Polynucleotides for treating GCase deficiency-related diseases

Abstract

The present disclosure provides codon-optimized GBA1 polynucleotides encoding GCase proteins, wherein portions of the coding sequence deviate from wild-type. The present disclosure also provides expression constructs, vectors, viral particles, or compositions containing the disclosed polynucleotides. Furthermore, methods and uses of these polynucleotides, expression constructs, vectors, viral particles, or compositions are provided, including for the treatment of diseases or conditions associated with GCase deficiency.

Description

[0001] priority

[0002] This application claims the benefit of and priority to PCT application No. PCT / CN2022 / 123891 filed on October 8, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to polynucleotides comprising a GBA1 (glucose ceramidase β1) nucleotide sequence encoding β-glucocerebrosidase (GCase), expression constructs, recombinant AAV vectors or viral particles comprising the polynucleotides, and treatments thereof. Background Art

[0004] Gaucher disease (GD) is an autosomal recessive lipid storage disorder characterized by the accumulation of glucocerebroside in cells of the macrophage-monocyte system. GD is caused by mutations in the housekeeping gene GBA1 that impair the activity and / or production of β-glucocerebrosidase (GCase). GCase deficiency leads to the accumulation of its substrate, glucosylceramide, in both visceral organs and the CNS.

[0005] Enzyme replacement therapy (ERT) has been successfully used to ameliorate the visceral pathology of GD. However, in addition to its high cost, ERT treatment for GD typically requires lifelong injections once or more every other week. There is an unmet need for effective therapeutic expression vectors for the treatment of GD, specifically vectors that allow for high-level and stable expression of GCase.

[0006] Adeno-associated virus (AAV) vectors are promising delivery vehicles for gene therapy. AAV has been widely used in preclinical and clinical studies targeting inherited genetic defects due to its high delivery efficiency, stable expression, good safety, and low immunogenicity.

[0007] The present disclosure relates to a gene therapy method for treating GD comprising administering viral particles comprising a GBA1 polynucleotide encoding GCase. The polynucleotides and viral vectors described herein provide higher levels of GCase expression than polynucleotides encoding wild-type GCase. Summary of the Invention

[0008] The present disclosure is based on the generation of optimized polynucleotide sequences and expression constructs thereof for expressing the GBA1 gene.

[0009] The present disclosure provides a highly expressed GBA1 nucleotide sequence, which is highly expressed in, for example, the liver and encodes a GCase polypeptide or a fragment thereof. As demonstrated in the Examples, the polynucleotides of the present invention exhibit higher levels of GCase activity than wild-type GBA1.

[0010] In a first aspect, a polynucleotide encoding human GBA1 is provided, wherein the polynucleotide is codon-optimized for expression in humans and comprises:

[0011] (a) a sequence that is 100% identical to the nucleotide sequence of SEQ ID NOs: 2-19, preferably any one of SEQ ID NOs: 2-7, 9-11, 13-19;

[0012] (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, at least 99.8% identical to the nucleotide sequence of any one of SEQ ID NOs: 2-19, preferably SEQ ID NOs: 2-7, 9-11, 13-19, and the human GBA1 comprises the amino acid sequence of SEQ ID NO: 39;

[0013] (c) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to the nucleotide sequence of SEQ ID NO: 1, or

[0014] (d) The functional fragment of (a), (b) or (c) that retains the function of human GBA1.

[0015] In certain embodiments, the GBA1 comprises a K321N mutation, and the GBA1 comprises the amino acid sequence of SEQ ID NO: 39.

[0016] In certain embodiments, at least a portion of the GBA1 nucleotide sequence is codon optimized.

[0017] In certain embodiments, a polynucleotide is provided, which further comprises a nucleotide sequence encoding a signal peptide, wherein the polynucleotide sequence comprises:

[0018] (a) a sequence that is 100% identical to the nucleotide sequence of SEQ ID NOs: 21-38, preferably any one of SEQ ID NOs: 21-26, 28-30, 32-38;

[0019] (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, at least 99.8% identical to the nucleotide sequence of any one of SEQ ID NOs: 21-38, preferably SEQ ID NOs: 21-26, 28-30, 32-38, and the human GBA1 comprises the amino acid sequence of SEQ ID NO: 40;

[0020] (c) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to the nucleotide sequence of SEQ ID NO: 20, or

[0021] (d) The functional fragment of (a), (b) or (c) that retains the function of human GBA1.

[0022] In certain embodiments, the GBA1 comprises a K321N mutation, and the GBA1 comprises the amino acid sequence of SEQ ID NO: 40.

[0023] In certain embodiments, a polynucleotide is provided that further comprises an untranslated intron region. The untranslated intron region comprises all or part of at least one sequence selected from the group consisting of SEQ ID NOs: 41-59 and 94, 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: 41-59 and 94. Preferably, the untranslated intron region comprises the first sequence set forth in SEQ ID NO: 41; preferably, the untranslated intron region comprises the first sequence set forth in SEQ ID NO: 41 and the second sequence set forth in SEQ ID NO: 45. Preferably, the untranslated intron region is operably linked to the 5' end of the coding region of the human GBA1 gene. Preferably, the untranslated intron region is located between 27-28bp, 115-116bp, 307-308bp, 454-455bp, 588-589bp, 761-762bp, 999-1000bp, 1224-1225bp, 1388-1389bp or 1505-1506bp of SEQ ID NO: 21-26, 28-30, 32-38.

