Constructs for enhancing gene expression

Abstract

The present disclosure provides an expression construct, vector, viral particle, or composition. The expression construct comprises a transcriptional control element operably linked to a polynucleotide sequence of interest, and the expression construct comprises a promoter, an enhancer located upstream of the promoter, and an optional intron. The present invention further provides uses of the expression construct, vector, viral particle, or composition.

Description

[Technical Field]

[0001] The present disclosure relates to a nucleic acid construct. The present invention also relates to said nucleic acid construct, an expression vector and uses thereof. [Background technology]

[0002] There remains an unmet need in the art for alternative, and preferably improved, methods for regulating transcription of transcripts and, optionally, expression of proteins or polypeptides of interest. Summary of the Invention

[0003] The present disclosure provides an expression construct comprising a transcriptional control element operably linked to a polynucleotide sequence of interest, the expression construct comprising a promoter and an enhancer located upstream of the promoter; The expression construct comprises, as elements, in the 5' to 3' direction: (a) any enhancer 3; (b) enhancer 2; (c) enhancer 1; (d) a promoter; the enhancer 1 comprises all or part of a sequence comprising at least one selected from the group consisting of SEQ ID NOs: 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 to SEQ ID NOs: 13 and 15, wherein the all or part of the sequence retains enhancer function; An expression construct is provided in which the enhancer 2 comprises all or part of a sequence containing at least one selected from the group consisting of SEQ ID NOs: 8 to 11 and 13, 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 to SEQ ID NOs: 8 to 11 and 13, and the all or part of the sequence retains enhancer function. In some embodiments, the enhancer 3 comprises all or part of a sequence comprising at least one selected from the group consisting of SEQ ID NOs:8 to 11 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 to SEQ ID NOs:8 to 11, wherein all or part of the sequence retains enhancer function.

[0004] In some embodiments, the promoter comprises all or part of a sequence selected from the group consisting of SEQ ID NOs: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 to SEQ ID NOs:2-6, wherein all or part of the sequence retains promoter function.

[0005] In some embodiments, the promoter comprises all or part of a sequence selected from the group consisting of SEQ ID NOs: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 to SEQ ID NOs:3-6, wherein all or part of the sequence retains promoter function.

[0006] In some embodiments, enhancer 1 comprises all or a portion of at least one sequence selected from the group consisting of SEQ ID NOs: 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 to SEQ ID NOs: 13 and 15, all or a portion of which retains enhancer function; and enhancer 2 comprises all or a portion of at least one sequence selected from the group consisting of SEQ ID NOs: 8 to 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 to SEQ ID NOs: 8 to 9, all or a portion of which retains enhancer function.

[0007] In some embodiments, the enhancer 1 comprises all or a portion of at least one sequence selected from the group consisting of SEQ ID NOs: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 to SEQ ID NOs:13 and 15, wherein all or a portion of the sequence retains enhancer function; and the enhancer 2 comprises all or a portion of SEQ ID NO:8, 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 to SEQ ID NO:8, wherein all or a portion of the sequence retains enhancer function.

[0008] In some embodiments, the expression construct further comprises an untranslated intron region.

[0009] In some embodiments, the untranslated intron region comprises all or part of a sequence selected from the group consisting of SEQ ID NOs: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 to SEQ ID NOs:24-43.

[0010] In some embodiments, the untranslated intron region is operably linked to the 5' end of the polynucleotide sequence of interest.

[0011] In some embodiments, the untranslated intron region is located between the 5' and 3' ends of the polynucleotide sequence of interest.

[0012] In another aspect, the present disclosure provides a vector comprising the expression construct of the present disclosure.The vector is a viral vector, preferably an AAV vector.The vector further comprises two adeno-associated virus inverted terminal repeat (ITR) sequences flanking the expression construct, and preferably further comprises a polyA sequence.

[0013] In another aspect, the present disclosure provides an adeno-associated virus (AAV) comprising the vector and capsid protein of the present disclosure. In some embodiments, the AAV is selected from the group consisting of serotypes AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrhlO, AAVhu37, or any one of the AAV serotypes isolated from a human or non-human mammal or variants thereof.

[0014] In another aspect, the disclosure provides a composition comprising an expression construct, vector, or AAV of the disclosure and a pharmaceutically acceptable excipient.

[0015] In another aspect, the disclosure provides an expression construct, vector, or AAV of the disclosure for use in a method of treatment.

[0016] In some embodiments, the disclosure provides the use of an expression construct, vector, or AAV of the disclosure in the manufacture of a medicament for treating a disease or condition in a subject.

[0017] In some embodiments, the present disclosure provides an expression construct, vector, or AAV of the present disclosure for use in a method for treating a disease or condition in a subject.

[0018] In some embodiments, the present disclosure provides a method of treating a disease or condition in a subject, comprising administering to said patient an effective amount of an expression construct, vector, or AAV of the present disclosure.

[0019] In another aspect, the disclosure provides an expression construct, vector, or AAV of the disclosure for use in a method of expressing the nucleotide sequence of interest in a subject.

[0020] In another aspect, the disclosure provides an expression construct, vector, or AAV of the disclosure for use in a method of expressing the nucleotide sequence of interest in a subject. [Brief explanation of the drawings]

