGGAGTTGGGTGTGCTGGGCCCCACCATCAAAGGATATGGCTGTGCTGGG

[0397] GTTTCGTCTGTGGCCTATGGGCTCCTGGCCCGAGAGCTGGAGCGGGTGGACAGTGGCTA

[0398] CAGGTCGGCGATGAGTGTCCAGTCCTCCCTCGTCATGCACCCTATCTATGCCTATGGCAG

[0399] CGAGGAACAGCGGCAGAAGTACCTGCCCCAGCTGGCCAAGGGGGAGCTCCTGGGCTGC

[0400] TTTGGCCTCACTGAGCCAAATTCTGGTTCAGACCCATCATCTATGGAAACAAGGGCCCAT

[0401] TACAATTCATCTAATAAGTCATACACTCTGAATGGTACTAAGACCTGGATCACCAACTCTC

[0402] CAATGGCAGACCTGTTTGTAGTTTGGGCAAGATGTGAAGATGGCTGTATTAGGGGTTTCC

[0403] TCCTGGAGAAGGGCATGAGAGGTCTCTCTGCACCAAGGATTCAGGGAAAATTCTCTCTG

[0404] AGAGCTTCTGCTACAGGCATGATTATTATGGATGGGGTGGAGGTTCCTGAAGAGAATGTC

[0405] CTGCCTGGAGCTTCATCACTGGGGGGCCCCTTTGGCTGTCTGAACAATGCCAGATATGGT

[0406] ATTGCATGGGGGGTTCTGGGGGCTAGTGAGTTCTGCCTGCACACAGCTAGACAGTATGC

[0407] TCTGGATAGGATGCAGTTTGGTGTTCCTCTGGCTAGGAACCAGCTGATTCAGAAAAAAC

[0408] TGGCTGATATGCTCACAGAGATTACACTGGGTCTGCATGCTTGTCTCCAGCTGGGTAGAC

[0409] TCAAAGATCAGGATAAGGCTGCTCCAGAAATGGTGTCACTCCTGAAGAGGAATAACTGT

[0410] GGCAAGGCTCTGGACATTGCTAGACAGGCTAGGGATATGCTGGGTGGTAATGGCATCTC

[0411] AGATGAATATCATGTTATTAGACATGCCATGAATCTGGAGGCTGTTAACACTTATGAAGGC

[0412] ACACATGATATTCATGCCCTCATCCTGGGGAGAGCTATTACAGGTATTCAGGCCTTTACTG

[0413] CTTCTAAGTGA

[0414] >C8(SEQ ID NO:18)