[0024] In certain embodiments, a polynucleotide is provided, comprising:

[0025] (a) a sequence that has 100% identity to the nucleotide sequence of any one of SEQ ID NOs: 60-71;

[0026] (b) a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or at least 99.8% identical to the nucleotide sequence of any one of SEQ ID NOs: 60-71, and the human GBA1 comprises the amino acid sequence of SEQ ID NO: 40; or

[0027] (c) The functional fragment of (a) or (b) that retains the function of human GBA1.

[0028] In another aspect, the present disclosure provides an expression construct comprising a transcriptional regulatory element operably linked to the aforementioned polynucleotide sequence, wherein the transcriptional regulatory element comprises a promoter and / or an enhancer. Preferably, the enhancer is upstream of the promoter.

[0029] In certain embodiments, the promoter comprises all or part of a sequence selected from the group consisting of SEQ ID NOs: 77-82 and 92-93, and sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or at least 99.8% identical to SEQ ID NOs: 77-82 and 92-93, and all or part of the sequence retains promoter function.

[0030] In certain embodiments, the enhancer comprises all or part of a sequence comprising at least one of SEQ ID NOs: 83-91 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 thereto, wherein all or part of the sequence retains enhancer function. The enhancer is a single copy or multi-copy sequence.

[0031] In certain embodiments, the transcriptional regulatory element comprises, in a 5' to 3' direction:

[0032] (a) enhancer 3, which is optional; (b) enhancer 2; (c) enhancer 1; and (d) a promoter; wherein

[0033] The promoter comprises all or part of a sequence selected from the group consisting of SEQ ID NOs: 78-82, and sequences at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or at least 99.8% identical to SEQ ID NOs: 78-82, wherein all or part of the sequence retains promoter function;

[0034] The enhancer 1 comprises all or part of a sequence comprising at least one selected from the group consisting of SEQ ID NOs: 89 and 91, 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: 89 and 91, wherein all or part of the sequence retains enhancer function.

[0035] The enhancer 2 comprises all or part of a sequence comprising at least one selected from the group consisting of SEQ ID NOs: 84-87 and 89, 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: 84-87 and 89, wherein all or part of the sequence retains the function of an enhancer, and

[0036] The enhancer 3 comprises all or part of a sequence comprising at least one selected from the group consisting of SEQ ID NOs: 84, 85, 86 and 87, 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: 84, 85, 86 and 87, and all or part of the sequence retains the function of an enhancer.

[0037] On the other hand, the present disclosure provides an expression vector comprising a polynucleotide sequence of the present disclosure or an expression construct of the present disclosure. The vector is a viral vector, preferably an AAV vector. The vector further comprises two AAV inverted terminal repeat (ITR) sequences flanking the expression construct, preferably further comprising a poly A sequence.

[0038] In another aspect, the present disclosure provides an adeno-associated virus (AAV) comprising the vector of the present disclosure and a capsid protein.

[0039] In certain 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, AAVrh10, AAVhu37, or any AAV serotype isolated from humans or non-human mammals, or variants thereof.

[0040] In another aspect, the present disclosure provides a composition comprising a polynucleotide, an expression construct, a vector or an AAV of the present disclosure and a pharmaceutically acceptable excipient.

[0041] In another aspect, the present disclosure provides a polynucleotide, expression construct, vector or AAV of the present disclosure for use in a method of treatment.

[0042] In certain embodiments, the present disclosure provides use of a polynucleotide, expression construct, vector, or AAV of the present disclosure in the preparation of a medicament for treating a disease or condition in a subject.

[0043] In certain embodiments, the present disclosure provides a polynucleotide, expression construct, vector, or AAV of the present disclosure for use in a method of treating a disease or disorder in a subject.

[0044] In certain embodiments, the present disclosure provides a method of treating a disease or condition in a subject, the method comprising administering to the patient an effective amount of a polynucleotide, expression construct, vector, or AAV of the present disclosure.

[0045] In certain embodiments, the disease or condition is associated with GCase deficiency.

[0046] In certain embodiments, the disease or condition is Parkinson's disease or Alzheimer's disease.

[0047] In certain embodiments, the disease or disorder is Gaucher disease, preferably

[0048] (i) the Gaucher disease is type I, II or III Gaucher disease or neuropathic Gaucher disease; and / or

[0049] (ii) The patient has an antibody or inhibitor against recombinant GCase used as part of the enzyme replacement therapy the patient has been receiving.

[0050] In another aspect, the present disclosure provides a polynucleotide, expression construct, vector or AAV of the present disclosure for use in a method of expressing the GBA1 nucleotide sequence and achieving stable GCase activity in a subject.

[0051] In another aspect, the present disclosure provides a polynucleotide, expression construct, vector, or AAV of the present disclosure for use in a method of expressing a GBA1 nucleotide sequence and providing increased GCase bioavailability in a subject compared to the bioavailability in a subject from GCase enzyme replacement therapy, wherein the bioavailability is measured over a period of 2 weeks from administration.

[0052] In certain embodiments, achieving stable GCase activity and / or providing increased GCase bioavailability facilitates treating a disease in the subject.

[0053] In certain embodiments, the disease is Gaucher disease, optionally wherein the Gaucher disease is type I, II, or III Gaucher disease or neuropathic Gaucher disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1Figure 2 shows the in vitro GCase activity of Huh7 cells transfected with various codon-optimized constructs containing the K321N mutation. P < 0.01 for the PG002-PG007, PG009-PG011, PG013-PG014, PG016-PG019, and PG021 groups compared to the PG129 group. Values ​​are expressed as mean ± SEM. N = 3 for each experimental group.