[0021] [Figure 1]Luciferase and GCase activities of chimeric HSRE constructs in HepG2 cells are shown. (A) Comparison of luciferase activity of different CHSREs based on the HSRE002 promoter. The PG135 group showed a P<0.05 compared to the PG130 group, and the PG139, PG140, PG131, PG132, PG141, PG142, and PG143 groups showed a P<0.01 compared to the PG130 group. (B) Comparison of luciferase activity of different CHSREs based on the HSRE005 promoter. The PG148 and PG151 groups showed a P<0.05 compared to the PG145 group, and the PG146, PG149, PG147, and PG150 groups showed a P<0.01 compared to the PG145 group. (C) Comparison of luciferase activity of different CHSREs based on the HSRE004 promoter. All groups showed a P<0.01 compared to the PG152 group. (D) Comparison of luciferase activity of different CHSREs based on the HSRE003 promoter. The PG165 group showed a P<0.01 comparison with the PG159 group, and the PG163, PG160, PG161, PG162, and PG164 groups showed a P<0.01 comparison with the PG159 group. (E) Comparison of GCase enzyme activity of different CHSREs based on the HSRE002 promoter. The PG025 group showed a P<0.05 comparison with the PG022 group, and the PG023, PG026, PG028, PG029, PG030, and PG031 groups showed a P<0.01 comparison with the PG022 group. (F) Comparison of GCase enzyme activity of different CHSREs based on the HSRE005 promoter. The PG035 group showed a P<0.05 comparison with the PG032 group, and the other groups showed a P<0.01 comparison with the PG032 group. (G) Comparison of GCase enzyme activity of different CHSREs based on the promoter HSRE004. The PG042 group showed a P<0.05 compared to the PG039 group, and the PG040, PG041, PG043, PG044, and PG045 groups showed a P<0.01 compared to the PG039 group. (H) Comparison of GCase enzyme activity of different CHSREs based on the promoter HSRE003. The PG049 and PG052 groups showed a P<0.05 compared to the PG046 group, and the PG047, PG048, and PG051 groups showed a P<0.01 compared to the PG046 group. Values ​​are expressed as mean ± SEM. N=3 for each experimental group. [Figure 2] Luciferase activity and GCase activity of HSRE012 constructs in HepG2 cells are shown. (A) Comparison of luciferase activity based on constructs with different HSRE012 positions flanked by the HSRE002 promoter. Significant differences were observed: PG133 vs. PG131 (P<0.01), PG134 vs. PG132 (P<0.01). (B) Comparison of luciferase activity based on constructs with different copy numbers of HSRE012 flanked by the HSRE002 promoter. No significant differences were observed: PG136 vs. PG131, PG137 vs. PG132, PG138 vs. PG135 (values: mean ± SEM, N=3 per group). (C) GCase enzyme activity based on constructs with different HSRE012 positions flanked by the HSRE002 promoter. Significant differences were observed: PG053 vs. PG025 (P<0.01), PG054 vs. PG026 (P<0.01). (B) Comparison of GCase enzyme activity based on constructs with different copy numbers of HSRE012 flanked by the promoter HSRE002. No significant differences were observed: PG055 vs. PG025, PG056 vs. PG026, PG057 vs. PG027 (Values: Mean ± SEM, N=3 per group). [Figure 3] In vitro GCase enzyme activity of constructs containing different exogenous introns flanked by HSREs is shown. (A) GCase enzyme activity of constructs containing endogenous introns in Huh7 cells. The PG059 group showed a P<0.05 increase compared to the PG058 group. (B) In vitro GCase enzyme activity of different constructs in HepG2 cells. The PG076, PG078, and PG081 groups showed a P<0.01 increase compared to the PG074 group. The PG090 group showed a P<0.01 increase compared to the PG082 group, and the PG091 group showed a P<0.05 increase compared to the PG082 group. The PG093, PG094, PG095, PG097, PG098, PG099, PG100, and PG101 groups showed a P<0.01 increase compared to the PG092 group. (C) GCase enzyme activity of different constructs in HEK293T cells. Error bars represent mean ± SEM. N = 3 for each experimental group. [Figure 4] The GCase enzyme activity of constructs with different introns flanked by chimeric HSREs is shown. (A) GCase enzyme activity of different constructs in HepG2 cells. The G107 and PG108 groups showed P<0.05 compared to the PG168 group. The PG114 and PG115 groups showed P<0.01 compared to the PG169 group. (B) GCase enzyme activity of different constructs in HEK293T cells. Error bars represent the mean ± SEM. N=3 for each experimental group. [Figure 5]This figure shows GCase enzyme activity in serum and tissue lysates of wild-type mice 2 weeks after injection of an AAV8 vector at a dose of 2E12 vg / kg. (A) GCase enzyme activity measured in serum. The PG026, PG037, PG051, and PG105 groups showed P<0.05 compared to the PG127 group, and the PG103, PG104, and PG105 groups showed P<0.01 compared to the PG127 group. The PG103 and PG104 groups showed P<0.01 compared to the PG102 group. The PG103 and PG104 groups showed P<0.01 compared to the PG026 group. (B) GCase enzyme activity measured in liver lysates. The PG026 group showed P<0.05 compared to the PG127 group, and the PG103, PG104, and PG105 groups showed P<0.01 compared to the PG127 group. The PG103 and PG104 groups showed a P<0.01 comparison with the PG102 group. The PG103 and PG104 groups showed a P<0.01 comparison with the PG026 group. The PG105 group showed a P<0.01 comparison with the PG051 group. (C) GCase enzyme activity measured in lung lysates. The PG105 and PG026 groups showed a P<0.05 comparison with the PG127 group, and the PG103 and PG104 groups showed a P<0.01 comparison with the PG127 group. The PG104 group showed a P<0.05 comparison with the PG102 group, and the PG103 group showed a P<0.01 comparison with the PG102 group. The PG103 group showed a P<0.05 comparison with the PG026 group. (D) GCase enzyme activity measured in spleen lysates. The PG026, PG103, PG104, and PG105 groups showed P<0.01 compared to the PG127 group. The PG103 and PG104 groups showed P<0.01 compared to the PG102 group. The PG103 group showed P<0.01 compared to the PG026 group. Values ​​are expressed as mean ± SEM. N=4 for each experimental group. [Figure 6]The significant efficacy of AAV8 gene therapy candidates controlled by chimeric HSREs in Gaucher disease mice is shown. (A) GCase enzyme activity in serum 8 weeks after injection of the AAV8 candidate at a dose of 2E12 vg / kg. P<0.05 for the PG118, PG119, PG120, and PG122 groups compared with the buffer control or cerezyme groups. P<0.05 for the PG118 and PG122 groups compared with the PG127 group, and P<0.01 for the PG119 and PG120 groups compared with the PG127 group. (B) Substrate accumulation in serum 8 weeks after injection of the AAV8 candidate at a dose of 2E12 vg / kg. P<0.01 for all groups compared with the buffer control or cerezyme group. The PG119 group showed a P<0.05 comparison with the PG127 group, and the PG118, PG120, and PG122 groups showed a P<0.01 comparison with the PG127 group. Serum glucosylsphingosine levels in PG119 were below the detection limit. The naive group represents the corresponding wild-type mice. Values ​​are expressed as mean ± SEM. N = 5 for each experimental group. [Figure 7]GCase enzyme activity in tissue lysates after 12 weeks of injection of AAV8 candidates at a dose of 2E12vg / kg in Gaucher disease mice. (A) GCase enzyme activity measured in liver lysates. The PG118-PG122 groups showed P<0.01 compared with the buffer control or cerezyme groups. The PG120 and PG122 groups showed P<0.05 compared with the PG127 group, and the PG118 and PG119 groups showed P<0.01 compared with the PG127 group. (B) GCase enzyme activity measured in lung lysates. The PG118-PG122 groups showed P<0.05 compared with the buffer control or cerezyme groups. The PG118 and PG122 groups showed P<0.05 compared with the PG127 group, and the PG119 and PG120 groups showed P<0.01 compared with the PG127 group. (C) GCase enzyme activity measured in spleen lysates. The PG118 to PG122 groups showed P<0.01 compared with the buffer control group. The PG120 group showed P<0.05 compared with the selezyme group, and the PG118, PG119, and PG122 groups showed P<0.01 compared with the selezyme group. The PG118 and PG122 groups showed P<0.05 compared with the PG127 group, and the PG119 and PG120 groups showed P<0.01 compared with the PG127 group. The naive group represents wild-type mice. Values ​​are expressed as mean ± SEM. N=5 for each experimental group. [Figure 8]Figure 1 shows the accumulation of glucosylsphingosine substrates in tissue lysates after 12 weeks of injection of AAV8 candidates at a dose of 2E12 vg / kg in Gaucher disease mice. (A) Substrate accumulation measured in liver lysates. P<0.01 for the PG001, PG011, PG119, and PG120 groups compared with the buffer control or selezyme group. P<0.01 for the PG119 and PG120 groups compared with the PG127 group. (B) Substrate accumulation measured in lung lysates. P<0.05 for the PG001 group compared with the buffer control or selezyme group, and P<0.01 for the PG011, PG119, and PG120 groups compared with the buffer control or selezyme group. P<0.05 for the PG119 and PG120 groups compared with the PG127 group. (C) Substrate accumulation measured in spleen lysates. P<0.01 for the PG001, PG011, PG119, and PG120 groups compared with the buffer control or Cerezyme group. P<0.01 for the PG119 and PG120 groups compared with the PG127 group. The naive group represents wild-type mice. Error bars represent the mean ± SEM. N=5 for each experimental group. Glucosylsphingosine levels in different tissue lysates were analyzed by LC-MS / MS. [Figure 9]GCase enzyme activity in serum and tissue lysates of wild-type mice 2 weeks after injection of an AAV9 candidate at a dose of 2E12 vg / kg is shown. (A) GCase enzyme activity measured in serum. The PG124 group showed a P<0.05 increase compared to the buffer control group, and the PG123, PG125, and PG126 groups showed a P<0.01 increase compared to the buffer control group. The PG124 and PG125 groups showed a P<0.05 increase compared to the PG001 group, and the PG123 and PG126 groups showed a P<0.01 increase compared to the PG001 group. The PG125 group showed a P<0.05 increase compared to the PG128 group, and the PG123 and PG126 groups showed a P<0.01 increase compared to the PG128 group. (B) GCase enzyme activity measured in liver lysates. The PG123 to PG126 groups showed a P<0.01 increase compared to the buffer control group. The PG124 and PG125 groups showed P<0.05 compared with the PG001 group, 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. The PG124 group showed P<0.05 compared with the buffer control group, 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 PG001, and the PG123, PG125, and PG126 groups showed P<0.01 compared with PG001. The PG123 and PG125 groups showed P<0.05 compared with the PG128 group, and the PG126 group showed P<0.01 compared with the PG128 group. (D) GCase enzyme activity measured in spleen lysates. The PG123 to PG126 groups showed P<0.01 compared with the buffer control group. The PG124 group showed P<0.05 compared with PG001, and the PG123, PG125, and PG126 groups showed P<0.01 compared with PG001. The PG124 group showed P<0.05 compared with PG128, and the PG123, PG125, and PG126 groups showed P<0.01 compared with PG128. Values ​​are expressed as mean ± SEM. N=4 for each experimental group. DETAILED DESCRIPTION OF THE INVENTION

