Recombinant AAV vectors for treatment of glutarinuria type I

Abstract

A codon optimized sequence encoding an hGCDH polypeptide and a recombinant adeno-associated virus (rAAV) vector comprising one of the sequences under the control of a promoter component are provided. Also provided herein are viral particles comprising the rAAV vector, pharmaceutical compositions comprising the rAAV vector or the viral particles, and uses thereof in the treatment of type I glutarinuria (GA-I).

Description

[0001] CROSS-REFERENCE 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] The present disclosure relates to the technical field of gene therapy. In particular, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising a nucleotide sequence encoding human glutaryl-CoA dehydrogenase (GCDH), which is used to treat disorders or conditions caused by the deficiency of glutaryl-CoA dehydrogenase (GCDH), especially glutaric aciduria type I (GA-I).

[0004] Sequence Listing

[0005] This disclosure includes a Sequence Listing as a part of this disclosure. Background Art

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

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

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

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

[0010] GA-I 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 causes in situ GA accumulation and, therefore, neurotoxicity [7]. GA-I patients may benefit from direct delivery of rAAV carrying a GCDH expression cassette to the CNS, 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 the conditions of acute encephalopathy crisis, GCDH knockout mice were challenged with a high-protein diet [9]. High-protein diet (HPD) challenge at 4 weeks of age - / - The mice were lethal. Under HPD stimulation, Gcdh - / - Mice develop GA accumulation, vasogenic edema, neuronal loss, paralysis, and epilepsy[9]. - / - The mouse may be a useful model for human GA-1, including developmentally dependent striatal vulnerability [9].

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

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

[0013] In a second aspect, the present application provides a promoter component having a nucleotide sequence selected from a group of nucleotide sequences consisting of 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, the present application provides an expression cassette comprising a coding sequence of a human GCDH polypeptide having an amino acid sequence as shown in SEQ ID NO: 33 operably linked to a promoter component, wherein the coding sequence has a nucleotide sequence selected from a group of nucleotide sequences consisting of SEQ ID NO: 11-18, and the promoter component has a nucleotide sequence selected from a group of nucleotide sequences consisting of SEQ ID NO: 2-9. In a preferred embodiment, the expression cassette comprises a coding sequence having a nucleotide sequence as shown in SEQ ID NO: 12 under the control of a promoter component having a nucleotide sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 9. In a more specific embodiment, the expression cassette comprises a nucleotide sequence as shown in SEQ ID NO: 20 or SEQ ID NO: 21 (V2 or V3).

[0015] In a fourth aspect, the present application provides a 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., a disease-related tissue in the CNS).

[0016] In a fifth aspect, the present application provides an AAV virus particle, the AAV virus 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, AAV214. In one embodiment, the AAV capsid is a capsid with CNS tropism (such as AAV9 or AAV PHP.B capsid).

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

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

[0019] In an eighth aspect, the present application provides use of the rAAV vector of the fourth aspect, the rAAV particles of the fifth aspect, or the pharmaceutical composition of the sixth aspect in treating GA-I patients. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0021] Figure 2 Shown is a comparison of GCDH protein expression from P0 to P8 in U87 MG cells.

[0022] Figure 3 Shown is a comparison of GCDH protein expression from P0 to P8 in HEK293 cells.

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

[0024] Figure 5 Shown is the evaluation of GCDH protein expression of C0-C8 in U87 MG cells.

[0025] Figure 6 Shown is the evaluation of GCDH protein expression of V1-V3 in U87 MG cells.

[0026] Figure 7 Shown is the evaluation of GCDH enzyme activity of V1-V3 in SH-sy5y cells.

[0027] Figure 8 It shows that after administration of rAAV9-V1, Gcdh - / - Survival curve of mice.

[0028] Fig. 9 It is shown that after administration of rAAV9-V2, Gcdh - / - Survival curve of mice.

[0029] Fig.10 Shown in Gcdh - / - Evaluation of GCDH protein expression of rAAV9-V1 and rAAV9-V2 in mice.