[0415] ATGGCCCTGAGAGGCGTCTCCGTGCGGCTGCTGAGCCGCGGACCCGGCCTGCACGTCC

[0416] TTCGCACGTGGGTCTCGTCGGCGGCGCAGACCGAGAAAGGCGGGAGAACACAGAGCC

[0417] AACTGGCTAAGTCCTCGCGTCCCGAGTTTGACTGGCAGGACCCGCTGGTGCTGGAGGA

[0418] GCAGCTGACCACAGATGAGATCCTCATCAGGGACACCTTCCGCACCTACTGCCAGGAGA

[0419] GACTCATGCCTCGCATCCTGTTGGCCAATCGCAACGAAGTTTTTCATCGGGAGATCATTT

[0420] CGGAGATGGGGGAGTTGGGTGTGCTGGGCCCCACCATCAAAGGATATGGCTGTGCTGGG

[0421] GTTTCGTCTGTGGCCTATGGGCTCCTGGCCCGAGAGCTGGAGCGGGTGGACAGTGGCTA

[0422] CAGGTCGGCGATGAGTGTCCAGTCCTCCCTCGTCATGCACCCTATCTATGCCTATGGCAG

[0423] CGAGGAACAGCGGCAGAAGTACCTGCCCCAGCTGGCCAAGGGGGAGCTCCTGGGCTGC

[0424] TTCGGGCTCACAGAGCCCAACAGCGGAAGTGACCCCAGCAGCATGGAGACCAGAGCCC

[0425] ACTACAACTCATCCAACAAGAGCTACACCCTCAATGGGACCAAGACCTGGATCACGAAC

[0426] TCGCCTATGGCCGATCTGTTTGTAGTGTGGGCTCGGTGTGAAGATGGCTGCATTCGGGGC

[0427] TTCCTGCTGGAGAAGGGGATGCGGGGTCTCTCGGCCCCCAGGATCCAGGGCAAGTTCTC

[0428] GCTGCGGGCCTCAGCCACAGGCATGATCATCATGGACGGTGTGGAGGTGCCAGAGGAG

[0429] AATGTGCTCCCTGGTGCATCCAGCCTGGGGGGTCCCTTCGGCTGTCTGAACAATGCCAG

[0430] ATATGGTATTGCATGGGGGGTTCTGGGGGCTAGTGAGTTCTGCCTGCACACAGCTAGAC

[0431] AGTATGCTCTGGATAGGATGCAGTTTGGTGTTCCTCTGGCTAGGAACCAGCTGATTCAGA

[0432] AAAAACTGGCTGATATGCTCACAGAGATTACACTGGGTCTGCATGCTTGTCTCCAGCTG

[0433] GGTAGACTCAAAGATCAGGATAAGGCTGCTCCAGAAATGGTGTCACTCCTGAAGAGGA

[0434] ATAACTGTGGCAAGGCTCTGGACATTGCTAGACAGGCTAGGGATATGCTGGGTGGTAAT

[0435] GGCATCTCAGATGAATATCATGTTATTAGACATGCCATGAATCTGGAGGCTGTTAACACTT

[0436] ATGAAGGCACACATGATATTCATGCCCTCATCCTGGGGAGAGCTATTACAGGTATTCAGG

[0437] CCTTTACTGCTTCTAAGTGA

[0438] >V1(SEQ ID NO:19)

