Constructs for enhancing gene expression

Abstract

The present disclosure provides an expression construct, a vector, a viral particle, or a composition. The expression construct comprises a transcriptional regulatory element operably linked to a polynucleotide sequence of interest, wherein the expression construct comprises a promoter, an enhancer upstream of the promoter, and optionally an intron. The present disclosure also provides uses of an expression construct, a vector, a viral particle, or a composition.

Description

[0001] This application claims the benefit and priority of PCT application PCT / CN2022 / 123892, filed on October 8, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure relates to a nucleic acid construct. The invention also relates to the said nucleic acid construct, expression vector, and its uses. Background Technology

[0003] There remains an unmet need in the art for alternative and preferred improved methods for regulating transcription of transcripts and optionally regulating the expression of proteins or peptides of interest. Summary of the Invention

[0004] This disclosure provides an expression construct comprising a transcriptional regulatory element operatively linked to a polynucleotide sequence of interest, wherein the expression construct includes a promoter and an enhancer, the enhancer being located upstream of the promoter.

[0005] The expression construct described herein includes the following elements in the 5' to 3' orientation:

[0006] a) Enhancer 3, which is optional;

[0007] b) Enhancer 2;

[0008] c) Enhancer 1; and

[0009] d) Promoter;

[0010] The enhancer 1 comprises all or part of a sequence selected from SEQ ID NO: 13 and 15 and a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or at least 99.8% identity with SEQ ID NO: 13 and 15, wherein all or part of the sequence retains the function of the enhancer, and

[0011] The enhancer 2 comprises all or part of a sequence containing at least one of SEQ ID NO: 8-11 and 13 and a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or at least 99.8% identity with SEQ ID NO: 8-11 and 13, wherein all or part of the sequence retains the function of the enhancer.

[0012] In some embodiments, the enhancer 3 comprises all or part of a sequence containing at least one of SEQ ID NO: 8-11 and a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or at least 99.8% identity with SEQ ID NO: 8-11, wherein all or part of the sequence retains the function of the enhancer.

[0013] In some embodiments, the promoter comprises all or part of a sequence selected from SEQ ID NO: 2-6 and sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or at least 99.8% identity with SEQ ID NO: 2-6, wherein all or part of the sequence retains the functionality of the promoter.

[0014] In some embodiments, the promoter comprises all or part of a sequence selected from SEQ ID NO: 3-6 and sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or at least 99.8% identity with SEQ ID NO: 3-6, wherein all or part of the sequence retains the functionality of the promoter.

[0015] In some embodiments, enhancer 1 comprises all or part of a sequence containing at least one of the sequences selected from SEQ ID NO: 13 and 15 and sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or at least 99.8% identity with SEQ ID NO: 13 and 15, wherein all or part of the sequence retains the function of the enhancer; enhancer 2 comprises all or part of a sequence containing at least one of the sequences selected from SEQ ID NO: 8-9 and sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or at least 99.8% identity with SEQ ID NO: 8-9, wherein all or part of the sequence retains the function of the enhancer.

[0016] In some embodiments, enhancer 1 comprises all or part of a sequence containing at least one of the sequences selected from SEQ ID NO: 13 and 15 and sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or at least 99.8% identity with SEQ ID NO: 13 and 15, wherein all or part of the sequence retains the function of the enhancer; enhancer 2 comprises all or part of a sequence containing SEQ ID NO: 8 and sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or at least 99.8% identity with SEQ ID NO: 8, wherein all or part of the sequence retains the function of the enhancer.

[0017] In some implementations, the expression construct further includes non-translated intron regions.

[0018] In some embodiments, the untranslated intron region comprises all or part of a sequence selected from the group consisting of SEQ ID NO: 24-43 and sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or at least 99.8% identity with SEQ ID NO: 24-43.

[0019] In some implementations, the untranslated intron region is operatively linked to the 5' end of the polynucleotide sequence of interest.

[0020] In some embodiments, the untranslated intron region is located between the 5' and 3' ends of the polynucleotide sequence of interest.

[0021] On the other hand, this disclosure provides a vector comprising the expression construct of this disclosure. The vector is a viral vector, preferably an AAV vector. The vector further comprises two adeno-associated virus inverted terminal repeat (ITR) sequences located flanking the expression construct, and preferably further comprises a polyA sequence.

[0022] On the other hand, this disclosure provides an adeno-associated virus (AAV) comprising the vector and capsid protein of this disclosure.

[0023] In some embodiments, the AAV is selected from serotypes AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, AAVhu37, or any of the AAV serotypes isolated from humans or non-human mammals, or a variant thereof.

[0024] On the other hand, this disclosure provides a composition comprising the expression construct, vector or AAV of this disclosure and a pharmaceutically acceptable excipient.

[0025] On the other hand, this disclosure provides expression constructs, vectors, or AAVs of this disclosure for use in therapeutic methods.

[0026] In some embodiments, this disclosure provides the use of the expression constructs, vectors, or AAVs of this disclosure in the preparation of medicaments for treating diseases or conditions in subjects.

[0027] In some embodiments, this disclosure provides an expression construct, vector, or AAV of this disclosure for use in methods of treating a disease or condition in a subject.

[0028] In some embodiments, this disclosure provides a method for treating a disease or condition in a subject, the method comprising administering an effective amount of an expression construct, vector, or AAV of this disclosure to the patient.

[0029] On the other hand, this disclosure provides expression constructs, vectors, or AAVs for use in methods of expressing the nucleotide sequences of interest in subjects.

[0030] On the other hand, this disclosure provides expression constructs, vectors, or AAVs for use in methods of expressing the nucleotide sequences of interest in subjects. Attached Figure Description

[0031] Figure 1The luciferase and GCase activities of chimeric HSRE constructs in HepG2 cells are shown. (A) Comparison of luciferase activities of different CHSREs based on promoter HSRE002. The PG135 group showed significantly higher luciferase activity than the PG130 group (P < 0.05), and the PG139, PG140, PG131, PG132, PG141, PG142, and PG143 groups showed significantly higher luciferase activity than the PG130 group (P < 0.01). (B) Comparison of luciferase activities of different CHSREs based on promoter HSRE005. The PG148 and PG151 groups showed significantly higher luciferase activity than the PG145 group (P < 0.05), and the PG146, PG149, PG147, and PG150 groups showed significantly higher luciferase activity than the PG145 group (P < 0.01). (C) Comparison of luciferase activities of different CHSREs based on promoter HSRE003. (D) Comparison of luciferase activity of different CHSREs based on promoter HSRE004. All groups compared with group PG165 showed P<0.01. (E) Comparison of GCase activity of different CHSREs based on promoter HSRE002. Group PG025 showed P<0.05 compared with group PG022. Groups PG023, PG026, PG028, PG029, PG030, and PG031 showed P<0.01 compared with group PG022. (F) Comparison of GCase activity of different CHSREs based on promoter HSRE005. (G) Comparison of GCase activity of different CHSREs based on promoter HSRE004. P<0.05 between group PG035 and group PG032, and P<0.01 between the other groups and group PG032. (H) Comparison of GCase activity of different CHSREs based on promoter HSRE003. P<0.05 between group PG042 and group PG039, and P<0.01 between groups PG040, PG041, PG043, PG044, and PG045 and group PG039. P<0.05 between groups PG049 and PG052 and group PG046, and P<0.01 between groups PG047, PG048, and PG051 and group PG046. Values ​​are expressed as mean ± SEM. N=3 for each experimental group.