[0030] Fig.11 Figure 3 shows the Gcdh of survival and death after administration of rAAV9-V2. - / - LC-MS analysis of GA levels in brain, liver, and plasma of mice.

[0031] Fig.12 Shown are 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) 4 weeks after AAV administration.

[0032] Fig.13 Shown are 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) at 13 weeks after AAV administration.

[0033] Fig.14 It was shown that at high doses (1.0 x 10 10 GCDH protein levels in different tissues of mice 4 and 13 weeks after administration of rAAV9-V2 (vg).

[0034] Fig.15 Shown are HE staining of mouse brains 13 weeks after administration of AAV or vehicle (200X). DETAILED DESCRIPTION

[0035] Unless specifically defined elsewhere in this document, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0036] As used herein, including the appended claims, singular forms such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0037] In the context of the present disclosure, unless otherwise stated, the word "comprise" and its variants such as "comprises" and "comprising" will be understood to imply the inclusion of the elements (e.g., amino acid sequences, nucleotide sequences, characteristics, steps or groups thereof), but not to exclude any other elements (e.g., amino acid sequences, nucleotide sequences, characteristics and steps). When used herein, the term "comprise" or any variant thereof can be replaced by the term "contain", "include" or sometimes "have" or its equivalent variants. In certain embodiments, the word "comprise" also includes the situation of "consisting of".

[0038] Coding sequence of hGCDH

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

[0040] "Isolated nucleic acid" means DNA or RNA removed from all or a portion of a polynucleotide, wherein the isolated polynucleotide is found in nature or is associated with polynucleotides that are not associated in nature. An isolated nucleic acid molecule "comprising" a particular nucleotide sequence may also contain, in addition to the specified sequence, operably linked regulatory sequences that control the expression of the coding region of the recited nucleic acid sequence. Due to codon degeneracy, those skilled in the art will appreciate that any particular amino acid sequence may be encoded by several different nucleotide sequences.

[0041] "Codon optimized coding sequence" herein refers to a nucleotide sequence encoding hGCDH protein, which is modified from its wild-type coding sequence adapted for codon bias. Optimization can be achieved by reducing sequence complexity, adjusting GC content, adjusting codon usage and / or avoiding rare codons. Codon optimized coding sequences generally show increased translation efficiency of the target gene (GOI), resulting in higher protein expression.

[0042] Compared to the wild-type coding sequence of hGCDH as shown in SEQ ID NO:10, the codon-optimized coding sequence of hGCDH of the present application has a reduced number of CpGs. "CpG content" or "number of CpGs" refers to the content or number of cytosine (C) guanine (G) dinucleotides connected by phosphate (p) in a DNA sequence. "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. In particular, a region containing at least 200 bp with a GC ratio of more than 50% and an observed / predicted ratio of CpG higher than 0.6 is called a "CpG island". The predicted value of CpG can be calculated as the number of Cs in the observation window multiplied by the number of Gs in the window divided by the window length. In mammals, unmethylated CpGs of exogenous genes are recognized by TLR9, resulting in CD8 + T cell activation is to remove infected cells, which is unfavorable for the long-term expression of foreign genes. Therefore, in order to more effectively express the GCDH coding gene, it is preferred to reduce the number of CpGs in the hGCDH coding sequence. The coding sequence of the present invention preferably has a lower CpG content. When CpG content is a factor to be considered in the codon optimization process, it further increases the complexity of sequence design and verification work.