[0055] Figure 2 Figure 2 shows the in vitro GCase activity of various codon-optimized constructs with the K321N mutation. Huh7 cells were transduced using an AAV9 vector at an MOI of 1E6 vg / cell. P < 0.05 for the PG002-PG007, PG009-PG011, PG013, PG019, and PG021 groups compared to the PG129 group. Values ​​are expressed as mean ± SEM. N = 3 for each experimental group.

[0056] Figure 3 Shown are serum GCase enzyme activities in Gaucher disease mice on day 56 after injection of vectors AAV9-PG001 and AAV9-PG011 at a dose of 2E12 vg / kg. Groups represent wild-type mice. P < 0.01 for the PG011 group compared with the PG001 and buffer control groups from day 7 to day 42. P < 0.05 for the PG011 group compared with the PG001 and buffer control groups on day 56. Values ​​are expressed as mean ± SEM. N = 4 per experimental group.

[0057] Figure 4 Figures show GCase enzyme activity in tissue lysates of Gaucher disease mice 8 weeks after injection with the vectors AAV9-PG001 and AAV9-PG011 at a dose of 2E12 vg / kg. (A) GCase enzyme activity measured in liver lysates. P < 0.01 for the PG011 group compared with the PG001 and buffer control groups. (B) GCase enzyme activity measured in lung lysates. P < 0.05 for the PG011 group compared with the buffer control group. (C) GCase enzyme activity measured in spleen lysates. P < 0.01 for the PG011 group compared with the PG001 and buffer control groups. Naive group represents the wild-type group. Values ​​are expressed as mean ± SEM. N = 4 per experimental group.

[0058] Figure 5Figures show the substrate accumulation levels of glucosphingosine in tissue lysates from Gaucher disease mice 8 weeks after injection of vectors AAV9-PG001 and AAV9-PG011 at a dose of 2E12 vg / kg, as measured by LC-MS / MS. (A) Substrate accumulation measured in liver lysates. Glucosphingosine levels in the PG011 2E12 vg / kg group were below the limit of detection. P < 0.01 for the PG011 and PG001 groups compared to the buffer control group. (B) Substrate accumulation measured in lung lysates. P < 0.01 for the PG011 group compared to the buffer control group. (C) Substrate accumulation measured in spleen lysates. P < 0.01 for the PG011 and PG001 groups compared to the buffer control group. Groups represent wild-type mice. Values ​​are expressed as mean ± SEM. N = 4 per experimental group.

[0059] Figure 6 Gaucher cell density in H&E-stained sections of Gaucher disease mice 8 weeks after injection of vectors AAV9-PG001 and AAV9-PG011 at doses of 2E13, 2E12, and 2E11 vg / kg. (A) Gaucher cell counts in liver sections. (B) Gaucher cell counts in lung sections. Groups represent wild-type mice. All Gaucher cells were judged by pale cytoplasm and multinuclear staining and manually counted throughout the tissue section.

[0060] Figure 7 Shown is immunohistochemistry of CD68 (a biomarker for macrophages) positive cells in liver and lung from Gaucher disease mice 8 weeks after injection of vectors AAV9-PG001 and AAV9-PG011 at a dose of 2E12 vg / kg detected by anti-CD68 antibody. Groups represent wild-type mice.

[0061] Figure 8Figures show the GCase activity of constructs containing chimeric HSREs transfected into HepG2 cells. (A) Comparison of GCase activity of different CHSREs based on the promoter HSRE002. P < 0.05 was observed for the PG025 group compared to the PG022 group, and P < 0.01 was observed for the PG023, PG026, PG028, PG029, and PG030 groups compared to the PG022 group. (B) Comparison of GCase activity of different CHSREs based on the promoter HSRE005. P < 0.05 was observed for the PG035 group compared to the PG032 group, and P < 0.01 was observed for the other groups compared to the PG032 group. (C) Comparison of GCase activity of different CHSREs based on the promoter HSRE004. P < 0.05 for the PG042 group compared with the PG039 group, and P < 0.01 for the PG040, PG041, PG043, PG044, and PG045 groups compared with the PG039 group. (D) Comparison of GCase activity of different CHSREs based on the promoter HSRE003. P < 0.05 for the PG049 and PG052 groups compared with the PG046 group, and P < 0.01 for the PG047, PG048, and PG051 groups compared with the PG046 group. Values ​​are expressed as mean ± SEM. N = 3 for each experimental group.

[0062] Figure 9 Figure 2 shows the GCase activity of constructs with different positions and copy numbers of HSRE012 in HepG2 cells. (A) GCase activity of constructs flanking the HSRE002 promoter with different positions of HSRE012. P < 0.01 for the PG053 group compared to the PG025 group. P < 0.01 for the PG054 group compared to the PG026 group. (B) Comparison of GCase activity of constructs flanking the HSRE002 promoter with different copy numbers of HSRE012. No significant differences were found between PG055 and PG025, PG056 and PG026, or PG057 and PG027. Values ​​are expressed as mean ± SEM. N = 3 for each experimental group.

[0063] Figure 10Figure 2 shows the in vitro GCase activity of constructs containing HSREs and different introns. (A) GCase activity of constructs containing endogenous introns in Huh7 cells. P < 0.05 for the PG059 group compared to the PG058 group. (B) GCase activity of constructs containing different exogenous introns in HepG2 cells. P < 0.01 for the PG076, PG078, and PG081 groups compared to the PG074 group. P < 0.01 for the PG090 group compared to the PG082 group, and P < 0.05 for the PG091 group compared to the PG082 group. P < 0.01 for the PG093, PG094, PG095, PG097, PG098, PG099, PG100, and PG101 groups compared to the PG092 group. (C) GCase activity of constructs containing different exogenous introns in HEK293T cells. Values ​​are expressed as mean ± SEM. N=3 for each experimental group.