[0032] Figure 2The luciferase and GCase activities of the HSRE012 construct in HepG2 are shown. (A) Comparison of luciferase activities of constructs at different positions of HSRE012 based on promoter HSRE002. Significant differences were observed: PG133 vs. PG131 (P<0.01), PG134 vs. PG132 (P<0.01). (B) Comparison of luciferase activities of constructs with different copy numbers of HSRE012 based on promoter HSRE002. No significant differences were observed: PG136 vs. PG131, PG137 vs. PG132, PG138 vs. PG135 (values: mean ± SEM, N = 3 per group). (C) GCase activity of constructs at different positions of HSRE012 based on promoter HSRE002. Significant differences were found: PG053 vs. PG025 (P<0.01), PG054 vs. PG026 (P<0.01). (D) Comparison of GCase activity of constructs with different copy numbers of HSRE012 based on promoter HSRE002. No significant differences: PG055 vs. PG025, PG056 vs. PG026, PG057 vs. PG027 (values: mean ± SEM, N = 3 for each group).

[0033] Figure 3 The in vitro GCase activity of HSREs binding to different exogenous intron constructs is shown. (A) GCase activity of constructs with endogenous introns in Huh7 cells. P < 0.05 for PG059 compared to PG058. (B) In vitro GCase activity of different constructs in HepG2 cells. P < 0.01 for PG076, PG078, and PG081 compared to PG074. P < 0.01 for PG090 compared to PG082, and P < 0.05 for PG091 compared to PG082. P < 0.01 for PG093, PG094, PG095, PG097, PG098, PG099, PG100, and PG101 compared to PG092. (C) GCase activity of different constructs in HEK293T cells. Error bars represent mean ± SEM. N = 3 for each experimental group.

[0034] Figure 4 GCase activity of chimeric HSREs binding to constructs with different introns is shown. (A) GCase activity of different constructs in HepG2 cells. P < 0.05 for G107 and PG108 groups compared to PG168 group. P < 0.01 for PG114 and PG115 groups compared to PG169 group. (B) GCase activity of different constructs in HEK293T cells. Error bars represent mean ± SEM. N = 3 for each experimental group.

[0035] Figure 5 The following figures show GCase activity in serum and tissue lysates 2 weeks after injection of AAV8 vector at a dose of 2E12 vg / kg in wild-type mice. (A) GCase activity measured in serum. PG026, PG037, PG051, and PG105 groups showed P < 0.05 compared to PG127, and PG103, PG104, and PG105 groups showed P < 0.01 compared to PG127. PG103 and PG104 groups showed P < 0.01 compared to PG102. PG103 and PG104 groups showed P < 0.01 compared to PG026. (B) GCase activity measured in liver lysates. PG026 group showed P < 0.05 compared to PG127, and PG103, PG104, and PG105 groups showed P < 0.01 compared to PG127. (C) GCase activity measured in lung lysates. P<0.01 between PG103 and PG104 groups and PG102 group. P<0.01 between PG103 and PG104 groups and PG026 group. P<0.01 between PG105 group and PG051 group. (D) GCase activity measured in spleen lysates. P<0.05 between PG103 and PG026 groups and PG127 group. P<0.05 between PG103 and PG026 groups and PG104 groups and PG127 group. P<0.05 between PG104 group and PG102 group. P<0.01 between PG103 group and PG026 group. P<0.05 between PG103 group and PG026 group. Compared with PG127, groups PG026, PG103, PG104, and PG105 showed P < 0.01. Compared with PG102, groups PG103 and PG104 showed P < 0.01. Compared with PG026, group PG103 showed P < 0.01. Values ​​are expressed as mean ± SEM. N = 4 for each experimental group.

[0036] Figure 6The significant efficacy of the AAV8 gene therapy candidate regulated by chimeric HSREs in Gaucher disease mice is demonstrated. (A) Serum GCase activity measured 8 weeks after injection of the AAV8 candidate at a dose of 2E12 vg / kg. P < 0.05 compared with buffer control or imiglucerase group in PG118, PG119, PG120 and PG122 groups. P < 0.05 compared with PG127 group in PG118 and PG122 groups, and P < 0.01 compared with PG127 group in G119 and PG120 groups. (B) Substrate accumulation in serum measured 8 weeks after injection of the AAV8 candidate at a dose of 2E12 vg / kg. P < 0.01 compared with buffer control or imiglucerase group in all groups. The PG119 group was significantly different from the PG127 group (P < 0.05), and the PG118, PG120, and PG122 groups were significantly different from the PG127 group (P < 0.01). Serum glucosamine levels in PG119 mice were below the detection limit. The Naïve group represents the corresponding wild-type mice. Values ​​are expressed as mean ± SEM. N = 5 for each experimental group.

[0037] Figure 7 GCase activity in tissue lysates of Gaucher disease mice 12 weeks after injection of the AAV8 candidate at a dose of 2E12 vg / kg is shown. (A) GCase activity measured in liver lysates. P < 0.01 for PG118-PG122 groups compared with buffer control or imiglucerase groups. P < 0.05 for PG120 and PG122 groups compared with PG127 groups, and P < 0.01 for PG118 and PG119 groups compared with PG127 groups. (B) GCase activity measured in lung lysates. P < 0.05 for PG118-PG122 groups compared with buffer control or imiglucerase groups. P < 0.05 for PG118 and PG122 groups compared with PG127 groups, and P < 0.01 for PG119 and PG120 groups compared with PG127 groups. (C) GCase activity measured in spleen lysates. The PG118-PG122 groups showed P < 0.01 compared to the buffer control group. The PG120 group showed P < 0.05 compared to the imiglucerase group, and the PG118, PG119, and PG122 groups showed P < 0.01 compared to the imiglucerase group. The PG118 and PG122 groups showed P < 0.05 compared to the PG127 group, and the PG119 and PG120 groups showed P < 0.01 compared to the PG127 group. The Naïve group represents wild-type mice. Values ​​are expressed as mean ± SEM. N = 5 for each experimental group.

[0038] Figure 8The accumulation of glucosamine substrate in tissue lysates in Gaucher disease mice 12 weeks after injection of the AAV8 candidate at a dose of 2E12 vg / kg is shown. (A) Substrate accumulation measured in liver lysates. P < 0.01 compared with buffer control or imiglucerase group in PG001, PG011, PG119, and PG120 groups. P < 0.01 compared with PG127 group in PG119 and PG120 groups. (B) Substrate accumulation measured in lung lysates. P < 0.05 compared with buffer control or imiglucerase group in PG001 group, and P < 0.01 compared with buffer control or imiglucerase group in PG011, PG119, and PG120 groups. P < 0.05 compared with PG127 group in PG119 and PG120 groups. (C) Substrate accumulation measured in spleen lysates. The PG001, PG011, PG119, and PG120 groups showed no significant difference compared to the buffer control or imiglucerase group (P < 0.01). The PG119 and PG120 groups showed no significant difference compared to the PG127 group (P < 0.01). Naïve mice represent wild-type mice. Error bars represent mean ± SEM. N = 5 for each experimental group. Glucosaminoglycine levels in different tissue lysates were analyzed using LC-MS / MS.