[0043] The number of CpGs in the wild-type coding sequence of hGCDH as shown in SEQ ID NO: 10 is 73. Preferably, the number of CpGs in the hGCDH coding sequence of the present application is less than 73. For example, the number of CpGs in the hGCDH coding sequence of the present application is no more than 65, no more than 55, no more than 50, no more than 45, no more than 40, no more than 35, no more than 30, no more than 25, no more than 20. For example, the number of CpGs in the hGCDH coding sequence of the present application is at least 10% less than the number of CpGs in the wild-type coding sequence of hGCDH (e.g., the nucleotide sequence as shown in SEQ ID NO: 10), preferably at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less than the number of CpGs in the wild-type coding sequence of hGCDH (e.g., the nucleotide sequence as shown in SEQ ID NO: 10). In a specific embodiment of the present application, the CpG number of the hGCDH coding sequence having the nucleotide sequence of 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 a nucleotide sequence selected from SEQ ID NOs: 11 to 18. In a particularly preferred embodiment, the codon-optimized coding sequence of the human GCDH protein comprises or consists of a nucleotide sequence as shown in SEQ ID NO: 12.

[0045] Adjustment sequence

[0046] In addition, in addition to the coding sequence, the expression cassette may contain one or more regulatory sequences. The regulatory sequence may be selected from one or more of a promoter, an enhancer, a polyadenylation sequence, and a translation termination signal. Certain combinations of regulatory sequences of the present disclosure may achieve unexpected results in improving the expression efficiency of the coding sequence.

[0047] On the one hand, the present application provides a series of novel promoter components. "Promoter component" refers to a sequence component located 5' upstream of a coding sequence, and is composed of a promoter and optionally one or more additional elements (such as an enhancer and / or an intron-derived fragment), wherein the enhancer is usually located upstream of the promoter, and the intron-derived fragment is usually located downstream of the promoter.

[0048] The term "promoter" refers to a DNA sequence that is capable of initiating 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. The promoter may be a naturally occurring promoter of a gene, a modified form of a naturally occurring promoter, or a synthetic promoter.

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

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

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

[0052] In some embodiments, the intron-derived fragment is derived from an 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 is composed of one or more fragments of one or more intron regions derived from human GCDH gene. In a preferred embodiment, the promoter component or expression cassette of the present application comprises an intron-derived fragment having a nucleotide sequence as shown in any one of SEQ ID NO: 27, 30 or 31 (respectively hGCDH intron 1, hGCDH intron 2 or hGCDH intron 3). 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 fragment is a hybrid intron. "Hybrid intron" refers to an intron fragment comprising at least two sequences from different origins or from the same origin but not continuous in nature. For example, the promoter component or expression cassette of the present application comprises a hybrid intron as an intron-derived fragment, wherein the hybrid intron comprises two intron-derived fragments derived from chicken beta-actin (CBA) and minute virus of mice (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, about or less than 400 bp.

[0056] In some embodiments, the promoter component of the present 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 a specific embodiment, 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 the present 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 hybrid intron derived from the hGCDH gene (e.g., SEQ ID NO: 32). In a specific embodiment, the promoter component comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 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 preferred embodiments, due to the limited packaging capacity of AAV, the length of the promoter component does not exceed 1,000 bp, does not exceed 900 bp, does not exceed 850 bp, does not exceed 800 bp, does not exceed 700 bp, does not exceed 600 bp, does not exceed 500 bp, or does not exceed 400 bp.

[0059] Expression cassette

[0060] The term "expression cassette" herein refers to a DNA component contained in a vector (eg, a rAAV vector) and consisting of a gene (eg, 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, particularly the promoter component, of the human GCDH gene, the expression cassette of hGCDH inserted into an AAV vector can provide desired expression levels and reduced immunogenicity following rAAV delivery into a subject.

[0062] In a specific embodiment, the expression cassette comprises any coding sequence having a nucleotide sequence as shown in any one of SEQ ID NOs: 11-18, preferably a nucleotide sequence as shown in SEQ ID NO: 12, operably linked to a promoter component having a nucleotide sequence as shown in SEQ ID NOs: 2-9, preferably a nucleotide sequence as shown in SEQ ID NOs: 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 the transcription of the coding sequence.