[0064] Figure 11 Figure 2 shows the in vitro GCase activity of constructs containing different introns of chimeric HSREs. (A) GCase activity of different constructs in HepG2 cells. P < 0.05 for the G107 and PG108 groups compared to the PG168 group. P < 0.01 for the PG114 and PG115 groups compared to the PG169 group. (B) GCase activity of different constructs in HEK293T cells. Error bars represent mean ± SEM. N = 3 for each experimental group.

[0065] Figure 12Figures show GCase enzyme activity in serum and tissue lysates 2 weeks after injection of AAV8 vectors at a dose of 2E12 vg / kg in wild-type mice. (A) GCase enzyme activity measured in serum. P < 0.05 for the PG026, PG037, PG051, and PG105 groups compared to the PG127 group, and P < 0.01 for the PG103, PG104, and PG105 groups compared to the PG127 group. P < 0.01 for the PG103 and PG104 groups compared to the PG102 group. P < 0.01 for the PG103 and PG104 groups compared to the PG026 group. (B) GCase enzyme activity measured in liver lysates. P < 0.05 for the PG026 group compared to the PG127 group, and P < 0.01 for the PG103, PG104, and PG105 groups compared to the PG127 group. P < 0.01 for the PG103 and PG104 groups compared with the PG102 group. P < 0.01 for the PG103 and PG104 groups compared with the PG026 group. P < 0.01 for the PG105 group compared with the PG051 group. (C) GCase enzyme activity measured in lung lysates. P < 0.05 for the PG105 and PG026 groups compared with the PG127 group, and P < 0.01 for the PG103 and PG104 groups compared with the PG127 group. P < 0.05 for the PG104 group compared with the PG102 group, and P < 0.01 for the PG103 group compared with the PG102 group. P < 0.05 for the PG103 group compared with the PG026 group. (D) GCase enzyme activity measured in spleen lysates. P < 0.01 for the PG026, PG103, PG104, and PG105 groups compared with the PG127 group. P < 0.01 for the PG103 and PG104 groups compared with the PG102 group. P < 0.01 for the PG103 group compared with the PG026 group. Values ​​are expressed as mean ± SEM. N = 4 for each experimental group.

[0066] Figure 13Figure 2 shows the significant efficacy of an AAV8 gene therapy candidate using chimeric HSREs in mice with Gaucher disease. (A) GCase enzyme activity in serum measured 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 imiglucerase group. P < 0.05 for the PG118 and PG122 groups compared with the PG127 group, and P < 0.01 for the G119 and PG120 groups compared with the PG127 group. (B) Substrate accumulation in serum measured 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 imiglucerase group. P < 0.05 for the PG119 group compared with the PG127 group, and P < 0.01 for the PG118, PG120, and PG122 groups compared with the PG127 group. Serum glucose sphingosine levels in PG119 were below the detection limit. Groups represent corresponding wild-type mice. Values ​​are expressed as mean ± SEM. N = 5 per experimental group.

[0067] Figure 14 Figure 2 shows GCase enzyme activity in tissue lysates from Gaucher disease mice 12 weeks after injection of an AAV8 candidate at a dose of 2E12 vg / kg. (A) GCase enzyme activity measured in liver lysates. P < 0.01 for the PG118-PG122 group compared to the buffer control or imiglucerase group. P < 0.05 for the PG120 and PG122 groups compared to the PG127 group, and P < 0.01 for the PG118 and PG119 groups compared to the PG127 group. (B) GCase enzyme activity measured in lung lysates. P < 0.05 for the PG118-PG122 group compared to the buffer control or imiglucerase group. P < 0.05 for the PG118 and PG122 groups compared to the PG127 group, and P < 0.01 for the PG119 and PG120 groups compared to the PG127 group. (C) GCase enzyme activity measured in spleen lysates. The PG118-PG122 groups showed a P < 0.01 difference compared with the buffer control group. The PG120 group showed a P < 0.05 difference compared with the imiglucerase group, and the PG118, PG119, and PG122 groups showed a P < 0.01 difference compared with the imiglucerase group. The PG118 and PG122 groups showed a P < 0.05 difference compared with the PG127 group, and the PG119 and PG120 groups showed a P < 0.01 difference compared with the PG127 group. Groups represent wild-type mice. Values ​​are expressed as mean ± SEM. N = 5 per experimental group.

[0068] Figure 15Figure 2 shows substrate accumulation of glucosphingosine in tissue lysates 12 weeks after 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 to the buffer control or imiglucerase group. P < 0.01 for the PG119 and PG120 groups compared to the PG127 group. (B) Substrate accumulation measured in lung lysates. P < 0.05 for the PG001 group compared to the buffer control or imiglucerase group, and P < 0.01 for the PG011, PG119, and PG120 groups compared to the buffer control or imiglucerase group. P < 0.05 for the PG119 and PG120 groups compared to the PG127 group. (C) Substrate accumulation measured in spleen lysates. The PG001, PG011, PG119, and PG120 groups were compared with the buffer control or imiglucerase group (P<0.01). The PG119 and PG120 groups were compared with the PG127 group (P<0.01). Representative of wild-type mice. Error bars represent mean ± SEM. N = 5 per experimental group. Glucose sphingosine levels in various tissue lysates were analyzed using LC-MS / MS.