[0022] Hereinafter, embodiments of the present disclosure will be described in more detail. However, each aspect of the present disclosure may be embodied in different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present invention to those skilled in the art. The terms used in the description of this specification are merely for the purpose of describing the embodiments and are not intended to be limiting.

[0023] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by those skilled in the art. Furthermore, it should be understood that terms as defined in common dictionaries should be interpreted as having a meaning consistent with the meaning in the context of this application and related art, and should not be interpreted in an idealized or overly formal sense, unless expressly defined herein.

[0024] [Definition] As used in the present description and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly indicates otherwise.

[0025] As used herein, the term "comprising" means that the compositions and methods include the described elements, but do not exclude other elements.

[0026] As used herein, the terms "nucleotide" and "polynucleotide" may be used interchangeably and refer to a polymeric form of nucleotides of any length, which may be ribonucleotides or deoxyribonucleotides. Thus, the terms include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising, consisting essentially of, or consisting of purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0027] As used herein, "expression" refers to the two-step process of transcribing a polynucleotide into mRNA and / or the subsequent translation of the transcribed mRNA into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in eukaryotic cells.

[0028] When applied to a polynucleotide, the term "encode" means that a polynucleotide is said to "encode" a polypeptide when it is transcribed to produce mRNA for that polypeptide and / or fragments thereof. The antisense strand is the complement of such a nucleic acid, from which the coding sequence can be deduced.

[0029] As used herein, the term "promoter" refers to a regulatory sequence that is a control coding sequence of a polynucleotide sequence, e.g., a region that controls the initiation and rate of transcription of a gene or transgene. Promoters may be constitutive, inducible, repressible, or tissue-specific. In embodiments, promoters are used in conjunction with enhancers to increase transcription efficiency. Enhancers are regulatory elements that increase expression of a target sequence.

[0030] The terms "protein," "peptide," and "polypeptide" can be used interchangeably and, in the broadest sense, refer to a compound of two or more amino acid, amino acid analog, or peptidomimetic subunits. The subunits may be linked by peptide bonds. Alternatively, the subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide comprises at least two amino acids, and there is no limit to the maximum number of amino acids that may be contained in, consist essentially of, or consist of a protein or peptide sequence. As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids, including glycine and D and L optical isomers, amino acid analogs, and peptidomimetics.

[0031] "Identity" refers to sequence similarity between two peptides or two nucleic acid molecules. Percentage identity can be determined by comparing a position in each sequence that is aligned for purposes of comparison. 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.

[0032] As used herein, the term "vector" refers to a nucleic acid comprising or consisting of a complete replicon such that the vector can replicate when placed into a cell, e.g., by transfection, infection, or transformation. It is understood in the art that once inside a cell, the vector may replicate as an extrachromosomal (adduct) element or may integrate into a host cell chromosome. Vectors can include nucleic acids derived from retroviruses, adenoviruses, herpesviruses, baculoviruses, modified baculoviruses, papoviruses, AAV viral vectors, lentiviral vectors, adenoviral vectors, alphavirus vectors, and the like. Alphavirus vectors, e.g., Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, e.g., Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827.