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

[0064] Recombinant AAV vectors and viral particles

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

[0066] In addition to the inserted nucleotide sequence as described above, the rAAV vector is in a self-complementary form. The rAAV vector is composed of two inverted terminal repeat (ITR) sequences at both ends of the inserted nucleotide sequence. The ITR of the present disclosure can be an ITR derived from any AAV serotype. When referring to the serotype of AAV ITR, the phrase "derived from" means that the ITR can be an ITR of a certain serotype or a variant with one or more modifications derived therefrom. In a preferred embodiment of the present 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 an AAV2 ITR lacking a truncated form of the C region or C' region. For example, the wild-type AAV2 ITR is located at the 5' end of the inserted nucleotide sequence, and 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 the present application has a nucleotide sequence as 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 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 a capsid suitable for intravenous (IV) delivery (e.g., IV injection) to peripheral tissues. The term "peripheral tissue" in the context of the present application refers to any tissue that is not a part of the brain or spinal cord. For example, the capsid can be a capsid suitable for nervous system delivery (e.g., intrathecal, intracerebellomedullary cisterna or intraventricular delivery (e.g., by injection)). In some embodiments, the AAV vector comprises a capsid or a variant thereof of an AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV PHP.B, AAV2.7m8 or AAVAnc80L65 serotype. 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 composition of the present disclosure comprises an isolated nucleic acid, a rAAV vector or a viral particle of the present disclosure and a pharmaceutically acceptable excipient. Conventional pharmaceutically acceptable excipients are known in the art and can be solid or liquid excipients. In one embodiment, the pharmaceutical composition can be a liquid for injection.

[0070] deliver

[0071] As used herein, the terms "administration", "administering", "treating", and "treatment" refer to contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with a subject, cell, tissue, organ, or biological fluid when applied to a subject (e.g., an animal (including a human)) or to a cell, tissue, organ, or biological fluid. Treatment of cells encompasses contact of an agent with a cell, as well as contact of an agent with a fluid, wherein the fluid is in contact with a cell. The terms "administration" and "treatment" also include in vitro and ex vivo treatment of, for example, a cell by an agent, a diagnostic agent, a binding compound, or by another cell.

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

[0073] The rAAV vector can be administered via a single dose or multiple doses. In a specific embodiment, 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 (eg, symptoms of GA-I).

[0076] Example

[0077] In order to facilitate understanding and utilization of the present invention, the advantages of the present invention will be described in more detail with reference to Examples and the accompanying drawings. However, it should be understood that the following examples are only intended to illustrate the present invention without any intention to limit the scope of the present invention. The scope of the present invention should be limited by the claims.

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

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

[0080] Table 1. Information of 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 by TrypLE. One day before transfection, cells were plated at 1×10 5 / cm 2Inoculate in 24-well plates. According to the user guide, plasmid was transfected into U87 MG or HEK293 cells using Lipofectamine3000 transfection reagent (Invitrogen, L3000008). 72 hours after transfection, cells were collected in RIPA lysis buffer (Beyotime, P0013C) with protease inhibitor mixture (Roche, 04693159001) and SDS-PAGE loading buffer (Cowin Bio, CW0027), denatured at 95°C for 10min, centrifuged at 12,000rpm for 10min. Supernatant was separated in 4%-10% SDS-PAGE gel (Cowin Bio, CW0022M), and blotted on 0.2 μm PVDF transfer membrane (Merck, ISEQ00010). The protein levels of GCDH and housekeeping gene GAPDH were detected by antibodies against GCDH (Abcam, ab232774) and GAPDH (Cell Signaling Technology, 2118S), respectively. Western blot images are shown in Figure 2 and Figure 3 Shown in.