[0439] CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCT

[0440] TTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGGGTTAAACGT

[0441] TGACATTGATTATTGCGGCCTCTAGACTCGAGGCGTTGACATTGATTATTGACTAGTTATT

[0442] AATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATA

[0443] ACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAA

[0444] TAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGG

[0445] AGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGC

[0446] CCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCT

[0447] TATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGAT

[0448] GCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAA

[0449] GTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTT

[0450] CCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTG

[0451] GGAGGTCTATATAAGCAGAGCTAAGCTTGCCGCCACCATGGCCCTGAGAGGCGTCTCCG

[0452] TGCGGCTGCTGAGCCGCGGACCCGGCCTGCACGTCCTTCGCACGTGGGTCTCGTCGGC

[0453] GGCGCAGACCGAGAAAGGCGGGAGAACACAGAGCCAACTGGCTAAGTCCTCGCGTCC

[0454] CGAGTTTGACTGGCAGGACCCGCTGGTGCTGGAGGAGCAGCTGACCACAGATGAGATC

[0455] CTCATCAGGGACACCTTCCGCACCTACTGCCAGGAGAGACTCATGCCTCGCATCCTGTT

[0456] GGCCAATCGCAACGAAGTTTTTCATCGGGAGATCATTTCGGAGATGGGGGAGTTGGGTG

[0457] TGCTGGGCCCCACCATCAAAGGATATGGCTGTGCTGGGGTTTCGTCTGTGGCCTATGGGC

[0458] TCCTGGCCCGAGAGCTGGAGCGGGTGGACAGTGGCTACAGGTCGGCGATGAGTGTCCA

[0459] GTCCTCCCTCGTCATGCACCCTATCTATGCCTATGGCAGCGAGGAACAGCGGCAGAAGT

[0460] ACCTGCCCCAGCTGGCCAAGGGGGAGCTCCTGGGCTGCTTCGGGCTCACAGAGCCCAA

[0461] CAGCGGAAGTGACCCCAGCAGCATGGAGACCAGAGCCCACTACAACTCATCCAACAAG

[0462] AGCTACACCCTCAATGGGACCAAGACCTGGATCACGAACTCGCCTATGGCCGATCTGTT

[0463] TGTAGTGTGGGCTCGGTGTGAAGATGGCTGCATTCGGGGCTTCCTGCTGGAGAAGGGGA

[0464] TGCGGGGTCTCTCGGCCCCCAGGATCCAGGGCAAGTTCTCGCTGCGGGCCTCAGCCAC

[0465] AGGCATGATCATCATGGACGGTGTGGAGGTGCCAGAGGAGAATGTGCTCCCTGGTGCAT

[0466] CCAGCCTGGGGGGTCCCTTCGGCTGCCTGAACAACGCCCGGTACGGCATCGCGTGGGG

[0467] CGTGCTTGGAGCTTCGGAGTTCTGCTTGCACACAGCCCGGCAGTACGCCCTCGACAGGA

[0468] TGCAGTTTGGTGTCCCACTGGCCAGGAACCAGCTGATTCAGAAGAAGCTGGCAGACAT

[0469] GCTCACTGAGATTACCCTGGGCCTTCACGCCTGCCTGCAGCTCGGCCGCTTGAAGGACC

[0470] AGGACAAGGCTGCCCCCGAGATGGTTTCTCTGCTGAAGAGGAATAACTGTGGGAAAGC

[0471] CCTGGACATCGCCCGCCAGGCCCGAGACATGCTGGGGGGGAATGGGATTTCTGACGAG

[0472] TATCACGTGATCCGGCACGCCATGAACCTGGAGGCCGTGAACACCTACGAAGGTACACA

[0473] TGACATTCACGCCCTGATCCTTGGGAGAGCTATCACGGGAATCCAGGCGTTCACGGCCA

[0474] GCAAGTAAGAATTCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTA

[0475] GAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAAC

[0476] CATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTT

[0477] CAGGGGGAGGTGTGGGAGGTTTTTTCGTTACTAGAGCATGGCTACGTAGATAAGTAGCA

[0478] TGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCT

[0479] GCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTT

[0480] GCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG

[0481] >V2(SEQ ID NO:20)

[0482] CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCT

[0483] TTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGGGTTATCTAG

[0484] ACCTAGGACTAGTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTC

[0485] CCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGC

[0486] GGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGG

[0487] AGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGC

[0488] CAGAACACAGGTCAGTGTGGGGTCGGGAGTGTGGAGGGAAGGAGGGAGGAACTGGGG

[0489] GTTTAGGGACTTTCCGGGGTGACTTTCCCGTTCTGTGCTTGCAGAAGCTTGCCGCCACC

[0490] ATGGCTCTGAGAGGGGTGAGCGTCAGACTGCTGAGCAGAGGCCCTGGCCTGCATGTGC

[0491] TGAGAACATGGGTGTCCAGCGCTGCTCAGACAGAGAAGGGGGGCAGAACACAGAGCC

[0492] AACTGGCCAAGAGCAGCAGACCTGAATTTGACTGGCAAGACCCCCTGGTCCTGGAGGA

[0493] GCAGCTGACCACAGATGAGATCCTGATCAGAGACACCTTCAGAACCTACTGCCAAGAG

[0494] AGACTGATGCCTAGAATCCTGCTGGCCAACAGAAATGAGGTCTTCCACAGAGAAATCAT

[0495] TAGCGAGATGGGGGAGCTGGGGGTGCTGGGCCCTACAATCAAGGGCTATGGCTGTGCTG

[0496] GGGTGAGCAGCGTGGCCTATGGCCTGCTGGCTAGAGAGCTGGAGAGAGTGGACAGCGG

[0497] GTACAGAAGCGCTATGAGCGTGCAGAGCAGCCTGGTCATGCACCCCATCTATGCCTATG

[0498] GCAGCGAGGAGCAGAGACAGAAATATCTCCCTCAGCTGGCCAAGGGGGAGCTGCTGGGG

[0499] CTGCTTTGGCCTCACAGAGCCCAATAGCGGCAGCGACCCTAGCAGCATGGAGACAAGA

[0500] GCCCACTACAACAGCAGCAACAAGAGCTACACCCTGAATGGCACCAAGACATGGATCA

[0501] CAAACAGCCCCATGGCTGATCTCTTGTGGTCTGGGCTAGATGTGAGGATGGCTGTATCA

[0502] GAGGCTTTCTCCTGGAGAAGGGCATGAGAGGCCTGAGCGCTCCTAGAATCCAAGGCAA

[0503] ATTCAGCCTCAGAGCTTCCGCCACCGGGATGATCATCATGGATGGGGTGGAGGTCCCTG

[0504] AGGAGAATGTGCTGCCTGGGGCTAGCTCCCTGGGGGGCCCCTTTGGCTGTCTCAATAAT

[0505] GCTAGATATGGCATTGCCTGGGGGGTGCTGGGGGCCAGCGAGTTCTGCCTGCATACAGC

[0506] TAGACAATATGCCCTGGACAGAATGCAGTTTGGGGTGCCCCTGGCTAGAAATCAGCTGA

[0507] TTCAGAAGAGCTGGCTGACATGCTGACAGAGATCACACTGGGCCTGCATGCCTGTCTG

[0508] CAGCTGGGGAGACTGAAGGACCAAGATAAGGCTGCCCCTGAGATGGTGAGCCTGCTGA

[0509] AGAGAAATAACTGTGGGAAAGCTCTGGACATTGCTAGACAAGCTAGAGACATGCTGGG

[0510] GGGCAATGGCATCTCCGATGAGTACCATGTCATCAGACATGCCATGAACCTGGAGGCTG

[0511] TGAACACCTATGAGGGCACACATGACATCCATGCCCTGATCCTGGGCAGAGCCATCACC

[0512] GGCATCCAAGCCTTCACAGCTAGCAAGTGAGAATTCCAGACATGATAAGATACATTGATG

[0513] AGTTTGGACAAACCACAACTAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGT

[0514] GATGCTATTGCTTTATTGTAACCATTAAAGCTGCAATAAACAAGTTAACAACAAAATT

[0515] GCATTCAGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCT

[0516] CTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCT

[0517] TTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG

[0518] >V3(SEQ ID NO:21)

[0519] CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCT

[0520] TTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGGGTTATGAAT

[0521] GCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCA

[0522] TCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACT

[0523] CATCAATGTATCTTATCATGTCTGGAATTCTCACTTGCTAGCTGTGAAGGCTTGGATGCCG

[0524] GTGATGGCTCTGCCCAGGATCAGGGCATGGATGTCATGTGTGCCCTCATAGGTGTTCACA

[0525] GCCTCCAGGTTCATGGCATGTCTGATGACATGGTACTCATCGGAGATGCCATTGCCCCCC

[0526] AGCATGTCTCTAGCTTGTCTAGCAATGTCCAGAGCTTTCCCACAGTTATTTCTCTTCAGC

[0527] AGGCTCACCATCTCAGGGGCAGCCTTATCTTGGTCCTTCAGTCTCCCCAGCTGCAGACA

[0528] GGCATGCAGGCCCAGTGTGATCTCTGTCAGCATGTCAGCCAGCTTCTTCTGAATCAGCT

[0529] GATTTCTAGCCAGGGGCACCCCAAACTGCATTCTGTCCAGGGCATATTGTCTAGCTGTAT

[0530] GCAGGCAGAACTCGCTGGCCCCCAGCACCCCCCAGGCAATGCCATATCTAGCATTATTG

[0531] AGACAGCCAAAGGGGCCCCCCAGGGAGCTAGCCCCAGGCAGCACATTCTCCTCAGGGA

[0532] CCTCCACCCCATCCATGATGATCATCCCGGTGGCGGAAGCTCTGAGGCTGAATTTGCCTT

[0533] GGATTCTAGGAGCGCTCAGGCCTCTCATGCCCTTCTCCAGGAGAAAGCCTCTGATACAG

[0534] CCATCCTCACATCTAGCCCAGACCACAAAGAGATCAGCCATGGGGCTGTTTGTGATCCAT

[0535] GTCTTGGTGCCATTCAGGGTGTAGCTCTTGTTGCTGCTGTTGTAGTGGGCTCTTGTCTCC

[0536] ATGCTGCTAGGGTCGCTGCCGCTATTGGGCTCTGTGAGGCCAAAGCAGCCCAGCAGCTC

[0537] CCCCTTGGCCAGCTGAGGGAGATATTTCTGTCTCTGCTCCTCGCTGCCATAGGCATAGAT

[0538] GGGGTGCATGACCAGGCTGCTCTGCACGCTCATAGCGCTTCTGTACCCGCTGTCCACTCT

[0539] CTCCAGCTCTCTAGCCAGCAGGCCATAGGCCACGCTGCTCACCCCAGCACAGCCATAGC

[0540] CCTTGATTGTAGGGCCCAGCACCCCCAGCTCCCCCATCTCGCTAATGATTTCTCTGTGGA

[0541] AGACCTCATTTCTGTTGGCCAGCAGGATTCTAGGCATCAGTCTCTCTTGGCAGTAGGTTC

[0542] TGAAGGTGTCTCTGATCAGGATCTCATCTGTGGTCAGCTGCTCCTCCAGGACCAGGGGG

[0543] TCTTGCCAGTCAAATTCAGGTCTGCTGCTCTTGGCCAGTTGGCTCTGTGTTCTGCCCCCC

[0544] TTCTCTGTCTGAGCAGCGCTGGACACCCATGTTCTCAGCACATGCAGGCCAGGGCCTCT

[0545] GCTCAGCAGTCTGACGCTCACCCCTCTCAGAGCCATGGTGGCGGCAAGCTTCTGAAAA

[0546] AAAGTGATTTCAGGCAGGTGCTCCAGGTAATTAAACATTAATACCCCACCAACCAACCAT

[0547] CCCTTAAACCCTTACCTCTTGCTCAGCTAATTACAGCCCGGAGGAGAAGGGCCGTCCCG

[0548] CCCGCTCACCTGTGGGAGTAACGCGGTCAGTCAGAGCCGGGGCGGGCGGCGCGAGGCG

[0549] GCGGCGGAGCGGGGCACGGGGCGAAGGCAGCGTCGCAGCGACTCCCTGTGTTCTGGC

[0550] GGCAAACCCGTTGCGAAAAAGAACGTTCACGGCGACTACTGCACTTATATACGGTTCTC

[0551] CCCCACCCTCGGGAAAAAGGCGGAGCCAGTACACGACATCACTTTCCCAGTTTACCCCG

[0552] CGCCACCTTCTCTAGGCACCCGTTCAATTGCCGACCCCTCCCCCCAACTTCTCGGGGACT

[0553] GTGGGCGATGTGCGCTCTGCCCACTGACGGGCACCGGAGCCACTAGTCCTAGGTCTAGA

[0554] GGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCG

[0555] CTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCG

[0556] GGCGGCCTCAGTGAGCGAGCGAGCGCGCAG

[0557] >L-ITR(SEQ ID NO:22)

[0558] CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCT

[0559] TTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGG

[0560] >R-ITR(SEQ ID NO:23)

[0561] AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAG

[0562] GCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCG

[0563] AGCGAGCGCGCAG

[0564] >CMV enhancer(SEQ ID NO:24) ​GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCA

[0566] TATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAAC

[0567] GACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACT

[0568] TTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAA

[0569] GTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGG

[0570] CATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAG

[0571] TCATCGCTATTACCATG

[0572] >CMV promoter (SEQ ID NO:25)

[0573] GTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTT

[0574] CCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGA

[0575] CTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTAC

[0576] GGTGGGAGGTCTATATAAGCAGAGCT

[0577] >Chicken β-actin promoter (SEQ ID NO:26)

[0578] TCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAA

[0579] TTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGG

[0580] GCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAG

[0581] GTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCG

[0582] GCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG

[0583] >HGCD intron 1 (SEQ ID NO: 27)

[0584] GTCAGTGTGGGGTCGGGAGTGTGGAGGGAAGGAGGGAGGAACTGGGGGTTTAGGGAC

[0585] TTTCCGGGGTGACTTTCCCGTTCTGTGCTTGCAG

[0586] >SV40 intron (SEQ ID NO: 28)

[0587] GTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGATCCGGTGGTGGTGCAAATCAA

[0588] AGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAG

[0589] >EF-1α core promoter (SEQ ID NO: 29)

[0590] GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGG

[0591] GGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGG

[0592] AAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATA

[0593] AGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG>HGCDH intron 2 (SEQ ID NO:30)

[0594] GTAAGGACCTCTGGTCGCACCGTGTGTCTGCTGCCCCTGTTCAGCTGTCTGTCTGCCGC

[0595] AGGTGGACTCTGTCCCAGAATCCGAGAGCTGCCCGAGCGGGGTGGCAGGGTCGTGGCC

[0596] AGGGTCAGAGGCACTAAGGCAGTGAGTGCGCTGTGCCTGCGGGGCCGGAGAAAAGTC

[0597] ACCTGATCAGTCTCGCTTGCAGCTCGCACTAGCCGGGGGGCGACATGGGTGTTGGGGGG

[0598] TAGGGCTGATGAGGGTCCGAGAAGGGAGGGCACAGTGATCTTGCGGACTGGACCGAGG

[0599] CGAATTCCCCTTCCCAG

[0600] >HGCDH intron 3 (SEQ ID NO:31)

[0601] [[ID=并]]GTGGGCGGGCTGGTGGGTGCCCTGAGACTGCTCCTCCGCCTGGAGCCATAGCCACCCCA

[0602] CCTCAAGGCCCCTCTGTCCTTGGGGCTGGGGCTTCCTGTGGCCTAGGCCTGGGCCTGAA

[0603] TTTGGGCACTGGTCCCTTTGCAG

[0604] >Heterozygous intron (SEQ ID NO:32)

[0605] It should be noted that there seems to be a possible error in the text you provided. The character "并" in line 28 is likely incorrect. I translated it as "GTGGGCGGGCTGGTGGGTGCCCTGAGACTGCTCCTCCGCCTGGAGCCATAGCCACCCCA" based on the context, but you may want to double-check the original content.GGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGTCCCGCCGCCGCTCCGGCCGCC