[0069] Figure 16Figures show GCase enzyme activity in serum and tissue lysates 2 weeks after injection of AAV9 candidates at a dose of 2E12 vg / kg in wild-type mice. (A) GCase enzyme activity measured in serum. P < 0.05 for the PG124 group compared to the buffer control group, and P < 0.01 for the PG123, PG125, and PG126 groups compared to the buffer control group. P < 0.05 for the PG124 and PG125 groups compared to the PG001 group, and P < 0.01 for the PG123 and PG126 groups compared to the PG001 group. P < 0.05 for the PG125 group compared to the PG128 group, and P < 0.01 for the PG123 and PG126 groups compared to the PG128 group. (B) GCase enzyme activity measured in liver lysates. P < 0.01 for the PG123-PG126 groups compared to the buffer control group. P < 0.05 for the PG124 and PG125 groups compared to the PG001 group, and P < 0.01 for the PG123 and PG126 groups compared to the PG001 group. P < 0.05 for the PG124 and PG125 groups compared to the PG128 group, and P < 0.01 for the PG123 and PG126 groups compared to the PG128 group. (C) GCase enzyme activity measured in lung lysates. P < 0.05 for the PG124 group compared to the buffer control group, and P < 0.01 for the PG123, PG125, and PG126 groups compared to the buffer control group. P < 0.05 for the PG124 group compared to the PG001 group, and P < 0.01 for the PG123, PG125, and PG126 groups compared to the PG001 group. P < 0.05 for the PG123 and PG125 groups compared to the PG128 group, and P < 0.01 for the PG126 group compared to the PG128 group. (D) GCase enzyme activity measured in spleen lysates. P < 0.01 for the PG123-PG126 groups compared to the buffer control group. P < 0.05 for the PG124 group compared to the PG001 group, and P < 0.01 for the PG123, PG125, and PG126 groups compared to the PG001 group. P < 0.05 for the PG124 group compared to the PG128 group, and P < 0.01 for the PG123, PG125, and PG126 groups compared to the PG128 group. Values ​​are expressed as mean ± SEM. N = 4 per experimental group. DETAILED DESCRIPTION

[0070] The following will be a more comprehensive description of embodiments according to the present disclosure. However, various aspects of the present disclosure may be embodied in different forms and should not be construed as being limited to the embodiments listed herein. On the contrary, these embodiments are provided to make the present disclosure comprehensive and complete and to fully convey the scope of the present invention to those skilled in the art. The terms used in the description herein are only used to describe the embodiments and are not intended to be limiting.

[0071] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should also be understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning consistent with their meaning in the context of this application and the related art, and should not be interpreted in an idealized or overly formal sense unless explicitly defined as such herein.

[0072] definition

[0073] As used in the description of the invention and the appended claims, singular forms are intended to include plural forms as well, unless the context clearly indicates otherwise.

[0074] As used herein, the term "comprising" means that the compositions and methods include the recited elements, but do not exclude other elements.

[0075] As used herein, the terms "nucleotide" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, 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 comprising, consisting essentially of, or consisting of purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derived nucleotide bases.

[0076] The polynucleotide can be DNA or RNA. In certain aspects, the present disclosure provides an isolated polynucleotide comprising an expression construct encoding GCase (e.g., the gene product of the GBA1 gene) or a portion thereof. GCase, also known as β-glucocerebrosidase or GBA1, refers to a lysosomal protein that cleaves the β-glucosidic bond of the chemical glucocerebroside, an intermediate in glycolipid metabolism. In humans, GCase is encoded by the GBA1 gene located on chromosome 1. In certain embodiments, GBA1 encodes a peptide having a K321N mutation as shown in SEQ ID NO: 39 or 40. In certain embodiments, the isolated polynucleotide comprises a codon-optimized GCase coding sequence (e.g., codon-optimized for expression in mammalian cells, such as human cells), such as a sequence set forth in SEQ ID NOs: 2-7, 9-11, 13-19, 21-26, 28-30, 32-38, or a sequence having at least 90%, at least 95%, at least 99%, at least 99.5%, or at least 99.8% identity to the nucleotide sequence of any one of SEQ ID NOs: 2-7, 9-11, 13-19, 21-26, 28-30, 32-38. The polynucleotide further comprises an untranslated intron region, such as a sequence set forth in SEQ ID NOs: 41-61.

[0077] As used herein, "expression" refers to a two-step process in which a polynucleotide is transcribed into mRNA and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression in eukaryotic cells may include splicing of the mRNA.

[0078] The term "encoding" when applied to a polynucleotide means that if a polynucleotide can be transcribed to produce mRNA for a polypeptide and / or fragment thereof, then the polynucleotide is said to "encode" the polypeptide. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be inferred therefrom.

[0079] As used herein, the term "promoter" refers to a control sequence that is a region of a polynucleotide sequence that controls the initiation and rate of transcription of a coding sequence, such as a gene or transgene. Promoters can 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 the expression of a target sequence.

[0080] The terms "protein," "peptide," and "polypeptide" are used interchangeably and in the broadest sense refer to a compound of two or more subunits of amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. Alternatively, the subunits may be linked by other bonds such as esters, ethers, and the like. A protein or peptide must contain at least two amino acids, and there is no limit on the maximum number of amino acids that a protein or peptide sequence may comprise, consist essentially of, or consist of. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including glycine and the D and L optical isomers, amino acid analogs, and peptidomimetics.

[0081] "Identity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Percent identity can be determined by comparing positions in each sequence that may 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.