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

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

[0085] It has been reported that CpG in AAV vectors induces immune response and exogenous gene silencing [10,11]. In this example, the coding sequence of GCDH was optimized to enhance expression and reduce the number of CpG, and the expression efficiency of the codon-optimized sequence was tested using the CMV promoter. Figure 4 A total of eight different optimized coding sequences, C1-C8, were synthesized, having nucleotide sequences as shown in SEQ ID NOs: 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. CpG numbers of 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 codon-optimized sequences C1-C8, U87 MG cells were maintained in DMEM + 10% FBS and passaged every 3 days by TrypLE. One day before transfection, cells were plated at 1×10 5 Pieces / cm 2 Inoculate in 24-well plates. According to the user guide, plasmids were transfected into U87 MG using Lipofectamine 3000 transfection reagent (Invitrogen, L3000008). 72 hours after transfection, cells were collected in RIPA lysis buffer (Beyotime, P0013C) with protease inhibitor cocktail (Roche, 04693159001) and SDS-PAGE loading buffer (Cowin Bio, CW0027), denatured at 95°C for 10 min, centrifuged at 12,000 rpm for 10 min. Supernatant was separated in 4%-10% SDS-PAGE gel (Cowin Bio, CW0022M), and blotted on 0.2 μm PVDF transfer membrane (Merck, ISEQ00010). The protein levels of GCDH and housekeeping gene GAPDH were detected by antibodies against GCDH (Abcam, ab232774) and β-tubulin (Proteintech, 66240-1), respectively. Western blot images are shown in Figure 5 Shown in.

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

[0090] Example 3. GCDH constructs 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 any 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 by TrypLE. One day before transfection, cells were plated at 1×10 5 Pieces / cm 2Inoculate into 24-well plates. According to the user guide, constructs V2 (P4-C2), V3 (P8-C2) and control V1 (P0-C0) plasmids were transfected into U87 MG using Lipofectamine 3000 transfection reagent (Invitrogen, L3000008). 72 hours after transfection, cells were collected in RIPA lysis buffer (Beyotime, P0013C) with protease inhibitor cocktail (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. Supernatant was separated in 4%-10% SDS-PAGE gel (Cowin Bio, CW0022M) and blotted onto 0.2 μm PVDF transfer membrane (Merck, ISEQ00010). The protein levels of GCDH and housekeeping gene GAPDH were detected by antibodies against GCDH (Abcam, ab232774) and β-tubulin (Proteintech, 66240-1), respectively. Western blot images are shown in Figure 6 Shown in.

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

[0094] Construct V2 was selected for further studies because V2 has a lower CpG number than V3.

[0095] The enzymatic activity of GCDH proteins expressed by constructs V1 and V2 in SH-sy5y cells was evaluated by incubating cell lysates with glutaconyl-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 by TrypLE. One day before transfection, cells were plated at 3 × 10 6 The cells were inoculated into 100 mm culture dishes. The plasmid was transfected into SH-sy5y using jetOptimus reagent (Polyplus, 117-15) according to the user guide. 48 hours after transfection, the cells were collected. The cells were adjusted to 3×10 7Cells were homogenized by ultrasound in a cell lysis buffer (0.2 mM flavin adenine dinucleotide disodium salt hydrate (Sigma, F6625-25MG), 1 mM L-cysteine ​​(Sangon Biotech, A600132-0100) in 1×PBS (Sangon Biotech, B540626-0500). Total protein in the cell lysis buffer was measured by 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 glutaryl-CoA lithium salt (Sigma G9510-5MG), 0.5 mM L-cysteine, 0.1 mM flavin adenine dinucleotide disodium salt hydrate, 1 mM phenazine methylsulfate (Sigma, P9625-1G) in 1×PBS] 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, 15 min, respectively, and terminated by 500 μL of 7 M trichloroacetic acid (Sigma, T9159-100G). GCDH activity was measured by the addition of crotonyl-CoA. The crotonyl-CoA produced in the reaction buffer was monitored and determined by LC-MS / MS. The time course of crotonyl-CoA production was Figure 7 Shown in.

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

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

[0099] Both constructs V1 and V2 were introduced into AAV9 vectors to obtain rAAV9-V1 and rAAV9-V2 for evaluation of their expression in Gcdh - / - In vivo efficacy in mouse models.

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

[0101] Within 24 hours after birth, 4.38×10 8 4.38×109 4.38×10 10 The dose of vg to Gcdh - / - Rats were injected intracerebroventricularly (single dose) with rAAV9-V1 or rAAV9-V2. Rats injected with PBS were used as a control group. HPD was administered for 2 consecutive days 4 weeks after administration, and the survival rate of each group was evaluated.