[0039] Figure 9The following figures illustrate GCase activity in serum and tissue lysates 2 weeks after injection of the AAV9 candidate at a dose of 2E12 vg / kg in wild-type mice. (A) GCase activity measured in serum. P<0.05 in the PG124 group compared to the buffer control group, and P<0.01 in the PG123, PG125, and PG126 groups compared to the buffer control group. P<0.05 in the PG124 and PG125 groups compared to the PG001 group, and P<0.01 in the PG123 and PG126 groups compared to the PG001 group. P<0.05 in the PG125 group compared to the PG128 group, and P<0.01 in the PG123 and PG126 groups compared to the PG128 group. (B) GCase activity measured in liver lysates. P<0.01 in the PG123-PG126 groups compared to the buffer control group. Compared with the PG001 group, the PG124 and PG125 groups showed P < 0.05, and the PG123 and PG126 groups showed P < 0.01 compared with the PG001 group. The PG124 and PG125 groups showed P < 0.05 compared with the PG128 group, and the PG123 and PG126 groups showed P < 0.01 compared with the PG128 group. (C) GCase enzyme activity measured in lung lysates. Compared with the buffer control group, the PG124 group showed P < 0.05, and the PG123, PG125, and PG126 groups showed P < 0.01 compared with the buffer control group. The PG124 group showed P < 0.05 compared with the PG001 group, and the PG123, PG125, and PG126 groups showed P < 0.01 compared with the PG001 group. P < 0.05 for PG123 and PG125 groups compared to PG128, and P < 0.01 for PG126 group compared to PG128. (D) GCase enzyme activity measured in spleen lysate. P < 0.01 for PG123-PG126 groups compared to buffer control group. P < 0.05 for PG124 group compared to PG001 group, and P < 0.01 for PG123, PG125, and PG126 groups compared to PG001 group. P < 0.05 for PG124 group compared to PG128 group, and P < 0.01 for PG123, PG125, and PG126 groups compared to PG128 group. Values ​​are expressed as mean ± SEM. N = 4 for each experimental group. Detailed Implementation

[0040] Embodiments according to this disclosure will now be described more fully. However, aspects of this disclosure may be embodied in different forms and should not be construed as limited to the embodiments listed herein. Rather, these embodiments are provided to make this disclosure comprehensive and complete, and to fully convey the scope of the invention to those skilled in the art. The terminology used in the description herein is for descriptive purposes only and is not intended to be limiting.

[0041] Unless otherwise defined, all terms used herein (including technical and scientific terms) shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be understood that terms, for example, those defined in common dictionaries, shall be interpreted as having the meaning consistent with their meaning in the context of this application and related technologies, and shall not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0042] definition

[0043] When used in the specification and appended claims of this invention, the singular form also means to include the plural form, unless the context clearly indicates otherwise.

[0044] As used herein, the term "comprising" means that a composition or method includes the described elements, but does not exclude other elements.

[0045] As used herein, the terms “nucleotide” and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, which may be ribonucleotides or deoxyribonucleotides. Therefore, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derived nucleotide bases, or substantially composed of or consisting of such bases.

[0046] As used in this article, “expression” refers to a two-step process of transcribing polynucleotides into mRNA and / or subsequently translating the transcribed mRNA into peptides, polypeptides, or proteins. If the polynucleotides are derived from genomic DNA, expression in eukaryotic cells may involve mRNA splicing.

[0047] When applied to polynucleotides, the term "encoding" means that if a polynucleotide can be transcribed to produce mRNA of a polypeptide and / or fragments thereof, then the polynucleotide is said to "encode" the polypeptide. An antisense strand is a complement to such a nucleic acid, and the coding sequence can be inferred from it.

[0048] As used herein, the term "promoter" refers to a control sequence, which is a region of a polynucleotide sequence that controls the initiation and rate of transcription of a gene or transgene. Promoters can be constitutive, inducible, repressive, or tissue-specific. In embodiments, promoters are used in conjunction with enhancers to improve transcription efficiency. Enhancers are regulatory elements that increase the expression of target sequences.

[0049] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably and, in the broadest sense, refer to compounds consisting of subunits of two or more amino acids, amino acid analogs, or peptide mimics. These subunits may be linked by peptide bonds. Alternatively, they may be linked by other bonds such as esters, ethers, etc. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids contained in, substantially composed of, or constituted by the protein or peptide sequence. As used herein, the term “amino acid” refers to natural and / or non-natural or synthetic amino acids, including glycine and its D and L optical isomers, amino acid analogs, and peptide mimics.

[0050] "Identity" refers to the sequence similarity between two peptides or two nucleic acid molecules. The percentage of identity can be determined by comparing positions in each sequence that could be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are identical at that position. The degree of identity between sequences is a function of the number of matching positions shared by the sequences.

[0051] As used herein, the term "vector" refers to a nucleic acid containing a complete replicon, or consisting substantially of or composed of a complete replicon, such that when the vector is placed inside a cell by processes such as transfection, infection, or transformation, it can be replicated. It should be understood in the art that once inside a cell, a vector can replicate as an extrachromosomal (epitaphthous) element or can integrate into the host cell chromosome. Vectors can include nucleic acids derived from retroviruses, adenoviruses, herpesviruses, baculoviruses, modified baculoviruses, papillomaviruses, AAV viral vectors, lentiviral vectors, adenoviral vectors, alphavirus vectors, etc. Alphavirus vectors, such as those based on Semliki Forest virus and those based on Sindbis virus, have also been developed for gene therapy and immunotherapy. See, for example, Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827.