[0082] As used herein, the term "vector" refers to a nucleic acid comprising a complete replicon, or consisting essentially of or consisting of a complete replicon, such that the vector can be replicated when placed in a cell by, for example, a transfection, infection, or transformation process. It should be understood in the art that once inside the cell, the vector can be replicated as an extrachromosomal (episomal) element, or can be integrated into the host cell chromosome. The vector can include nucleic acids derived from retroviruses, adenoviruses, herpes viruses, baculoviruses, modified baculoviruses, papillomaviruses, AAV viral vectors, lentiviral vectors, adenoviral vectors, alphaviral vectors, etc. Alphaviral vectors, such as vectors based on Semliki Forest virus and vectors 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.

[0083] The term "adeno-associated virus" or "AAV" as used herein refers to members of the class of viruses associated with that name and belonging to the genus Dependoparvovirus of the family Parvoviridae. Adeno-associated virus is a single-stranded DNA virus that grows only in cells in which certain functions are provided by a co-infecting helper virus. All AAV serotypes exhibit very similar replication characteristics mediated by homologous rep genes; and all carry three related capsid proteins. At least 13 sequentially numbered naturally occurring AAV serotypes are known in the art. Non-limiting exemplary serotypes for use 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 comprise, consist essentially of, or consist of the three major viral proteins VP1, VP2, and VP3. In embodiments, the AAV particles comprise an AAV capsid protein selected from the group consisting of AAVPHP.B, AAVrh74, AAV110, AAV204, AAV214, AAV214A, AAV214e, AAV214e8, AAV214e9, AAV214e10, AAV1TB102-45, and AAV214AB. In embodiments, AAV refers to 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 human and non-human mammals, or variants thereof. In embodiments, the AAV particles comprise an AAV selected from the group consisting of 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、AAV 223.6、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.6、AAV3-9 / rh.61 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、AAV14 5.1 / 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、AAVh. u.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.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、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.17、AAVhu.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.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.40、AAVhu.41、AAVhu.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.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 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 SMAAV54.1 / 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-12, AAV CLv1-3, AAV CLv-13, AAV CLv1-4, AAV Clv1-7, AAV Clv1-8, AAV Clv1-9, AAV CLv-2AAV CLv-3、AAV CLv-4、AAV CLv-6、AAV CLv-8、AAV CLv-D1、AAVCLv-D2、AAV CLv-D3、AAV CLv-D4、AAV CLv-D5、AAV CLv-6、AAV CLv-D5 CLv-D7, AAV CLv-D8, AAVCLv-E1, AAV CLv-K1, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, MAAVCLv-AAV CLv-M11, AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLv-M7, AAV CLv-M8, AAVCLv-M9, AAV CLv-R1, AAV CLv-R2, AAV-RAV-RAV3 CLv-R4、AAV CLv-R5、AAV CLv-R6、AAVCLv-R7、AAV CLv-R8、AAV CLv-R9、AAV CSp-1、AAV CSp-10、AAV CSp-11、AAV CSp-10、AAV CSp-11、AAV CSp-2、AAV Cp-3 CSp-4、AAV CSp-6、AAV CSp-7、AAV CSp-8、AAV CSp-8.10、AAV CSp-8.2、AAVCSp-8.4、AAV CSp-8.5、AAV CSp-8.6、AAV CSp-8.7. 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, AAVF13 / HSC131 / AAVF14 HSC14,AAVF15 / HSC15,AAVF16 / HSC16,AAVF17 / HSC17,AAVF2 / HSC2,AAVF3 / HSC3,AAVF4 / HSC4,AAVF5 / HSC5,AAVF6 / HSC6, VF7 / 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、AAVPH. P.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, 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, AAVG2A15 / G2A3, AAVG2B4, AAVG2B5 and variants thereof.

[0084] In embodiments, AAV refers to serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13. In embodiments, the AAV particle comprises an AAV capsid protein selected from the group consisting of AAVPHP.B, AAVrh74, AAV110, AAV204, AAV214, AAV214A, AAV214e, AAV214e8, AAV214e9, AAV214e10, AAVITB102-45, and AAV214AB.

[0085] " AAV vector " used herein refers to a vector comprising one or more heterologous nucleic acid (HNA) sequences and one or more AAV inverted terminal repeats (ITRs). Such AAV vectors can replicate in host cells that provide the functions of rep and cap gene products, and allow the nucleic acid between ITR and ITR to be packaged in infectious viral particles. In an embodiment, within the ITR flanking the infectious AAV particles, the AAV vector comprises a promoter, at least one nucleic acid sequence and / or enhancer and / or terminator that can encode at least one protein or RNA. The nucleic acid between ITR and ITR can be wrapped in an AAV capsid, and this encapsidated nucleic acid can be referred to as an "AAV vector genome." In addition to the encapsidated part, the AAV vector can contain other elements such as antibiotic resistance genes or other elements known in the art, which are contained in a plasmid for manufacturing purposes, but are not packaged into AAV particles.

[0086] As used herein, the term "viral capsid" or "capsid" refers to the protein shell or capsid of the viral particle. The capsid has the function of encapsidating, protecting, transporting and / or releasing the viral genome into the host cell. The capsid is generally composed of oligomeric structural subunits of proteins ("capsid proteins"). The viral capsid of AAV is composed of a mixture of three viral capsid proteins: VP1, VP2, and VP3.

[0087] "AAV virion" or "AAV viral particle" or "AAV particle" refers to a viral particle composed of at least one AAV capsid protein and the encapsidated polynucleotide from an AAV vector (referred to herein as the AAV vector genome).