[0102] After intracerebroventricular administration of PBS, Gcdh - / - The survival rate of mice was 46% ( Figure 8 and Fig. 9 ). In the case of 4.38×10 8 4.38×10 9 4.38×10 10 After intraventricular administration of rAAV9-V1 at a dose of vg, Gcdh - / - The survival rates of mice were 46%, 83% and 83% respectively. Figure 8 ). In the case of 4.38×10 8 4.38×10 9 4.38×10 10 After intraventricular administration of rAAV9-V2 at a dose of vg, Gcdh - / - The survival rates of mice were 83%, 81% and 100% respectively ( Fig. 9 ). Compared with treatment with rAAV9-V1, treatment with rAAV9-V2 resulted in HPD-induced Gcdh - / - The mice had a significantly higher survival rate.

[0103] 8 weeks after AAV administration, surviving mice were killed 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) with protease inhibitor cocktail (Roche, 04693159001) and SDS-PAGE loading buffer (Cowin Bio, CW0027), denatured at 95°C for 10min, and centrifuged at 12,000rpm for 10min. The supernatant was separated in 4%-10% SDS-PAGE gel (Cowin Bio, CW0022M) and blotted onto 0.2 μm PVDF transfer membrane (Merck, ISEQ00010). The protein levels of GCDH and housekeeping gene COXIV were detected by antibodies against GCDH (Abcam, ab232774) and COXIV (Abcam, ab16056), respectively. Western blot images are available at Fig.10The brain GCDH expression was detected in a dose-dependent manner ( Fig.10 Since the mouse blood-brain barrier (BBB) ​​is immature when AAV is administered, hepatic GCDH expression can be detected in the highest dose group treated with rAAV9-V2, indicating that AAV is transferred from the CNS across the BBB to peripheral tissues and organs ( Fig.10 rAAV9-V2 mediated higher levels of GCDH protein expression than rAAV9-V1, suggesting that higher levels of GCDH protein expression protect more Gcdh - / - Mice were protected from HPD challenge-induced mortality.

[0104] For Gcdh cells that received rAAV9-V2 administration - / - Mice, liquid chromatography-mass spectrometry (LC-MS) analysis for measuring GA levels showed that GA levels were significantly reduced in the brain in a dose-dependent manner (analysis of variance using a two-way ANOVA test) ( Fig.11 There were no significant differences in liver and plasma GA levels between the different treatment groups ( Fig.11 ). Brain and liver GA levels of the dead mice were also examined, and the results showed dramatically elevated GA levels compared to those of the surviving mice ( Fig.11 ). Considering the mice that died, ICV administration of rAAV9-V2 effectively reduced GA accumulation and improved survival after HPD challenge. It was also observed that 4.38×10 8 The vg-treated groups showed no significant difference in brain GA levels, but had significantly improved survival, suggesting that even a modest reduction in brain GA levels can protect Gcdh - / - Mice were protected from the acute encephalopathy crisis induced by HPD challenge, indicating that lowering GA levels in the CNS is more important than lowering GA levels in peripheral tissues.

[0105] Example 5. Long-term efficacy study

[0106] A long-term study was conducted to test the durability of rAAV9-V2 efficacy. AAV9-V2 was administered at 0, 5×10 8 , 2.5×10 9 , 1×10 10 The dose of vg was administered intraventricularly to newborn Gcdh - / - In mice. Unlike the study described in Example 4, HPD challenge was not used in this study. At 4 and 13 weeks after AAV administration, animals were sacrificed for biochemical and histopathological analysis.