[0052] As used herein, the term "adeno-associated virus" or "AAV" refers to a member of the viral class associated with that name and belonging to the genus Dependoparvovirus in the family Parvoviridae. Adeno-associated virus is a single-stranded DNA virus that grows only in cells where certain functions are provided by a co-infected helper virus. All AAV serotypes exhibit remarkably similar replication characteristics mediated by homologous rep genes; and all carry three associated capsid proteins. At least 13 naturally occurring AAV serotypes are known in the art by serial number. Non-limiting exemplary serotypes used in the methods disclosed herein include any of these 13 serotypes, such as AAV2, AAV8, AAV9, or variant serotypes such as AAV-DJ and AAV PHP.B. AAV particles contain three major viral proteins, VP1, VP2, and VP3, and are substantially composed of or consist of said proteins. In this embodiment, AAV refers to serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13. In this embodiment, the AAV particle comprises an AAV capsid protein selected from the group consisting of AAVPHP.B, AAVrh74, AAV 110, AAV 204, AAV 214, AAV 214A, AAV 214e, AAV 214e8, AAV214e9, AAV 214e10, AAV ITB102_45, and AAV 214AB. In this embodiment, AAV refers to serotypes AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13, AAVrh10, AAVhu37, or any AAV serotype isolated from humans and non-human mammals, or a variant thereof. In this embodiment, AAV particles comprise a subset selected from AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9... .24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a,AAV42-5b、AAV42-6b、AAV42-8、AAV42-10、AAV42-11、AAV42-12、AAV42-13、AAV42-15、AAV42-aa、AAV43-1、AAV43-12、AAV43-20、AAV43-21、AAV43-23、 AAV43-25、AAV43-5、AAV44.1、AAV44.2、AAV44.5、AAV223.1、AAV223.2、AAV223.4、AAV223.5、AAV223.6、AAV223.7、AAV1-7 / rh.48、AAV1-8 / rh.49、AAV 2-15 / rh.62、AAV2-3 / rh.61、AAV2-4 / rh.50、AAV2-5 / rh.51、AAV3.1 / hu.6、AAV3.1 / hu.9、AAV3-9 / rh.52、AAV3-11 / rh.53、AAV4-8 / rh.64、AAV4-9 / rh. .54、AAV4-19 / rh.55、AAV5-3 / rh.57、AAV5-22 / rh.58、AAV7.3 / hu.7、AAV16.8 / hu.10、AAV16.12 / hu.11、AAV29.3 / bb.1、AAV29.5 / bb.2、AAV106.1 / hu.3 7、AAV114.3 / hu.40、AAV127.2 / hu.41、AAV127.5 / hu.42、AAV128.3 / hu.44、AAV130.4 / hu.48、AAV145.1 / hu.53、AAV145.5 / hu.54、AAV145.6 / hu.55、AAV161.10 / hu.60、AAV161.6 / hu.61、AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu.16、AAV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAVA3.3、AAVA 3.4、AAVA3.5、AAVA3.7、AAVC1、AAVC2、AAVC5、AAV-DJ、AAV-DJ8、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AA Vpi.2、AAVrh.60、AAVrh.44、AAVrh.65、AAVrh.55、AAVrh.47、AAVrh.69、AAVrh.45、AAVrh.59、AAVhu.12、AAVH6、AAVLK03、AAVH-1 / hu.1、AAVH-5 / hu.3AAVLG-10 / rh.40、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVN721-8 / rh.43、AAVCh.5、AAVCh.5R1、AAVcy.2、AAVcy .3、AAVcy.4、AAVcy.5、AAVCy.5R1、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVcy.6、AAVhu.1、AAVhu.2、AAVhu.3 、AAVhu.4、AAVhu.5、AAVhu.6、AAVhu.7、AAVhu.9、AAVhu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu u.17、AAVhu.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23.2、AAVhu.24、AAVhu.25、AAVhu.27、AAVhu.28、AAV hu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.40、AAVhu.41、AA Vhu.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu .48、AAVhu.48R1、AAVhu.48R2、AAVhu.48R3、AAVhu.49、AAVhu.51、AAVhu.52、AAVhu.54、AAVhu.55、AAVhu.56 、AAVhu.57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu.67、AAVhu.14 / 9、AAVhu.t 19、AAVrh.2、AAVrh.2R、AAVrh.8、AAVrh.8R、AAVrh.10、AAVrh.12、AAVrh.13、AAVrh.13R、AAVrh.14、AAVrh.17、AAVrh.18、AAVrh.19、AAVrh.20、AAVrh.21、AAVrh.22、 AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh.34、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37R2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.46AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, AAAV, BAAV, goat AAV, bovine AAV, AAVhE1.1, AAVhEr1.5, AAVhER1.14, AAVhEr1.8, AAVhEr1.16, AAVhEr1.18, AAVhEr1.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAVShuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPENN AAV 10, Japanese AAV 10 serotype, AAV CBr-7.1, AAVCBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-E1, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAVCBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAVCHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-P1, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAVCKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-B1, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-H1, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-F1, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-1, AAV CLv1-1, AAV Clv1-10, AAV CLv1-2, AAV CLv-12AAV CLv1-3, AAV CLv-13, AAV CLv1-4, AAV Clv1-7, AAV Clv1-8, AAV Clv1-9, AAV CLv-2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8 CLv-D1、AAVCLv-D2、AAV CLv-D3、AAV CLv-D4、AAV CLv-D5、AAV CLv-D6、AAV CLv-D7、AAV CLv-D8、AAVCLv-E1、AAV-CAV1、AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAVCLv-M1, AAV CLv-M11, AAV CLv-M2, AAV CLv-M1, AAV CLv-M2, AAV CLv-M5, AAV-CLv-AAV CLv-M7, AAV CLv-M8, AAVCLv-M9, AAV CLv-R1, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, RAVCLv-AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAVCSp-3, AAV CSp-4, AAV CSp-6, AAV Csp-7, AAV Cp-8 CSp-8.10、AAV CSp-8.2、AAVCSp-8.4、AAV CSp-8.5、AAV CSp-8.6、AAV CSp-8.7、AAV CSp-8.8、AAV CSp-8.9、AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12, AAV F13 / HSC13、AAVF14 / HSC14、AAVF15 / HSC15、AAVF16 / HSC16、AAVF17 / HSC17、AAVF2 / HSC2、AAVF3 / HSC3、A AVF4 / HSC4、AAVF5 / HSC5、AAVF6 / HSC6、AAVF7 / HSC7、AAVF8 / HSC8、AAVF9 / HSC9、AAV-PHP.B (PHP.B)、AAV -PHP.A(PHP.A), G2B-26, G2B-13, TH1.1-32, TH1.1-35, AAVPHP.B2, AAVPHP.B3, AAVPHP.N / PHP.B-DGT.AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SG S, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP (3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, The AAV capsid proteins comprised of AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S / G2A12, AAVG2A15 / G2A3, AAVG2B4, AAVG2B5, and their variants.

[0053] As used herein, "AAV vector" refers to a vector containing one or more heterologous nucleic acid (HNA) sequences and one or more AAV inverted terminal repeat (ITR) sequences. Such AAV vectors can replicate in host cells that provide the functions of rep and cap gene products and allow the packaging of nucleic acids between the ITRs into infectious viral particles. In embodiments, within the ITRs flanking the infectious AAV particle, the AAV vector contains a promoter, at least one nucleic acid encoding at least one protein or RNA, and / or an enhancer and / or a terminator. The nucleic acids between the ITRs and ITRs can be encapsulated in an AAV capsid, and this capsidated nucleic acid can be referred to as the "AAV vector genome." In addition to the capsidated portion, the AAV vector may contain other elements, such as antibiotic resistance genes or other elements known in the art, which are included in plasmids for manufacturing purposes but are not packaged into AAV particles.

[0054] As used herein, the term "viral capsid" or "capsid" refers to the protein outer shell or capsid of a viral particle. The capsid functions to encapsulate, protect, transport the viral genome, and / or release it into host cells. The capsid is typically composed of oligomeric subunits of proteins ("capsid proteins"). The AAV viral capsid is composed of a mixture of three viral capsid proteins: VP1, VP2, and VP3.

[0055] "AAV virion" or "AAV virus particle" or "AAV particle" refers to a viral particle composed of at least one AAV capsid protein and capsidized polynucleotides from an AAV vector (referred to herein as the AAV vector genome).

[0056] The “subject” for diagnosis or treatment is an animal, such as a mammal or a human. Subjects are not limited to a specific species and include non-human animals undergoing diagnosis or treatment, as well as non-human animals or animal models of infection, including but not limited to apes, mice, rats, canines, or rabbits, and other livestock, livestock, or pets. In this implementation, the subject is a human.

[0057] When used herein, “treatment” of a disease in a subject means: (1) preventing the onset of symptoms or disease in a subject who is susceptible to the disease or who has not yet shown symptoms of the disease; (2) suppressing the disease or preventing its development; or (3) improving the disease or disease symptoms or causing their resolution. As understood in the art, “treatment” is a method of obtaining a beneficial or desired outcome (including clinical outcomes). For the purposes of this art, a beneficial or desired outcome may include, but is not limited to, one or more of the following: reduction or improvement of one or more symptoms, reduction of the severity of a condition (including the disease), stabilization of the state of a condition (including the disease) (i.e., preventing its deterioration), delay or slowing the progression of a condition (including the disease), improvement or relief of the state of a condition (including the disease), and relief (whether partial or complete), whether detectable or undetectable.

[0058] As used herein, the term "effective amount" means an amount sufficient to achieve the desired effect. In the case of therapeutic or preventative applications, the effective amount may depend on the type and severity of the disease in question, as well as the characteristics of the individual subject such as general health, age, sex, weight, and tolerance to the pharmaceutical composition. In the case of gene therapy, in one embodiment, the effective amount is an amount sufficient to induce partial or full function of the defective gene in the subject. In other embodiments, the effective amount of AAV viral particles is an amount sufficient to cause gene expression in the subject. Those skilled in the art will be able to determine a suitable amount based on these and other factors.