[0606] CGCCCCGGCTCTGACTGACCGCGTTACTCCCCACAGGTGAGCGGGCGGGACGGCCCTTCT

[0607] CCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGG

[0608] GGTATTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTCAG

[0609] >GCDH protein (SEQ ID NO:33)

[0610] MALRGVSVRLLSRGPGLHVLRTWVSSAAQTEKGGTQSQLAKSSRPEFDWQDPLVLEEQL

[0611] TTDEILIRDTFRTYCQERLMPRILLANRNNEVFHREIISEMGELGVLGPTKGYGCAGVSSVAY

[0612] GLLARELERVDSGYRSAMSVQSSLVMHPIYAGSEEQRQKYLPQLAKGELLGCFGLTEPNS

[0613] GSDPSSMETRAHYNSSNKYTLNGTKTWITNSPMADLFVVWARCEDGCIRGFLLEKGMRG

[0614] LSAPRIQGKFSLRASATGMIIMDGVEVPEENVLPGASSLGGPFGCLNNARYGIAWGVLGASE

[0615] FCLHTARQYALDRMQFGVPLARNQLIQKKLADMLTEITLGLHACLQLGRLKDQDKAAPEM

[0616] VSLLKRNNCGKALDIARQARDMLGGNGGISDEYHVIRHAMNLEAVNTYEGTHDIHALILGRAITGIQAFTASK*

Claims

1. An expression cassette comprising the nucleotide sequence shown in SEQ ID NO: 12, wherein the nucleotide sequence encodes a human glutaryl-CoA dehydrogenase (hGCDH) polypeptide having the amino acid sequence shown in SEQ ID NO: 33, the expression cassette further comprising a promoter component operatively linked to the 5' of the nucleotide sequence encoding the hGCDH polypeptide, wherein the promoter component comprises: (a) a constitutive promoter that is a human elongation factor-1 alpha (EF-1 alpha) core promoter having a nucleotide sequence as set forth in SEQ ID NO: 29, and (b) an intron-derived fragment; wherein the intron-derived fragment consists of one or more fragments derived from one or more intron regions of a human GCDH GAPDH gene, the intron-derived fragment having a nucleotide sequence as set forth in SEQ ID NO: 27, the intron-derived fragment being downstream of the promoter.

2. An expression cassette comprising the nucleotide sequence shown in SEQ ID NO: 12, wherein the nucleotide sequence encodes a human glutaryl-CoA dehydrogenase (hGCDH) polypeptide having the amino acid sequence shown in SEQ ID NO: 33, the expression cassette further comprising a promoter component operatively linked to the 5' of the nucleotide sequence encoding the hGCDH polypeptide, wherein the promoter component comprises: (a) a constitutive promoter that is a human elongation factor-1 alpha (EF-1 alpha) core promoter, and (b) an intron-derived fragment; wherein the intron-derived fragment consists of one or more fragments derived from one or more intronic regions of a human GCDH gene, the intron-derived fragment having a nucleotide sequence as set forth in SEQ ID NO: 27, the intron-derived fragment being downstream of the promoter, wherein the promoter component consists of a nucleotide sequence as set forth in SEQ ID NO:

5.

3. The expression cassette of claim 2, wherein the expression cassette consists of the nucleotide sequence as set forth in SEQ ID NO:

20.

4. An rAAV vector comprising the expression cassette of any one of claims 1-3.

5. The rAAV vector of claim 4, further comprising two AAV inverted terminal repeats (ITRs).

6. The rAAV vector of claim 5, wherein the 5’ ITR has the nucleotide sequence of SEQ ID NO: 22 and the 3’ ITR has the nucleotide sequence of SEQ ID NO:

23.

7. A viral particle comprising the rAAV vector of any one of claims 4-6 packaged into an AAV capsid.

8. The viral particle of claim 7, wherein the capsid has CNS tropism.

9. The viral particle of claim 8, wherein the capsid is an AAV9 or AAV PHP.B capsid.

10. A pharmaceutical composition comprising the rAAV vector of any one of claims 4-6 or the viral particle of any one of claims 7-9 and a pharmaceutically acceptable excipient.

11. Use of the rAAV vector of any one of claims 4-6, the viral particle of any one of claims 7-9, or the pharmaceutical composition of claim 10 for the manufacture of a medicament for treating glutaric aciduria type I (GA-I) in a subject in need thereof.

12. The use of claim 11, wherein the subject is a mammal.

13. The use of claim 12, wherein the subject is a human.

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