[0033] As used herein, the term "adeno-associated virus" or "AAV" refers to a member of the genus Dependopavovirus in the family Parvoviridae. Adeno-associated viruses are single-stranded DNA viruses that grow only in cells in which some functions are provided by a commonly infected helper virus. All AAV serotypes apparently exhibit very similar replication characteristics mediated by homologous rep genes and all possess three related capsid proteins. At least 13 consecutively numbered naturally occurring AAV serotypes are known in the art. Non-limiting exemplary serotypes in the methods disclosed herein include any of these 13 serotypes, e.g., AAV2, AAV8, AAV9, or variant serotypes, e.g., AAV-DJ and AAV-PHP.B. AAV particles comprise, consist essentially of, or consist of the three major viral proteins VP1, VP2, and VP3. In embodiments, AAV refers to serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV 13. In embodiments, 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, AAV 214e9, AAV 214el 0, AAV ITB102_45, and AAV 214AB. In embodiments, the AAV is serotype AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13, AAVrh10, AAVhu37, or any of the AAV serotypes isolated from humans and non-human mammals or variants thereof. In embodiments, the AAV particles are 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、AAV42-11 AV43-1、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、AAV22 3.6、AAV223.7、AAV1-7 / rh.48、AAV1-8 / rh.49、AAV2-15 / rh.62、AAV2-3 / rh.61、AAV2-4 / rh.50、AAV2-5 / rh.51、AAV3.1 / rh.6、AAV3.1 / rh.9、AAV3-9 / rh. .52、AAV3-11 / rh.53、AAV4-8 / r11.64、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.37、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、AAVA3.4、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、AAVpi.2、AAVrh.60、AAVrh.44、AAVrh.65、AAVrh.55、AAVrh.47、AAVrh.69、AAVrh.45、AAVrh. rh.59、AAVhu.12、AAVH6、AAVLK03、AAVH-1 / hu.1、AAVH-5 / hu.3、AAVLG-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、AAV hu.2、AAVhu.3、AAVhu.4、AAVhu.5、AAVhu.6、AAVhu.7、AAVhu.9、AAVhu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu. u.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23.2、AAVhu.24、AAVhu.25、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu. hu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.40、AAVhu.41、AAVhu.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AA Vhu.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.3 4、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37R2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.46、AAVrh.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 mutation、AAAV、BAAV、ヤギAAV、ウシAAV、AAVhE1.1、AAVhEr1.5、AAVhEr1.14、AAVhEr1.8、AAVhEr1.16、AAVhEr1.18、AAVhEr1.35、AAVh Er1.7、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, AAV Shuffle 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.11, AAVhu.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, AAV CBr-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、AAV CBr-E5、AAV CBr-e5、AAV CBr-E6、AAV CBr-E7、AAV CBr-E8、AAV CHt-1、AAV CHt-2、AAV CHt-3、AAV CHt-6.1、AAV CHt-6.1、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、AAV CKd-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-12, AAV CLv1-3, AAV CLv-13, AAV CLv1-4, AAV Clv1-7, AAV Clv1-8, AAV Lv1-9, AAV Clv-2 CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-D1, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-6, AAV CLv-7 CLv-D8, AAV CLv-E1, AAV CLv-K1, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-M1, AAV-AAV1, AAV CLv1 CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-R1, AAV CLv-R2, AAV CLv-R3, AAV-CLv-RAV4 CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-4, AAV CSp-4. CSp-6、AAV CSp-7、AAV CSp-8、AAV CSp-8.10、AAV CSp-8.2、AAV CSp-8.4、AAV CSp-8.5、AAV CSp-8.6、AAV CSp-8.7、AAV CSp-8.8. CSp-8.9、AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF1 2 / HSC12、AAVF13 / HSC13、AAVF14 / HSC14、AAVF15 / HSC15、AAVF16 / HSC16、AAVF17 / HSC17、A AVF2 / HSC2,AAVF3 / HSC3,AAVF4 / HSC4,AAVF5 / HSC5,AAVF6 / HSC6,AAVF7 / HSC7,AAVF8 / HSC 8、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-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, The AAVPHP.B capsid protein is selected from the group consisting of AAVPHP.B-EGS, 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, AAVVG2A15 / G2A3, AAVPHP2B4, AAVPHP2B5, and variants thereof.

[0034] 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 repeats (ITRs). Such AAV vectors are capable of replicating in a host cell that provides the functions of the rep and cap gene products, allowing the nucleic acid between the ITRs to be packaged into infectious viral particles. In embodiments, an AAV vector comprises a promoter, at least one nucleic acid sequence capable of encoding at least one protein or RNA, and / or an enhancer and / or terminator, flanked by ITRs that are packaged into infectious AAV particles. The nucleic acid between the ITRs may be packaged into an AAV capsid, and such encapsidated nucleic acid may be referred to as an "AAV vector genome." In addition to the encapsulated portion, AAV vectors may also contain other elements, such as antibiotic resistance genes and other elements known in the art, that are included in a plasmid for manufacturing purposes but are not packaged into AAV particles.

[0035] As used herein, the term "viral capsid" or "capsid" refers to the protein shell or coating of a viral particle. The capsid functions to package, protect, transport, and / or release the viral genome into the host cell. Capsids are typically composed of oligomeric structural subunits of proteins ("capsid proteins"). The viral capsid of AAV consists of a mixture of three viral capsid proteins: VP1, VP2, and VP3. An "AAV virion" or "AAV viral particle" or "AAV particle" refers to a viral particle consisting of at least one AAV capsid protein and an encapsidated polynucleotide derived from an AAV vector (referred to herein as an AAV vector genome).

[0036] A "subject" of diagnosis or treatment is an animal, e.g., a mammal or a human. Subjects are not limited to a particular species and include non-human animals to be diagnosed or treated, and non-human animals or animal models to be infected, including, but not limited to, monkey, mouse, rat, dog, or rabbit species, as well as other farm animals, sport animals, or pets. In an embodiment, the subject is a human.

[0037] As used herein, "treating" a disease in a subject means (1) preventing the onset of symptoms or disease in a subject who is susceptible to or does not develop symptoms of the disease; (2) suppressing or arresting the progression of a disease; or (3) ameliorating or causing the attenuation of a disease or symptoms of a disease. As understood in the art, "treatment" is an approach for obtaining beneficial or desired results (including clinical results). For purposes of the present technology, beneficial or desired results include, but are not limited to, alleviation or amelioration of one or more symptoms, reduction in the severity of a condition (including a disease), stabilization (i.e., not worsening) of the condition (including a disease), delay or alleviation of the progression of a condition (including a disease), improvement or alleviation of the condition (including a disease), and alleviation (whether partial or complete), whether detectable or undetectable.

[0038] As used herein, the term "effective amount" refers to an amount sufficient to achieve a desired effect. For therapeutic or prophylactic uses, the effective amount may depend on the type and severity of the disease in question, as well as individual subject characteristics such as general health, age, sex, weight, and tolerance to the pharmaceutical composition. For gene therapy, in embodiments, the effective amount is an amount sufficient to allow a defective gene in a subject to regain some or all of its function. In other embodiments, an effective amount of AAV viral particles is an amount sufficient to express the gene in a subject. Those skilled in the art can determine the appropriate amount based on these and other factors.

[0039] In embodiments, the effective amount depends on the scale and nature of the intended use. It also depends on the characteristics and sensitivity of the intended subject and the method of use. Those skilled in the art can determine the effective amount based on these and other considerations. Depending on the embodiment, the effective amount may comprise, consist essentially of, or consist of one or more administrations of the composition.

[0040] As used herein, the term "administration" means delivering a substance to a subject, such as an animal or human. Administration can be single, continuous, or intermittent throughout the course of treatment. Methods for determining the most effective means of administration and dosage are known to those skilled in the art and will vary depending on the composition used for treatment, the purpose of the treatment, and the age, health, or sex of the subject being treated. Single or multiple administrations are possible, with dosage levels and patterns being selected by the treating physician, or, in the case of pets and other animals, by the treating veterinarian. [Example]

[0041] The embodiments described below follow the following general methodology unless otherwise specified.