[0059] In implementation, the effective amount will depend on the scale and nature of the application under discussion. It also depends on the characteristics and sensitivity of the target subject and the method used. Those skilled in the art will be able to determine the effective amount based on these and other considerations. The effective amount may include, substantially constitute, or consist of the composition, depending on the implementation method.

[0060] As used herein, the term "administration" means the delivery of a substance to a subject, such as an animal or a human. Administration can be performed once, continuously, or intermittently throughout the treatment. Methods for determining the most effective manner and dosage of administration are known to those skilled in the art and vary depending on the composition used for treatment, the purpose of treatment, and the age, health, or sex of the subject being treated. Administration can be single or multiple times, and the dosage level and pattern can be selected by the treating physician, or, in the case of pets and other animals, by the treating veterinarian.

[0061] Example

[0062] Unless otherwise specified, the following general approach is followed in the embodiments described below.

[0063] rAAV production

[0064] AAV8 and AAV9 viral particles were generated by transient transfection of HEK293T cells or HEK293 suspension cells with a plasmid encoding the AAV Rep and Cap genes, a plasmid containing an adenovirus helper gene, and a construct plasmid containing the GBA1 gene. rAAV particles were purified using iodixanol-based density gradient ultracentrifugation. Subsequently, rAAV was quantified by probe-based ddPCR (Biorad) and purity was characterized by silver staining.

[0065] In vitro transfection and rAAV potency assay

[0066] One day prior to transfection, HEK293T or hepatocyte lines HepG2 and Huh7 were added to 24-well plates at a density of 1.5E5 cells / well. 500 μl of complete cell culture medium was added to each well. Transfection was performed using a PEI-based transfection reagent. Specifically, 0.15 μg of a plasmid containing the transgenic sequence and 0.15 μg of a plasmid containing the luciferase reporter gene were co-transfected into each well. Forty-eight hours post-transfection, 300 μl of fresh complete cell culture medium was added to each well, and the cells were incubated for another 24 hours.

[0067] For the dual-luciferase reporter assay, cells were lysed using TransGen lysate buffer, and firefly luciferase and Renida luciferase were detected using a TransGen luciferase detection system. 96-well plates containing cell lysates and assay reagents were read on a Varioskan LUX plate reader (ThermoFisher). Firefly luciferase intensities were first normalized to Renida luciferase levels and then normalized to control levels.

[0068] For GCase activity assays, enzyme activity was determined in cell culture supernatants according to the method described below. Cells were lysed using cell lysis buffer (Promega), and luciferase activity was detected using the Steady-Glo luciferase detection system (Promega). 96-well plates containing cell lysates and assay reagents were read on a Varioskan LUX plate reader (ThermoFisher). The data shown are enzyme activities normalized to firefly luciferase intensity and then normalized to the control group. rAAV biopotency assays were performed using HEK293T, Huh7, or HepG2 cells via cell transduction. Twenty-four hours prior to transduction, cells were seeded in 24-well plates at a density of 1.5E5 cells / well. rAAV transduction was performed at a multiplicity of infection (MOI) of 1E5 or 1E6. Forty-eight hours post-infection, 300 μl of fresh complete cell culture medium was added to each well, and the cells were incubated for another 24 hours. 72 h post-infection, enzyme activity was tested in the cell culture supernatant according to the method described below.

[0069] Wild-type mouse study design

[0070] Will contain GBA1 The transgenic AAV vector was administered to 8-9 week old wild-type (C57BL / 6) male mice via tail vein injection. The AAV injection dose was 1E12 vg / ml. To assess the kinetics and persistence of transgene expression, serum GCase levels were measured at various time intervals (1, 2, or 4 weeks) post-injection. Mice were followed up for up to 4 weeks after AAV treatment and then sacrificed for biochemical and pathological analysis.

[0071] Gaucher disease mouse study design

[0072] Will contain GBA1 The transgenic AAV vector was administered via tail vein injection to 7-12 week old Gaucher disease (a combination of two different types of GBA1 mutations) mice. All mice were housed in pathogen-free, specially designed environments in individually ventilated cages. All cages, corn cob bedding, and water were sterilized before use. Cages, corn cob bedding, food, and water were changed twice weekly.

[0073] AAV injection doses ranged from 2E11 to 5E13 vg / mL. To assess the kinetics and persistence of transgene expression, serum GCase levels and substrate accumulation were measured at various time intervals following injection. Mice were followed until the endpoint study and then sacrificed for biochemical and pathological analysis.

[0074] Preparation and application of AAV / imiglucosidase

[0075] Store aliquots of rAAV at -80°C. Before injection, thaw the aliquots on ice and dilute with AAV preparation buffer. Place the diluted AAV on ice before injection and use within 2 hours.

[0076] Resuspend the imiglucerase according to the manufacturer's instructions, aliquot into equal portions (40 IU / ml), and store at -80°C. Before injection, thaw and dilute the aliquots on ice, mixing gently but thoroughly.

[0077] Serum and tissue collection

[0078] Serum was separated from fresh blood (without anticoagulant) at 4°C over 0.5 hours by centrifugation at 12,000 rpm for 15 minutes. The serum was stored at -80°C. For the imiglucerase group, serum was collected 1.5 hours after injection.

[0079] Mice were anesthetized and euthanized. Tissue was collected from mice after perfusion with saline and stored at -80°C. For the imiglucerase group, tissue samples were collected 1.5 hours after injection. The tissue samples were aliquoted into four fractions, three of which were frozen in separate tubes and stored at -80°C for GCase activity assays, glucosamine analysis, and mRNA analysis. The remaining fractions were fixed in 10% neutral buffered formalin (NBF, pH 7.4) at room temperature for approximately 24–48 h for histological analysis. Bone marrow cells were collected from the femur and tibia of both legs of mice.

[0080] Assay of GCase activity in mouse serum and tissues

[0081] Serum samples were obtained from mouse blood and stored at -80°C. Tissues were lysed using a homogenizer (Shanghai Jingxin) in tissue lysis buffer (citrate-phosphate buffer, pH 5.0, 0.25% sodium taurocholate, 1% TX-100, and a mixture of protease inhibitors) under a specific program (50 Hz, 30 s on and 30 s off, for a total of 4 min). For enzyme activity assays, β-glucocerebrosidase (acid β-glucosidase; GCase) activity was determined by a fluorescence-based assay. 4-Methylumbelliferone-β-D-glucopyranoside (4MU-Glc, Carbosynth) was used as the substrate for GCase. On the day of assay, serum was diluted 1:100 using enzyme assay buffer (citrate-phosphate buffer, pH 5.0, 0.25% sodium taurocholate, 0.25% TX-100). Tissue lysates were diluted 1:40 using lysis buffer (citrate-phosphate buffer, pH 5.0, 0.25% sodium taurocholate, 1% TX-100, and a mixture of protease inhibitors). All samples were measured at 37°C for 1 hour in citrate-phosphate buffer, pH 5.0, 0.25% sodium taurocholate, 0.25% TX-100, and 1 mM 4MU-Glc. The reaction was terminated by adding three volumes (150 μL) of stop solution (0.5 M glycine, pH 10.8). Relative fluorescence levels (RFU) were assessed using a Varioskan LUX plate reader (ThermoFisher) at excitation and emission wavelengths of 360 nm and 460 nm, respectively. Protein concentrations in the tissue lysate samples were also determined using a BCA kit (ThermoFisher). The fluorescence levels were then converted to nmol / h / ml (serum) or nmol / h / mg total protein (liver, spleen, lung, bone marrow, and brain) based on a standard curve of 4-methylumbelliferone (4-MU, Sigma-Aldrich).