[0088] A "subject" for diagnosis or treatment is an animal, such as a mammal or a human. The subject is not limited to a particular species and includes non-human animals undergoing diagnosis or treatment and non-human animals or animal models for infection, including but not limited to simian, murine, rat, canine, or lagomorphic species, as well as other livestock, sports animals, or pets. In an embodiment, the subject is a human.

[0089] As used herein, "treatment" of a disease in a subject means: (1) preventing the occurrence of symptoms or disease in a subject susceptible to the disease or not yet showing symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating the disease or disease symptoms or causing their regression. As understood in the art, "treatment" is a method of obtaining beneficial or desired results (including clinical results). For the purposes of the present technology, beneficial or desired results may include, but are not limited to, one or more of the following: alleviation or improvement of one or more symptoms, alleviation of the extent of a condition (including a disease), stabilization (i.e., non-exacerbation) of the state of a condition (including a disease), delaying or slowing the progression of a condition (including a disease), improvement or alleviation of the state of a condition (including a disease), and remission (whether partial or complete), whether detectable or undetectable.

[0090] As used herein, the term "effective amount" means an amount sufficient to achieve the desired effect. In the case of therapeutic or prophylactic 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 overall health, age, sex, weight, and tolerance to the pharmaceutical composition. In the case of gene therapy, in an embodiment, an effective amount is an amount sufficient to enable the defective gene in the subject to gain partial or full function. In other embodiments, the effective amount of AAV viral particles is an amount sufficient to cause the gene to be expressed in the subject. Those skilled in the art will be able to determine a suitable amount based on these and other factors.

[0091] In embodiments, the effective amount will depend on the scale and nature of the application in question. It will also depend on the characteristics and sensitivity of the target subject and the method of use. A skilled artisan will be able to determine an effective amount based on these and other considerations. An effective amount may comprise, consist essentially of, or consist of one or more administrations of the composition, depending on the embodiment.

[0092] As used herein, the term "administer" means delivering a substance to a subject, such as an animal or a human. Administration can be performed once, continuously, or intermittently throughout the course of treatment. Methods for determining the most effective means and dosage of administration are known to those skilled in the art and vary with the composition used for treatment, the purpose of treatment, and the age, health, or sex of the subject being treated. Single or multiple administrations can be performed, with the dosage level and pattern being selected by the treating physician or, in the case of pets and other animals, by the treating veterinarian.

[0093] Example

[0094] Unless otherwise specified, the following general procedures were followed in the examples described below.

[0095] rAAV production

[0096] AAV8 and AAV9 viral particles were produced by transient transfection of HEK293T cells or HEK293 suspension cells with a plasmid encoding the AAV Rep and Cap genes, a plasmid encoding the adenoviral helper genes, and a construct plasmid containing the GBA1 gene. rAAV particles were purified using an iodixanol-based density gradient ultracentrifugation method. Subsequently, rAAV was quantified by a probe-based ddPCR (Biorad) assay and characterized for purity by silver staining.

[0097] In vitro transfection and r-AAV potency assay

[0098] One day before transfection, HEK293T cells or hepatocyte cell lines HepG2 and Huh7 were added to a 24-well plate at a cell density of 1.5E5 cells / well. 500 μL complete cell culture medium was added to each well. Transfection used a PEI-based transfection reagent. Specifically, 0.15 μg of a plasmid containing a transgenic sequence and 0.15 μg of a plasmid containing a luciferase reporter gene were co-transfected into each well. 48 h after transfection, 300 μL of fresh complete cell culture medium was added to each well, and the cells were incubated for a further 24 hours. 72 hours after transfection, cell culture supernatants were collected. Cell lysates were obtained using cell lysis buffer (Promega). GCase enzyme activity was determined for the cell culture supernatants according to the method described below. The cell lysates were diluted and luciferase assays were performed using the Steady-Glo luciferase assay system (Promega). 96-well plates containing cell lysates and detection reagents were read on a Varioskan LUX plate reader (ThermoFisher). Data shown are enzyme activities normalized to firefly luciferase intensity and then to a control group.

[0099] The rAAV bioefficacy assay was performed by cell transduction using HEK293T, Huh7 or HepG2 cell lines. 24 hours before transduction, these cells were plated in 24-well plates at a density of 1.5E5 cells / well. rAAV transduction was performed at a defined 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 incubated for an additional 24 hours. 72 hours after infection, the cell culture supernatant was assayed for enzyme activity as described above.

[0100] Wild-type mouse study design

[0101] An AAV vector containing the GBA1 transgene was administered via tail vein injection into 8-9 week old wild-type (C57BL / 6) male mice. The AAV injection dose was 2E12 vg / kg. To assess the kinetics and persistence of transgene expression, serum GCase levels were measured at various time intervals, specifically 1 and 2 weeks post-injection. Mice were monitored for up to 2 weeks following AAV treatment and then sacrificed for biochemical and pathological analyses.

[0102] Gaucher disease mouse study design

[0103] An AAV vector containing the GBA1 transgene was administered via tail vein injection into 7-12 week-old mice with Gaucher disease (a combination of two different GBA1 mutations). All mice were housed in a pathogen-free environment in individually ventilated cages. All cages, corncob bedding, and water were sterilized before use. Cages, corncob bedding, food, and water were changed twice weekly.