[0107] Glutaric acid (GA) and 3-hydroxyglutaric acid (3-OHGA) levels were determined by liquid chromatography tandem mass spectrometry (LC-MS / MS). Fig.12Results measured 4 weeks after AAV administration are shown. Fig.12 As shown in Figure 3, brain GA and 3-OHGA levels were significantly reduced in a dose-dependent manner. CSF GA, serum GA and serum 3-OHGA levels showed a dose-dependent decrease trend, but not significant. GA and 3-OHGA levels in liver and urine did not show significant changes. Fig.13 LC-MS / MS results measured 13 weeks after AAV administration are shown. Fig.13 As shown in Figure 2, brain GA and 3-OHGA levels were significantly reduced in a dose-dependent manner. Serum GA and 3-OHGA levels showed a dose-dependent decrease trend, among which 3-OHGA level decreased more significantly. GA and 3-OHGA in liver and urine did not show significant changes. CSF GA showed a dose-dependent decrease trend ( Fig.13 ). GA and 3-OHGA levels in brain and serum and GA levels in CSF showed even greater decreases 13 weeks after AAV administration compared to the results at 4 weeks.

[0108] The expression of GCDH was determined by ELISA. After AAV administration, GCDH expression was most abundant in the brain, followed by the heart, spinal cord, and liver ( Fig.14 ). At 4 weeks after AAV administration, the mean brain GCDH concentration was 6,178.5 ng / mg total protein, and at 13 weeks after AAV administration, the mean brain GCDH concentration was 12,143.5 ng / mg total protein. At 4 weeks after AAV administration, the mean liver GCDH concentration was 227.0 ng / mg total protein, and at 13 weeks after AAV administration, the mean liver GCDH concentration was 92.2 ng / mg total protein.

[0109] Brain histopathology was evaluated using hematoxylin-eosin (HE) staining. - / - Vacuolization was observed in the cortex and striatum of mice. After AAV administration, vacuolization was reduced in a dose-dependent manner ( Fig.15 ).

[0110] The results of this long-term study validated the role of AAV9-V2 in enhancing GCDH expression, especially in the brain, and in reducing GA and 3-OHGA levels in CNS and peripheral tissues. In addition, the results showed that long-term expression of GCDH achieved by AAV9-V2 therapy can protect CNS tissues from GCDH deficiency-induced vacuolation. Therefore, this 13-week study provides strong evidence that AAV9-V2 can be an effective therapy for a sustained period of time.

[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] AGCGAGCGAGCGCGCAG

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

[0565] 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] >HGCDH 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] GTGGGCGGGCTGGTGGGTGCCCTGAGACTGCTCCTCCGCCTGGAGCCATAGCCACCCCA

[0602] CCTCAAGGCCCCTCTGTCCTTGGGGCTGGGGCTTCCTGTGGCCTAGGCCTGGGCCTGAA

[0603] TTTGGGCACTGGTCCCTTTGCAG

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

[0605] 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] VSLLKRNNCGKALDIARQARDMLGGGNGISDEYHVIRHAMNLEAVNTYEGTHDIHALILGRAITGIQAFTASK*

Claims

1. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11-18, wherein the nucleotide sequence encodes a human glutaryl-CoA dehydrogenase (hGCDH) polypeptide having an amino acid sequence as shown in SEQ ID NO:

33.

2. The isolated nucleic acid molecule of claim 1, wherein the nucleotide sequence is SEQ ID NO:

12.

3. An expression cassette comprising the isolated nucleic acid molecule according to claim 1 or claim 2.

4. The expression cassette of claim 3, further comprising a promoter component operably linked to the 5' of the nucleotide sequence encoding the hGCDH polypeptide.

5. The expression cassette of claim 4, wherein the promoter component comprises (a) a constitutive promoter, and (b) an enhancer and / or an intron-derived fragment.

6. The expression cassette of claim 5, wherein the constitutive promoter is a cytomegalovirus (CMV) promoter, a chicken β-actin (CBA) promoter, or a human elongation factor-1α (EF-1α) promoter.

7. The expression cassette according to claim 6, wherein the promoter is a CMV promoter having a nucleotide sequence as shown in SEQ ID NO: 25, a CBA promoter having a nucleotide sequence as shown in SEQ ID NO: 26, or an EF-1α core promoter having a nucleotide sequence as shown in SEQ ID NO:

29.