[0082] Vector genome copy number, relative RNA transcription level

[0083] To determine the vector genome copy number in tissue samples following rAAV injection, DNA was isolated from frozen liver tissue using the DNeasy Blood and Tissue Kit (QIAGEN) according to the manufacturer's instructions. Following DNA isolation, probe-based qPCR (Roche) was performed to determine the vector genome copy number / reaction. The cell number / reaction ratio was calculated based on the quantification of DNA quantity. The vector genome copy number / cell ratio was then calculated by normalizing the genome copy number / reaction ratio to the cell number / reaction ratio.

[0084] To determine the relative RNA transcription levels in tissue samples following rAAV injection, RNA was isolated from frozen liver samples using the RNeasy kit (QIAGEN) according to the manufacturer's instructions. After RNA isolation, cDNA was synthesized using the Primescript RT master mix (TAKARA). 300–500 ng of RNA was added to each RT reaction. The cDNA was then diluted and applied to a probe-based qPCR (Roche) assay.

[0085] Immunohistochemistry

[0086] Mouse macrophages were detected using rabbit anti-mouse CD68 antibody (1:25 Abcam AB53444). Formalin-fixed mouse tissue was dewaxed with xylene and washed with fractionated ethanol, then the antigen was recovered using pepsin according to product usage instructions. Sections were counterstained with hematoxylin. Detection was performed using a biotin-labeled secondary antibody. Signal development was performed using a streptavidin-HRP and Tyramide signal amplification kit according to instructions.

[0087] Storage cell count

[0088] Tissue sections were stained with hematoxylin and eosin (H&E). The stained tissues were scanned using an Aperio AT2 (Leica, 40X). Tissue images were processed using Aperio ImageScope (V12.4.3.5008). All Gaucher cells on entire tissue sections of the liver and lungs of each mouse were manually counted. Gaucher cell counts from the entire section were normalized to the tissue section area (square centimeters) for use in data plots.

[0089] Glucosaminoglycine (Lyso-GL1) analysis

[0090] Tissue homogenates were prepared by homogenizing with 9 volumes (w:v) of PBS buffer. Aliquots (10 pL) of tissue lysates or serum samples were analyzed by LC / MS. Quantitative tissue Lyso-GL1 levels were normalized by tissue weight, and substrate levels in serum were normalized by serum volume. In the corresponding plots, values ​​below 10 ng / g (for tissue) and 1 ng / mL (for serum or plasma) for Lyso-GL1 quantification (LLOQ) were labeled as BQL.

[0091] Statistical analysis

[0092] Data are expressed as mean ± standard error of the mean (mean ± SEM). Statistical analysis of differences between groups was performed using GraphPad Prism software. A p-value ≤ 0.05 was considered statistically significant.

[0093] Example 1: Construct

[0094] To improve the therapeutic efficacy of gene therapy for Gaucher disease caused by metabolic disorders, this disclosure outlines a comprehensive method for designing and screening novel expression cassettes that efficiently and selectively express therapeutic GCases in the liver.

[0095] The first step involves combining the firefly luciferase gene and GBA1 The nucleotide sequence of the gene was cloned into the target vector. To confine the expression of luciferase and GCase proteins to the liver, the firefly luciferase gene and... GBA1 The nucleotide sequence of the gene is driven by a series of chimeric hepatic specific regulatory elements (CHSREs), which are rationally designed with combinations of different promoters and various regulatory elements. The promoters described herein are HSRE001, HSRE002, HSRE003, HSRE004, HSRE005, HSRE015, and HSRE016. The regulatory elements described herein are HSRE006, HSRE007, HSRE008, HSRE009, HSRE010, HSRE011, HSRE012, HSRE013, and HSRE014. In some embodiments, introns are also used to further enhance GCase expression. Table 1 shows the different promoter and various regulatory element IDs used in this disclosure and their nucleotide sequences. Codon-optimized with K321N mutation. GBA1 A gene is an example of a nucleotide sequence of interest.

[0096] Table 1. Different control elements used in this disclosure

[0097]

[0098] Table 2. Nucleotide sequences of luciferase and human GBA1

[0099]

[0100] A codon-optimized human GBA1 polypeptide sequence (SEQ ID) containing the K321N mutation but lacking the signal peptide moiety. NO: 22)

[0101] ARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDFLAPANATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSIHTYLWRRQ*

[0102] A codon-optimized polypeptide sequence of human GBA1 containing a signal peptide moiety and having a K321N mutation (SEQ ID NO: 1). NO: 23)

[0103] MEFSSPSREECPKPLSRVSIMAGSLTGLLLLQAVSWASGARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMA SCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCL GFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDFLAPANATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLY HVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSIHTYLWRRQ*

[0104] Example 2:

[0105] In vitro screening of chimeric liver-specific promoters

[0106] To restrict the expression of luciferase or GCase to the liver, a series of chimeric liver-specific regulatory elements (CHSREs) containing a core promoter and one, two, three, or more regulatory elements were specifically designed, and in vitro or in vivo screening was performed by comparing the expression efficiency of luciferase or GCase in HepG2 cells. Firefly luciferase or mGBA-C110 (codon-optimized with a K321N mutation) was also considered. GBA1 Versions) are expressed under various CHSRE controls.

[0107] Chimeric liver regulatory elements (CHSREs) consist of 1, 2, 3, or more copies of enhancers selected from HSRE010, HSRE014, HSRE012, HSRE006, and HSRE009. These enhancers are then combined with four promoters—HSRE002, HSRE004, HSRE003, or HSRE005—to drive the luciferase gene or... GBA1Gene expression was studied, and the resulting constructs PG130-PG165 and PG023, PG024, PG025, PG026, PG027, PG028, PG029, PG030, PG033, PG034, PG035, PG036, PG037, PG038, PG040, PG041, PG042, PG043, PG044, PG045, PG047, PG048, PG049, PG050, PG051, and PG052 were used to further investigate the expression of luciferase or GCase in HepG2 cells. Information on these constructs is shown in Table 3.

[0108] Table 3. Build Information

[0109]

[0110] Compared to the corresponding promoter-only versions, all luciferase constructs with CHSRE exhibited higher luciferase activity. Figure 1 A, B, C, D). When the luciferase gene is used... GBA1 When gene substitution occurs, compared to the corresponding promoter-only versions, all except PG024-CHSRE003, PG027-CHSRE006, and PG050-CHSRE029 are... GBA1 All constructs showed significantly higher GCase activity. Figure 1 E, F, G, H).

[0111] To determine the effects of these enhancers on luciferase and GBA1 To investigate whether there was a further additive effect on expression efficiency, enhancers with different copy numbers or their further tandem combinations were constructed in CHSRE. The results showed that further increases in the copy number of HSRE010 (PG141-CHSRE007 compared to PG131-CHSRE004) or HSRE007 (PG142-CHSRE008 compared to PG132-CHSRE005) did not affect luciferase expression. Figure 1 A). Similarly, further increases in the copy number of HSRE010 (PG028-CHSRE007 compared to PG025-CHSRE004) and HSRE007 (PG029-CHSRE008 compared to PG026-CHSRE005) had no effect on GCase expression. Figure 1 E). Compared to PG132-CHSRE005, CHSRE009, which further tandemly adds HSRE010 to CHSRE005 in PG143, significantly enhanced luciferase expression. Figure 1 A), however, compared with the corresponding control PG026-CHSRE005, no effect on GCase expression was observed in CHSRE009 in PG030 ( Figure 1 E).