[0042] [rAAV production] AAV8 and AAV9 viral particles were produced by transient triple-plasmid transfection of HEK293T cells or HEK293 suspension cells with plasmids encoding recombinant genomes containing the AAV Rep and Cap proteins, adenovirus helper genes, and the GBA1 construct. rAAV particles were purified using iodixanol-based density gradient ultracentrifugation. rAAV was then quantified by probe-based ddPCR (Biorad) assay and characterized by silver staining.

[0043] In vitro transfection and rAAV potency assay The day before transfection, HEK293T or hepatocyte cell lines HepG2 and Huh7 were added to 24-well plates at a cell density of 1.5E5 cells / well. 500 μL of complete cell culture medium was added to each well. Transfection was performed using a PEI transfection reagent. Specifically, 0.15 μg of a plasmid containing the transgene sequence and 0.15 μg of a plasmid containing a luciferase reporter gene were co-transfected into each well. 48 hours after transfection, 300 μL of fresh complete cell culture medium was added to each well, and the cells were cultured for an additional 24 hours.

[0044] For the dual luciferase reporter assay, cells were lysed using cell lysis buffer (TransGen) and firefly and Renilla luciferases were detected using a luciferase detection system (TransGen). The 96-well plates containing cell lysates and detection reagents were read on a Varioskan LUX reader (ThermoFisher). All firefly luciferase activity results were first normalized to the corresponding Renilla luciferase intensity. Results were then normalized to the control group.

[0045] For GCase activity assays, enzyme activity assays were performed on cell culture supernatants as described below. Cells were lysed using cell lysis buffer (Promega), and luciferase detection was performed using the Steady-Glo Luciferase Detection System (Promega). 96-well plates containing cell lysates and detection reagents were read on a Varioskan LUX reader (Thermo Fisher). Data shown in the figures are enzyme activity normalized to luciferase intensity and further normalized to the control group.

[0046] rAAV biopotency assays were performed by cell transduction using HEK293T, Huh7, or HepG2 cells. 24 hours before transduction, cells were plated 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. 48 hours after infection, 300 μl of fresh complete cell culture medium was added to each well, and the cells were cultured for an additional 24 hours. 72 hours after infection, the cell culture supernatant was subjected to an enzyme activity test as described below.

[0047] [Wild-type mouse study design] An AAV vector containing the GBA1 transgene was injected via the tail vein into 8-9 week-old wild-type (C57BL / 6) male mice. The AAV injection dose was 1E12 vg / ml. To evaluate the kinetics and persistence of transgene expression, serum GCase levels were measured at various time intervals (1, 2, or 4 weeks) after injection. Mice were monitored for 4 weeks after AAV treatment and then sacrificed for biochemical and pathological analysis.

[0048] [Research design for Gaucher disease mice] An AAV vector containing the GBA1 transgene was injected via the tail vein into 7- to 12-week-old mice with Gaucher disease (a combination of two different types of GBA1 mutations). All mice were housed in individually ventilated cages in a specific pathogen-free environment. All cages, cob bedding, and water were sterilized before use. Cages, cob bedding, food, and water were changed twice a week.

[0049] The injected dose of AAV 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 after injection. Mice were followed until end-point testing and then sacrificed for biochemical and pathological analysis.

[0050] [Preparation and administration of AAV / Cerezyme] Aliquots of rAAV were stored at -80°C. Prior to injection, aliquots were thawed on ice and diluted with AAV preparation buffer. The diluted AAV was kept on ice prior to injection and used within 2 hours.

[0051] Cerezyme was resuspended according to the manufacturer's instructions, dispensed into aliquots (40 IU / ml) and stored at −80° C. Before injection, the aliquots were thawed on ice, diluted and mixed gently and thoroughly.

[0052] [Serum and tissue collection] Serum was isolated from fresh anticoagulant-free blood by centrifugation at 12,000 rpm for 15 minutes at 4°C within 0.5 hours and stored at -80°C. For the Cerezyme group, serum was collected 1.5 hours after injection. Mice were anesthetized and euthanized. After perfusion with saline, tissues were collected from the mice and stored at -80°C. For the Cerezyme group, tissue samples were collected 1.5 hours after injection. The tissue samples were divided into four portions, three of which were frozen in separate tubes and stored at -80°C for use in GCase activity assays, glucosylsphingosine analysis, and mRNA analysis. The remaining portions were fixed in 10% neutral buffered formalin (NBF, pH 7.4) at room temperature for approximately 24–48 hours for histological analysis. Bone marrow cells were collected from the femurs and tibias of both mouse legs.

[0053] [GCase activity assay in mouse serum and tissues] Serum samples were collected from mouse blood and stored at -80°C. Using a homogenizer (Shanghai Jingxin), tissues were lysed in tissue lysis buffer (citrate-phosphate buffer, pH 5.0, 0.25% sodium taurocholate, 1% TX-100, and protease inhibitor cocktail) using a specific program (50 Hz for 30 s, cooled for 30 s, total 4 min). For enzyme activity assays, β-glucocerebrosidase (acid β-glucosidase; GCase) activity was measured by a fluorometric assay. 4-methylumbelliferyl β-D-glucopyranoside (4MU-Glc, Carbosynth) was used as a GCase substrate. On the day of the assay, serum was diluted 1:100 with enzyme assay buffer (citrate-phosphate buffer, pH 5.0, 0.25% sodium taurocholate, 0.25% TX-100). Tissue lysates were diluted 1:40 with lysis buffer (citrate-phosphate buffer, pH 5.0, 0.25% sodium taurocholate, 1% TX-100 and protease inhibitor cocktail).

[0054] All samples were measured in citrate-phosphate buffer, pH 5.0, 0.25% sodium taurocholate, 0.25% TX-100, and 1 mM 4MU-Glc at 37°C for 1 hour. The reaction was terminated by adding a threefold volume (150 μl) of stop solution (0.5 M glycine, pH 10.8). Relative fluorescence levels (RFU) were assessed using a Varioskan LUX reader (Thermo Fisher) at excitation and emission wavelengths of 360 nm and 460 nm, respectively. Protein concentration assays were also performed on tissue lysate samples using a BCA kit (Thermo Fisher). 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).

[0055] [Vector genome copy number, relative RNA transcription level] To determine the vector genome copy number in tissue samples after rAAV injection, DNA was isolated from frozen liver samples using the DNeasy Blood and Tissue Kit (QIAGEN) according to the manufacturer's instructions. After DNA isolation, probe-based qPCR (Roche) was performed to determine the vector genome copy number / reaction. The cell number / reaction was calculated from the DNA quantification results. The vector genome copy number / cell was then calculated by normalizing the genome copy number / reaction to the cell number / reaction.

[0056] To determine the relative RNA transcription levels in tissue samples after 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 Primescript RT master mix (TAKARA). 300–500 ng of RNA was added to each RT reaction. The cDNA was then diluted and subjected to qPCR (Roche) with a probe.

[0057] [Immunohistochemistry] Mouse macrophages were detected using rabbit anti-mouse CD68 antibody (1:25 Abcam AB53444). Formalin-fixed mouse tissues were dewaxed in xylene and washed with graded ethanol, followed by antigen retrieval using pepsin according to the manufacturer's recommendations. Sections were counterstained with hematoxylin. Detection was performed using a biotin-conjugated secondary antibody. Signals were visualized using streptavidin-HRP and a Tyramide signal amplification kit according to the manufacturer's recommendations.