8. The expression cassette of claim 5, wherein the enhancer is a CMV enhancer.

9. The expression cassette according to claim 8, wherein the CMV enhancer has the nucleotide sequence shown in SEQ ID NO:

24.

10. The expression cassette of claim 5, wherein the intron-derived fragment is derived from an intron of SV40.

11. The expression cassette according to claim 10, wherein the intron-derived fragment has the nucleotide sequence as shown in SEQ ID NO:

28.

12. The expression cassette of claim 5, wherein the intron-derived fragment consists of one or more fragments derived from one or more intron regions of the human GCDH gene.

13. The expression cassette according to claim 12, wherein the intron-derived fragment comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 27, 30, 31.

14. The expression cassette according to claim 13, wherein the intron-derived fragment comprises or consists of any two or three nucleotide sequences selected from the group consisting of the nucleotide sequences shown in any one of SEQ ID NOs: 27, 30 and 31, such as a combination of SEQ ID NO: 27 and SEQ ID NO:

30.

15. The expression cassette of claim 5, wherein the intron-derived fragment is a hybrid intron.

16. The expression cassette according to claim 15, wherein the hybrid intron has a nucleotide sequence as shown in SEQ ID NO:

32.

17. The expression cassette according to any one of claims 4 to 16, wherein the promoter components comprise a CMV enhancer, a CBA promoter and an intron-derived fragment, and the intron-derived fragment is derived from the hGCDH gene or SV40.

18. The expression cassette according to any one of claims 4 to 16, wherein the promoter component comprises an EF-1α promoter, such as an EF-1α core promoter, and an intron-derived fragment or a hybrid intron derived from a hGCDH gene.

19. The expression cassette according to any one of claims 4 to 18, wherein the promoter component comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NOs: 2 to 9.

20. The expression cassette of claim 19, wherein the promoter component comprises or consists of the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO:

9.

21. The expression cassette according to any one of claims 3 to 20, wherein the expression cassette comprises or consists of the nucleotide sequence as shown in SEQ ID NO: 20 or SEQ ID NO:

21.

22. An rAAV vector comprising the nucleic acid molecule of claim 1 or claim 2 or the expression cassette of any one of claims 3-21.

23. The rAAV vector of claim 22, further comprising two AAV inverted terminal repeats (ITRs).

24. The rAAV vector of claim 23, 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.

25. A viral particle comprising the rAAV vector according to any one of claims 22-24 packaged into an AAV capsid.

26. The viral particle of claim 25, wherein the capsid has CNS tropism.

27. The viral particle of claim 26, wherein the capsid is an AAV9 or AAV PHP.B capsid.

28. A pharmaceutical composition comprising the rAAV vector according to any one of claims 22-24 or the viral particle according to any one of claims 25-27 and a pharmaceutically acceptable excipient.

29. A method for treating glutaric aciduria type I (GA-I) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective dose of the rAAV vector of any one of claims 22-24, the viral particle of any one of claims 25-27, or the pharmaceutical composition of claim 28.

30. The method of claim 29, wherein the subject is a mammal.

31. The method of claim 30, wherein the subject is a human.

32. The method of any one of claims 29-31, wherein the rAAV vector, the viral particle or the pharmaceutical composition is administered to peripheral tissue and / or the nervous system.

33. The method of claim 32, wherein the administration to a peripheral tissue is via an intravenous (IV), intramuscular (IM), subcutaneous (SC), intraarterial, intraperitoneal (IP), intradermal, transdermal, oral, nasal, or rectal route.

34. The method of claim 32, wherein the administration to the nervous system is via the intracerebroventricular (ICV), intrathecal, or intracisternal (ICM) route.

35. Use of the rAAV vector according to any one of claims 22-24 or the viral particle according to any one of claims 25-27 for the manufacture of a medicament for the treatment of GA-I.

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