[0112] The position of HSRE012 relative to the HSRE002 promoter was tested for its effect on luciferase or GBA1 The effects on expression were investigated, and the results showed that when HSRE012 moved downstream from upstream of the HSRE002 promoter (PG131-CHSRE004-luci and PG132-CHSRE005-luci, PG025-CHSRE004- and PG026-CHSRE005-) to downstream (PG133-CHSRE032-luci and PG134-CHSRE033-luci, PG053-CHSRE032- and PG054-CHSRE033-), luciferase or GCase expression was significantly reduced. Figure 2 A, C). As shown in PG136-CHSRE034-luci, PG137-CHSRE035-luci, PG138-CHSRE036-luci, PG055-CHSRE034-GBA1, PG056-CHSRE035-GBA1, and PG057-CHSRE036-GBA1, the increased copy number of HSRE012 in CHSRE does not further increase the expression of luciferase or GCase compared to the corresponding constructs with a single copy of HSRE012 (PG131-CHSRE004-luci, PG132-CHSRE005-luci, PG135-CHSRE006-luci, PG025-CHSRE004-GBA1, PG026-CHSRE005-GBA1, and PG027-CHSRE006-GBA1). Figure 2 B, D).

[0113] Example 3:

[0114] In vitro screening of different introns

[0115] To further enhance GCase expression, mGBA-C110 was used to extract GCase from human cells. GBA1The source endogenous introns Int001 (inserted between 27-28 bp of SEQ ID NO: 21), Int002 (inserted between 115-116 bp of SEQ ID NO: 21), Int003 (inserted between 307-308 bp of SEQ ID NO: 21), Int004 (inserted between 454-455 bp of SEQ ID NO: 21), Int005 (inserted between 588-589 bp of SEQ ID NO: 21), Int006 (inserted between 761-762 bp of SEQ ID NO: 21), Int007 (inserted between 999-1000 bp of SEQ ID NO: 21), Int008 (inserted between 1224-1225 bp of SEQ ID NO: 21), and Int009 (inserted between SEQ ID NO: 21) are: Int001 (inserted between 27-28 bp of SEQ ID NO: 21), Int002 (inserted between 115-116 bp of SEQ ID NO: 21), Int003 (inserted between 307-308 bp of SEQ ID NO: 21), Int004 (inserted between 454-455 bp of SEQ ID NO: 21), Int005 (inserted between 588-589 bp of SEQ ID NO: 21), Int006 (inserted between 761-762 bp of SEQ ID NO: 21), Int007 (inserted between 999-1000 bp of SEQ ID NO: 21), Int008 (inserted between 1224-1225 bp of SEQ ID NO: 21 Introns of SEQ ID NO: 21 (between 1388-1389 bp) and Int0010 (intercalated between 1505-1506 bp) were cloned under the HSRE002 promoter to obtain constructs PG059, PG060, PG061, PG062, PG063, PG064, PG065, PG066, PG067, and PG068. The construct without any introns is PG058. Endogenous introns selected from Int002, Int003, and Int005, and exogenous introns selected from Int011 and Int020 were cloned under the HSRE002 promoter using mGBA-C110 to obtain constructs PG069-PG073. These constructs, along with PG058 and PG059, were transfected into Huh7 cells to study GCase activity in the cell culture supernatant. It was demonstrated that the PG059 construct with Int001 insertion exhibited higher GCase expression compared to the control without introns. Figure 3 C).

[0116] Simultaneously, using mGBA-C110, exogenous introns selected from Int012, Int013, Int014, Int015, Int016, Int017, Int018, Int019, and Int011 were cloned into the liver-specific promoters HSRE001, HSRE015, and HSRE016, respectively, to obtain constructs of PG074-101. These introns with insertion into... GBA1 Constructs containing exogenous introns upstream of the coding sequence were transfected into HepG2 and HEK293T cells to investigate GCase activity in the cell culture supernatant. Results showed that all constructs efficiently expressed GCase in HepG2 cells, but expressed it at very low levels in HEK293T cells. Figure 3(A and 3B).

[0117] The above results demonstrate that when using liver-specific promoters, exogenous introns and GBA1 Endogenous introns exhibited high GCase expression in HepG2 cells, but all showed very weak GCase activity in HEK293T cells. These results demonstrate that expression cassettes constructed using liver-specific promoters combined with endogenous or exogenous introns, as described in this paper, can be specifically transcribed in liver tissue. GBA1 .

[0118] Table 4. Intron sequences

[0119]

[0120] Table 5. Builder Information

[0121]

[0122] Table 6. Codon-optimized GBA1 nucleotide sequences with introns

[0123]

[0124] Example 4: In vitro testing of chimeric HSREs with introns

[0125] Based on the above results, when constructed with different CHSREs, it exhibits endogenous characteristics. GBA1 Constructs of intron 1 (Int001, i1) and the chimeric intron (Int011, iC) showed higher GCase expression in HepG2 cells; therefore, these two introns were cloned into a GCase expression cassette containing CHSRE and mGBA-C110. GBA1The constructs of Int001 between 27 bp and 28 bp of SEQ ID NO: 21 are PG103, PG104, PG105, PG106, PG168, PG107, and PG108. These constructs have insertions in the chimeric HSRE and GBA1 The constructs containing exogenous chimeric introns between coding sequences were PG110, PG111, PG112, PG113, PG169, PG114, and PG115. HepG2 and HEK293T cells were transfected, and the culture supernatant was harvested for GCase activity measurement. Results showed that all constructs containing a combination of chimeric HSREs and both endogenous and exogenous chimeric introns efficiently expressed GCase in HepG2 cells. Figure 4 A). All of these constructs showed very weak GCase activity in HEK293T cells compared to the PG011 construct driven by promoter CRE001, which was used as a universal expression control. Figure 4 B).

[0126] Table 7. Builder Information

[0127]

[0128] Example 5:

[0129] In vivo studies of constructs with chimeric HSREs and introns using the AAV8 vector in wild-type mice

[0130] Based on the above results, constructs PG102, PG026, PG103, PG104, PG037, PG107, PG108, PG051, PG105, and PG106 were selected, packaged with AAV8, and used for further studies in wild-type mice. These AAV8 products, along with the reference product PG127 constructed using the sequence combination of SEQ ID NO: 14, SEQ ID NO: 5, and SEQ ID NO: 23 as described in patent WO2020161483A1, were injected into wild-type mice at a dose of 2E12 vg / kg. Figure 5 This study also included a buffer control group and an enzyme replacement therapy (imiglucerase) group. Results showed that all these AAV8 products effectively increased GCase activity in serum, liver, spleen, and lung. Serum enzyme activity results were highly consistent with those in different tissue lysates. Most AAV8 products, PG102, PG026, PG103, PG104, PG037, PG107, PG108, PG051, PG105, and PG106, performed significantly better than or at least comparable to the imiglucerase group.

[0131] In summary, constructs using AAV8-packaged HSREs or chimeric HSREs combined with Int001, which contain the HSREs or chimeric HSREs described herein, can... GBA1 It is specifically delivered to the liver and systemically increases GCase activity in serum and all other target tissues.