[0058] [Stored cell count] Tissue sections were stained with hematoxylin and eosin (H&E). Stained tissues were scanned with an Aperio AT2 (Leica, 40X). Tissue images were processed with an Aperio ImageScope (V12.4.3.5008). Total Gaucher cells in entire tissue sections of mouse liver and lung were manually counted. Gaucher cell counts from all sections were normalized to the tissue section area (cm2) and used for data mapping.

[0059] [Glucosylsphingosine (Lyso-GL1) Analysis] Tissue homogenates were prepared by homogenization with 9 volumes (w:v) of PBS buffer. Aliquots (10 pL) of tissue lysates or serum samples were subjected to LC / MS analysis. Quantified tissue Lyso-GL1 was normalized to tissue weight, and serum substrate levels were normalized to serum volume. In the corresponding figures, Lyso-GL1 lower limit of quantification (LLOQ) values ​​of <10 ng / g (for tissue) and <1 ng / mL (for serum or plasma) were denoted as BQL.

[0060] [Statistical analysis] Data were expressed as mean ± standard error of the mean (mean ± SEM). Statistical analysis of differences between different groups was performed using GraphPad Prism software. A p-value ≤ 0.05 was considered statistically significant.

[0061] Example 1: Constructs This disclosure outlines a comprehensive method for designing and screening novel expression cassettes that efficiently and selectively express therapeutic GCase in the liver to improve the therapeutic efficacy of gene therapy for Gaucher disease, a metabolic disorder.

[0062] The first step was to clone the nucleotide sequences of the firefly luciferase gene and the GBA1 gene into a targeting vector. To restrict expression of the luciferase and GCase proteins to the liver, the nucleotide sequences of the firefly luciferase gene and the GBA1 gene were driven by a series of rationally designed chimeric hepatic-specific regulatory elements (CHSREs) by combining different promoters with various regulatory elements. These 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, an intron was further used to enhance GCase expression. The IDs of the different promoters and various regulatory elements used in this disclosure, along with their nucleotide sequences, are listed in Table 1. A codon-optimized GBA1 gene with a K321N mutation is one exemplary nucleotide sequence of interest.

[0063] [Table 1]

[0064] TIFF2025534900000002.tif216166

[0065] TIFF2025534900000003.tif219166

[0066] TIFF2025534900000004.tif158165

[0067] [Table 2]

[0068] TIFF2025534900000006.tif215166

[0069] TIFF2025534900000007.tif235166

[0070] TIFF2025534900000008.tif140166

[0071] Polypeptide sequence of codon-optimized human GBA1 without the signal peptide portion and with the K321N mutation (SEQ ID NO:22) ARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMA SCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCL GFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDFLAPANATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLY HVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSIHTYLWRRQ*

[0072] Polypeptide sequence of codon-optimized human GBA1 containing the signal peptide portion and carrying the K321N mutation (SEQ ID NO:23) *

[0073] Example 2: In vitro screening of chimeric liver-specific promoters To restrict luciferase or GCase expression to the liver, we designed a series of chimeric liver-specific regulatory elements (CHSREs) containing a core promoter and one, two, or three or more regulatory elements. We then performed in vitro and in vivo screening by comparing the expression efficiency of luciferase or GCase in HepG2 cells. Firefly luciferase or mGBA-C110 (a codon-optimized version of GBA1 with a K321N mutation) was expressed under the control of various CHSREs.

[0074] The chimeric liver-specific regulatory element (CHSRE) consists of one, two, three or multiple copies of an enhancer selected from HSRE010, HSRE014, HSRE012, HSRE006, and HSRE009, and is constructed in combination with four promoters, HSRE002, HSRE004, HSRE003, or HSRE005, to drive the expression of the luciferase gene or the GBA1 gene. The resulting construct is designated PG Constructs 130 to 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 study the expression of luciferase or GCase in HepG2 cells. Information about these constructs is shown in Table 3.

[0075] [Table 3]

[0076] TIFF2025534900000010.tif238166

[0077] TIFF2025534900000011.tif225166

[0078] TIFF2025534900000012.tif105166

[0079] All of the luciferase constructs containing CHSREs exhibited higher luciferase activity compared to the corresponding promoter-only versions (Figure 1A, B, C, D). When the luciferase gene was replaced with the GBA1 gene, all other GBA1 constructs, except for PG024-CHSRE003, PG027-CHSRE006, and PG050-CHSRE029, exhibited significantly higher GCase activity compared to the corresponding promoter-only versions (Figure 1E, F, G, H).

[0080] To determine whether these enhancers had any additive effect on luciferase and GBA1 expression, we constructed enhancers with different copy numbers or in tandem with them in the CHSRE. Further increases in the copy number of HSRE010 (PG141-CHSRE007 vs. PG131-CHSRE004) or HSRE007 (PG142-CHSRE008 vs. PG132-CHSRE005) did not affect luciferase expression (Figure 1A). Similarly, further increases in the copy number of HSRE010 (PG028-CHSRE007 vs. PG025-CHSRE004) and HSRE007 (PG029-CHSRE008 vs. PG026-CHSRE005) did not affect GCase expression (Figure 1E). Furthermore, CHSRE009, which was the addition of HSRE010 in tandem to CHSRE005 in PG143, significantly enhanced luciferase expression compared to PG132-CHSRE005 (Figure 1A), whereas CHSRE009 in PG030 had no effect on GCase expression compared to the corresponding control in PG026-CHSRE005 (Figure 1E).

[0081] We examined the effect of HSRE012 on luciferase or GBA1 expression relative to the HSRE002 promoter position. When HSRE012 was relocated from upstream (PG131-CHSRE004-luci and PG132-CHSRE005-luci, PG025-CHSRE004- and PG026-CHSRE005-luci) to downstream (PG133-CHSRE032-luci and PG134-CHSRE033-luci, PG053-CHSRE032- and PG054-CHSRE033-luci) of the HSRE002 promoter, luciferase or GCase expression was significantly reduced (Fig. 2A, C). Increasing the copy number of HSRE012 in CHSRE constructs such as PG136-CHSRE034-luci, PG137-CHSRE035-luci, PG138-CHSRE036-luci, PG055-CHSRE034-GBA1, PG056-CHSRE035-GBA1, and PG057-CHSRE036-GBA1 did not further increase luciferase or GCase expression compared with the corresponding constructs with one copy of HSRE012 (PG131-CHSRE004-luci, PG132-CHSRE005-luci, PG135-CHSRE006-luci, PG025-CHSRE004-GBA1, PG026-CHSRE005-GBA1, and PG027-CHSRE006-GBA1) (Figure 2B, D).

[0082] Example 3: In vitro screening of different introns To further enhance GCase expression, endogenous introns from human GBA1 were inserted: Int001 (inserted between bp 27 and 28 of SEQ ID NO:21), Int002 (inserted between bp 115 and 116 of SEQ ID NO:21), Int003 (inserted between bp 307 and 308 of SEQ ID NO:21), Int004 (inserted between bp 454 and 455 of SEQ ID NO:21), Int005 (inserted between bp 588 and 589 of SEQ ID NO:21), Int006 (inserted between bp 761 and 762 of SEQ ID NO:21), Int007 (inserted between bp 999 and 1000 of SEQ ID NO:21), Int008 (inserted between bp 1224 and 1225 of SEQ ID NO:21), and Int009 (inserted between bp 1224 and 1225 of SEQ ID NO:21). Introns selected from Int002 (inserted between bp 1388 and 1389 of SEQ ID NO:21) and Int0010 (inserted between bp 1505 and 1506 of SEQ ID NO:21) were cloned under the HSRE002 promoter in mGBA-C110 to obtain constructs PG059, PG060, PG061, PG062, PG063, PG064, PG065, PG066, PG067, and PG068. The construct without any introns was PG058. Endogenous introns selected from the group consisting of Int002, Int003, and Int005 and exogenous introns selected from Int011 and Int020 were cloned under the HSRE002 promoter in mGBA-C110 to obtain constructs PG069 to PG073. The above constructs, as well as PG058 and PG059, were transfected into Huh7 cells, and GCase activity in the cell culture supernatant was investigated. The PG059 construct with the Int001 inserted demonstrated higher GCase expression than the intron-free control (Figure 3C).

[0083] Additionally, exogenous introns selected from the group consisting of Int012, Int013, Int014, Int015, Int016, Int017, Int018, Int019, and Int011 were cloned into mGBA-C110 under the liver-specific promoters HSRE001, HSRE015, and HSRE016, respectively, to obtain constructs PG074-101. Constructs containing these exogenous introns inserted upstream of the GBA1 coding sequence were transfected into HepG2 cells and HEK293T cells, and GCase activity in the cell culture supernatants was examined. The results showed that all constructs efficiently expressed GCase in HepG2 cells, but very low expression in HEK293T cells (Figures 3A and 3B). These results demonstrate that when constructed with a liver-specific promoter, the exogenous intron and the GBA1 endogenous intron show high GCase expression in HepG2 cells, but both show very weak GCase activity in HEK293T cells. As is clear from these results, the expression cassettes constructed with the liver-specific promoter described herein and endogenous or exogenous introns can specifically transcribe GBA1 in liver tissue.

[0084] [Table 4]

[0085] TIFF2025534900000014.tif241165

[0086] TIFF2025534900000015.tif223166

[0087] TIFF2025534900000016.tif218166

[0088] [Table 5]

[0089] TIFF2025534900000018.tif192165

[0090]

Table 6

[0091] TIFF2025534900000020.tif218166

[0092] TIFF2025534900000021.tif218166

[0093] TIFF2025534900000022.tif213166

[0094] TIFF2025534900000023.tif217166

[0095] TIFF2025534900000024.tif217166

[0096] TIFF2025534900000025.tif213166

[0097] TIFF2025534900000026.tif218166

[0098] TIFF2025534900000027.tif218166

[0099] TIFF2025534900000028.tif215166

[0100] TIFF2025534900000029.tif218166

[0101] TIFF2025534900000030.tif218166

[0102] TIFF2025534900000031.tif129166

[0103] Example 4: In vitro testing of chimeric HSREs with introns Based on the results above, constructs containing the endogenous GBA1 intron 1 (Int001, i1) and chimeric intron (Int011, iC) showed higher GCase expression in HepG2 cells when constructed with different CHSREs. Therefore, these two introns were cloned into a GCase expression cassette containing a CHSRE and mGBA-C110. Constructs in which Int001 was inserted between bp 27 and 28 of GBA1 SEQ ID NO:21 were PG103, PG104, PG105, PG106, PG168, PG107, and PG108. Constructs in which an exogenous chimeric intron was inserted between the chimeric HSRE and the GBA1 coding sequence were PG110, PG111, PG112, PG113, PG169, PG114, and PG115. HepG2 and HEK293T cells were transfected, and the culture supernatants were harvested and assayed for GCase activity. As can be seen, the constructs containing both the chimeric HSRE and the endogenous intron 1 and the exogenous chimeric intron efficiently expressed GCase in HepG2 cells (Figure 4A). These constructs also showed very weak GCase activity in HEK293T cells compared with the universal expression control, the PG011 construct, in which mGBA-C110 was driven by the CRE001 promoter (Figure 4B).

[0104] [Table 7]

[0105] TIFF2025534900000033.tif25166

[0106] Example 5: In vivo study of constructs with chimeric HSRE and introns using AAV8 vectors in wild-type mice Based on these results, constructs PG102, PG026, PG103, PG104, PG037, PG107, PG108, PG051, PG105, and PG106 were selected and packaged in AAV8 for further study in wild-type mice. Reference product PG127, constructed by combining these AAV8 products with SEQ ID NO:14, SEQ ID NO:5, and SEQ ID NO:23 described in patent WO2020161483A1, was 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 (Cerezyme) group. Results showed that all of these AAV8 products effectively increased GCase activity in serum, liver, spleen, and lung. The enzyme activity results in serum were highly consistent with those in different tissue lysates. Most of the AAV8 products PG102, PG026, PG103, PG104, PG037, PG107, PG108, PG051, PG105 and PG106 showed higher or at least equivalent activity compared to the Cerezyme group.

[0107] In summary, constructs containing the HSREs described herein or chimeric HSREs in combination with Int001 packaged in AAV8 delivered GBA1 specifically to the liver and systematically increased GCase activity in serum and all other target tissues.

[0108] Example 6: In vivo study of the therapeutic potential of AAV8 candidates against Gaucher disease The above constructs PG011, PG103, PG104, PG107, and PG105 all possess ampicillin resistance, and were renamed PG117, PG118, PG119, PG120, PG121, and PG122 after replacing the ampicillin resistance with kanamycin. To examine the long-term therapeutic efficacy of these AAV8 candidates for Gaucher disease gene therapy, the CHSRE-driven mGBAi1-C110 constructs PG119, PG120, PG121, and PG122, the CHSRE-driven GBAi1-C110 construct PG118, and the CRE001-driven mGBA-C110 construct PG117 were packaged into AAV8. These AAV8 candidates, along with the wild-type GBA1 control PG001 and the reference product PG127, were administered at a dose of 2E12 vg / kg via tail vein injection into Gaucher disease mice for a 12-week therapeutic efficacy study. The study also included control groups, including a wild-type control (Naive), a buffer control, and enzyme replacement therapy (Cerezyme). Serum enzyme activity and glucosylsphingosine accumulation were monitored at intervals after injection (weeks 1, 2, 4, 6, 8, and 12). All AAV8-injected groups showed high and stable serum GCase activity. PG117, PG118, PG119, PG120, PG121, and PG122 showed higher GCase activity than the buffer control, Cerezyme, and PG127 reference products (Figure 6A). Following injection of the AAV8 products, serum glucosylsphingosine levels rapidly decreased. Eight weeks after injection, AAV8 candidates PG117, PG118, PG119, PG120, PG121, and PG122 reduced glucosylsphingosine levels to levels similar to those in naive wild-type mice (FIG. 6B).