[0132] Example 6:

[0133] In vivo studies of the therapeutic potential of AAV8 candidates for Gaucher disease

[0134] The aforementioned constructs PG011, PG103, PG104, PG107, and PG105 all exhibited ampicillin resistance. They were all replaced with kanamycin and renamed PG117, PG118, PG119, PG120, PG121, and PG122, respectively. To investigate the long-term therapeutic effects of these AAV8 candidates for gene therapy against Gaucher disease, constructs of PG119, PG120, PG121, and PG122 using CHSRE-driven mGBAi1-C110, PG118 using CHSRE-driven GBAi1-C110, and PG117 using CRE001-driven mGBA-C110 were packaged into AAV8. These AAV8 candidates, along with wild-type... GBA1 PG001 (control) and PG127 (reference) were administered to Gaucher disease mice via tail vein injection at a dose of 2E12 vg / kg for a 12-week treatment study. Several control groups were also included in this study, including a wild-type control (Naïve), a buffer control, and an enzyme replacement therapy (imiglucosidase) group. Serum enzyme activity and glucosamine accumulation were monitored at post-injection time points (weeks 1, 2, 4, 6, 8, and 12). Based on the results, all AAV8 injection groups showed high and stable GCase activity in serum. Compared with the buffer control, imiglucosidase group, and PG127 reference product, PG117, PG118, PG119, PG120, PG121, and PG122 showed higher GCase activity. Figure 6 A). Following injection of AAV8 products, serum glucosphosphingosine levels decreased rapidly. Eight weeks post-injection, AAV8 candidates PG117, PG118, PG119, PG120, PG121, and PG122 reduced glucosphosphingosine levels to near those of Naïve wild-type mice. Figure 6 B).

[0135] Tissue samples were collected at the end of the study for GCase activity and glucosamine analysis. Increased GCase activity was observed in the liver, lungs, and spleen in all AAV-injected groups. Consistent with the serum results above, the AAV8 product groups of PG117, PG118, PG119, PG120, PG121, and PG122 showed higher GCase activity and lower glucosamine accumulation in all tested tissues compared to the imiglucerase group and the PG127 reference product group. Figure 7 and Figure 8 ).

[0136] In summary, the AAV8 products of PG117, PG118, PG119, PG120, PG121, and PG122 have been demonstrated to be promising therapeutic candidates for Gaucher disease and have outperformed existing imiglucerase therapies.

[0137] Table 8. Builder Information

[0138]

[0139] Example 7:

[0140] In vivo studies of intron-containing constructs using the AAV9 vector in wild-type mice

[0141] Similar to AAV8 products, to further enhance the therapeutic potential of AAV9 products expressing GCase, endogenous [products] such as Int001, Int002, Int005, or a combination of Int001 and Int005 are used. GBA1Intron insertion into the universal promoter CRE001 within the expression cassette of mGBA-C110 yielded mGBAi1-C110 (PG123), mGBAi2-C110 (PG124), mGBAi5-C110 (PG125), and mGBAi1i5-C110 (PG126). The constructs of PG001, PG123, PG124, PG125, and PG126 were packaged into AAV9 and evaluated in vivo in wild-type mice. These AAV9 products, along with a reference product of PG128 constructed using the sequence (149 bp-3806 bp) of SEQ ID NO: 1 as described in patent US10837028B2, were administered to wild-type mice via tail vein injection at a dose of 2E12 vg / kg. This study also included a buffer control group and an enzyme replacement therapy (imiglucerase) group. Compared to the buffer control and the reference product groups of PG001 and PG128, the AAV9 products of PG123, PG124, PG125, and PG126 showed higher GCase activity in both serum and tissue lysates. GCase activity was highly consistent across all groups from serum and different tissue lysates. Figure 9 Therefore, the newly designed AAV9 product candidates enhanced GCase activity in both serum and tissues, demonstrating their superior therapeutic potential for Gaucher disease, as well as Parkinson's and Alzheimer's diseases. AAV9 can cross the blood-brain barrier and... GBA1 Efficient gene delivery to the CNS to achieve GCase expression has the potential to alleviate neurological symptoms and benefit type II and III Gaucher disease, as well as Parkinson's disease and Alzheimer's disease.

Claims

1. An expression construct comprising a transcriptional regulatory element operatively linked to a polynucleotide sequence of interest, wherein the expression construct comprises a promoter and an enhancer, the enhancer being located upstream of the promoter. The expression construct described herein includes the following elements in the 5' to 3' orientation: a) Enhancer 3, which is optional; b) Enhancer 2; c) Enhancer 1; and d) Promoter; The sequence of enhancer 1 is SEQ ID NO: 13 or 15, the sequence of enhancer 2 is SEQ ID NO: 8, enhancer 3 is not present, and the sequence of the promoter is SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; The sequence of enhancer 1 is SEQ ID NO: 13, the sequence of enhancer 2 is SEQ ID NO: 9, enhancer 3 is not present, and the sequence of the promoter is SEQ ID NO: 3; The sequence of enhancer 1 is SEQ ID NO: 13, the sequence of enhancer 2 is SEQ ID NO: 8, the sequence of enhancer 3 is SEQ ID NO: 11, and the sequence of the promoter is SEQ ID NO: 3; or The sequence of enhancer 1 is SEQ ID NO: 14, the sequence of enhancer 2 is SEQ ID NO: 8, enhancer 3 is not present, and the sequence of the promoter is SEQ ID NO:

3.

2. The expression construct of claim 1, wherein the expression construct further comprises a non-translated intron region.

3. The expression construct of claim 2, wherein the untranslated intron region comprises all or part of a sequence selected from the group consisting of SEQ ID NO: 24-43.

4. The expression construct of claim 2, wherein the untranslated intron region is operatively linked to the 5' end of the polynucleotide sequence of interest.

5. The expression construct according to claim 2, wherein the untranslated intron region is located between the 5' and 3' ends of the polynucleotide sequence of interest.

6. The expression construct according to any one of claims 1-5, wherein the polynucleotide sequence of interest is a polynucleotide sequence expressing GCase.

7. A vector comprising the expression construct according to any one of claims 1-6.

8. The vector according to claim 7, wherein the vector is a viral vector.

9. The carrier according to claim 8, wherein the carrier is an AAV carrier.

10. The vector of claim 8, wherein the vector further comprises two adeno-associated virus inverted terminal repeat (ITR) sequences located flanking the expression construct.

11. The vector of claim 10, wherein the vector further comprises a polyA sequence.

12. An adeno-associated virus (AAV) comprising a vector and a capsid protein according to any one of claims 7-11.

13. The AAV according to claim 12, wherein the AAV is selected from the group consisting of serotypes AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, and AAVhu37.

14. A composition comprising: The expression construct according to any one of claims 1-6, the vector according to any one of claims 7-11, or the AAV according to claim 12 or 13, and a pharmaceutically acceptable excipient.

15. Use of the expression construct according to any one of claims 1-6, the vector according to any one of claims 7-11, the AAV according to claim 12 or 13, or the composition according to claim 14 in the preparation of a medicament for treating a disease or condition in a subject, wherein the disease or condition is Gaucher disease or Parkinson's disease, and the polynucleotide sequence of interest is a polynucleotide sequence expressing GCase.

16. The use according to claim 15, wherein the polynucleotide sequence of interest is SEQ ID NO:

21.

17. The use according to claim 15, wherein the treatment comprises administering to a subject an effective amount of the expression construct according to any one of claims 1-6, the vector according to any one of claims 7-11, the AAV according to claim 12 or 13, or the composition according to claim 14.

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