[0109] At the end of the study, tissue samples were collected and analyzed for GCase enzyme activity and glucosylsphingosine. All AAV-injected groups showed increased GCase enzyme activity in the liver, lung, and spleen. Similar to the serum results, the PG117, PG118, PG119, PG120, PG121, and PG122 AAV8 product groups showed higher GCase activity and lower glucosylsphingosine accumulation in all tested tissues compared with the cerezyme and PG127 reference product groups (Figures 7 and 8).

[0110] In summary, the AAV8 products PG117, PG118, PG119, PG120, PG121 and PG122 were proven to be ideal candidates for treating Gaucher disease and showed superior efficacy to conventional Cerezyme therapy.

[0111] [Table 8]

[0112] Example 7: In vivo studies of intron-bearing constructs using AAV9 vectors in wild-type mice To further improve the therapeutic potential of AAV9 products expressing GCase, similar to AAV8 products, we inserted the endogenous GBA1 introns Int001, Int002, Int005, or a combination of Int001 and Int005 under the universal promoter CRE001 in the mGBA-C110 expression cassette, resulting in mGBAi1-C110(PG123), mGBAi2-C110(PG124), mGBAi5-C110(PG125), and mGBAi1i5-C110(PG126). The PG001, PG123, PG124, PG125, and PG126 constructs were packaged into AAV9 and evaluated in vivo in wild-type mice. These AAV9 products and the PG128 reference product, constructed using the sequence of SEQ ID NO:1 (149 bp to 3806 bp) described in patent US10837028B2, were injected into wild-type mice via the tail vein at a dose of 2E12 vg / kg. A buffer control group and an enzyme replacement therapy (Cerezyme) group were also included in this study. The PG123, PG124, PG125, and PG126 AAV9 products showed higher GCase activity in both serum and tissue lysates compared to the buffer control, PG001, and PG128 reference product groups. The GCase activity in serum and different tissue lysates was highly consistent across all groups (Figure 9). Therefore, the newly designed AAV9 product candidates improved GCase activity in both serum and tissues, demonstrating their potential for the treatment of Gaucher disease, Parkinson's disease, and Alzheimer's disease. AAV9 can cross the blood-brain barrier and efficiently deliver the GBA1 gene to the CNS, potentially alleviating neurological symptoms and contributing to types II and III Gaucher disease, Parkinson's disease, and Alzheimer's disease.

[0113] This application claims the benefit of and priority to PCT application PCT / CN2022 / 123892, filed October 8, 2022, the entire contents of which are incorporated herein by reference.

Claims

1. An expression construct comprising a transcriptional control element operably linked to a polynucleotide sequence of interest, said expression construct comprising a promoter and an enhancer located upstream of said promoter; The expression construct comprises, as elements, in the 5' to 3' direction: (a) an optional enhancer 3; and (b) enhancer 2; and (c) enhancer 1; and (d) a promoter; The enhancer 1 comprises all or part of a sequence comprising at least one selected from the group consisting of SEQ ID NOs: 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 to SEQ ID NOs: 13 and 15, wherein all or part of the sequence retains enhancer function; an expression construct, wherein the enhancer 2 comprises all or a part of a sequence containing at least one selected from the group consisting of SEQ ID NOs: 8 to 11 and 13, 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 to SEQ ID NOs: 8 to 11 and 13, and the all or a part of the sequence retains enhancer function.

2. 2. The expression construct of claim 1, wherein the enhancer 3 comprises all or part of a sequence comprising at least one selected from the group consisting of SEQ ID NOs: 8 to 11 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 to SEQ ID NOs: 8 to 11, and the all or part of the sequence retains enhancer function.

3. 2. The expression construct of claim 1, wherein the promoter comprises all or part of a sequence selected from the group consisting of SEQ ID NOs: 2 to 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 to SEQ ID NOs: 2 to 6, and the all or part of the sequence retains promoter function.

4. 4. The expression construct of claim 3, wherein the promoter comprises all or part of a sequence selected from the group consisting of SEQ ID NOs: 3 to 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 to SEQ ID NOs: 3 to 6, and the all or part of the sequence retains promoter function.

5. The enhancer 1 comprises all or part of a sequence comprising at least one selected from the group consisting of SEQ ID NOs: 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 to SEQ ID NOs: 13 and 15, wherein all or part of the sequence retains enhancer function; 2. The expression construct of claim 1, wherein the enhancer 2 comprises all or part of a sequence comprising at least one selected from the group consisting of SEQ ID NOs: 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 to SEQ ID NOs: 8-9, and the all or part of the sequence retains enhancer function.

6. The enhancer 1 comprises all or part of a sequence comprising at least one selected from the group consisting of SEQ ID NOs: 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 to SEQ ID NOs: 13 and 15, wherein all or part of the sequence retains enhancer function; 6. The expression construct of claim 5, wherein the enhancer 2 comprises all or part of SEQ ID NO: 8 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 to SEQ ID NO: 8, and the all or part of the sequence retains enhancer function.

7. The expression construct of claim 1 , wherein the expression construct further comprises an untranslated intron region.

8. 8. The expression construct of claim 7, wherein the untranslated intron region comprises all or part of a sequence selected from the group consisting of SEQ ID NOs: 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 to SEQ ID NOs: 24-43.

9. 8. The expression construct of claim 7, wherein the untranslated intron region is operably linked to the 5' end of the polynucleotide sequence of interest.

10. 8. The expression construct of claim 7, wherein the untranslated intron region is located between the 5' and 3' ends of the polynucleotide sequence of interest.

11. A vector comprising the expression construct according to any one of claims 1 to 10.

12. The vector according to claim 11, wherein the vector is a viral vector, preferably an AAV vector.

13. The vector of claim 12, further comprising two adeno-associated virus inverted terminal repeat (ITR) sequences flanking the expression construct, preferably further comprising a polyA sequence.

14. An adeno-associated virus (AAV) comprising the vector according to any one of claims 11 to 13 and a capsid protein.

15. 15. The AAV of claim 14, 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, AAVrhlO, AAVhu37, or any one of the AAV serotypes isolated from a human or non-human mammal or a mutant thereof.

16. 10. A composition comprising the polynucleotide, expression construct, vector or AAV of any one of the preceding claims and a pharmaceutically acceptable excipient.

17. 17. Use of an expression construct according to any one of claims 1 to 10, a vector according to any one of claims 11 to 13, an AAV according to any one of claims 14 to 15, or a composition according to claim 16 in the manufacture of a medicament for treating a disease or condition in a subject.

18. The use according to claim 17, wherein the treatment comprises administering to a subject an effective amount of an expression construct according to any one of claims 1 to 10, a vector according to any one of claims 11 to 13, an AAV according to any one of claims 14 to 15, or a composition according to claim 16.

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