[0104] AAV injection doses ranged from 2E11 to 2E13 vg / kg. To assess the kinetics and persistence of transgene expression, serum GCase levels and substrate accumulation were measured at various time intervals post-injection. Mice were followed until study endpoint and sacrificed for biochemical and pathological analyses.

[0105] Preparation and administration of AAV / imiglucerase

[0106] Store rAAV aliquots at -80°C. Thaw aliquots on ice and dilute with AAV formulation buffer before injection. Place diluted AAV on ice before injection and use within 2 hours.

[0107] Imiglucerase was resuspended according to the manufacturer's instructions, divided into aliquots (40 IU / mL) and stored at -80° C. Prior to injection, the aliquots were thawed on ice and diluted with gentle but thorough mixing.

[0108] Serum and tissue collection

[0109] Serum was separated from fresh blood without anticoagulant by centrifugation at 12,000 rpm for 15 minutes at 4° C. within 0.5 hours and stored at −80° C. For the imiglucerase group, serum was collected 1.5 hours after injection.

[0110] Mice were anesthetized and euthanized. Tissues were collected from mice after saline perfusion and stored at -80°C. For the imiglucerase group, tissue samples were collected 1.5 hours after injection. Each tissue sample was divided into 4 parts, 3 of which were frozen in separate tubes and stored at -80°C. These samples were intended for GCase activity assay, glucose sphingosine analysis, and mRNA analysis. The remaining part was fixed in 10% neutral buffered formalin solution (NBF, pH 7.4) at room temperature for approximately 24-48 hours for histological analysis. Bone marrow cells were collected from the femur and tibia of both legs of the mice.

[0111] GCase activity assay in mouse serum and tissue

[0112] Serum samples were obtained from mouse blood and stored at -80 ° C. Homogenizer (Shanghai jingxin) was used to crack tissue in tissue lysis buffer (citrate-phosphate buffer, pH 5.0, 0.25% sodium taurocholate, 1% TX-100 and protease inhibitor cocktail) under a specific program (50 Hz homogenization 30 s and cooling 30 s, 4 min in total). For enzyme activity determination, β-glucocerebrosidase (acid β-glucosidase; GCase) activity was measured by a fluorescence-based assay. The substrate for GCase was 4-methylumbelliferyl β-D-glucopyranoside (4MU-Glc, Carbosynth). On the same day of determination, serum samples were 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 protease inhibitor cocktail).

[0113] All samples were measured at 37 ℃ for 1 hour in citrate-phosphate buffer, pH 5.0, 0.25% sodium taurocholate, 0.25% TX-100, 1mM 4MU-Glc. The reaction was terminated by adding three times the volume of stop solution (0.5M glycine, pH 10.8) of 150ul. Varioskan LUX was used to read a plate reader (ThermoFisher), and excitation and emission wavelengths of 360nm and 460nm were used to assess relative fluorescence levels (RFU). Tissue lysate samples were also measured by BCA test kit (Thermofisher) for protein concentration. Fluorescence levels were then converted to nmol / h / mL (serum) or nmol / h / mg total protein (liver, spleen, bone marrow and brain) based on the standard curve of 4-methylumbelliferone (4-MU, Sigma-Aldrich).

[0114] Vector genome copy number, relative RNA transcription level

[0115] To determine the vector genome copy number in tissue samples after rAAV injection, DNA was isolated from frozen liver tissue 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 number of cells / reaction was calculated based on the quantitative results of the DNA amount. The vector genome copy number / cell was then calculated by normalizing the genome copy number / reaction to the number of cells / reaction.

[0116] To determine relative RNA transcript 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 applied to a probe-based qPCR (Roche) assay.

[0117] Immunohistochemistry

[0118] Mouse macrophages were detected using a rabbit anti-mouse CD68 antibody (Abcam AB53444, 1:25). Formalin-fixed mouse tissues were deparaffinized with xylene and washed with graded ethanol. Antigen retrieval was then performed using pepsin according to the manufacturer's instructions. Sections were counterstained with hematoxylin. Detection was performed using a biotinylated secondary antibody. Signal development was performed using a streptavidin-HRP and Tyramide signal amplification kit according to the manufacturer's instructions.

[0119] Storage cell count

[0120] 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 an Aperio ImageScope (V12.4.3.5008). All Gaucher cells were manually counted on the entire tissue section of the liver and lung of each mouse. Gaucher cell counts from the entire section were normalized to the tissue section area (square centimeters) for data plots.

[0121] Glucose sphingosine analysis

[0122] 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. Quantitative tissue glucose sphingosine levels were normalized by tissue weight, and substrate levels in serum were normalized by serum volume. In the corresponding figures, values ​​below the lower limit of quantification (LLOQ) of glucose sphingosine of 10 ng / g (for tissue) and 1 ng / mL (for serum or plasma) will be labeled BQL.

[0123] Statistical analysis

[0124] Data are presented 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.

[0125] Example 1: GBA1 construct

[0126] To improve the therapeutic efficacy of gene therapy for Gaucher disease caused by metabolic disorders, this disclosure outlines a comprehensive approach for designing and screening novel expression cassettes that efficiently and selectively express therapeutic GCase in the liver.

[0127] The first step involves cloning a codon-optimized GBA1 gene with the K321N mutation into an AAV vector under the control of the universal promoter CRE001. To restrict GCase expression to the liver, the GBA1 gene is driven by a series of chimeric liver-specific promoters strategically designed using combinations of different promoters and various regulatory elements. These promoters, described herein, are HSRE001, HSRE002, HSRE003, HSRE004, HSRE005, HSRE015, and HSRE016. In addition, several regulatory elements are used, designated HSRE006, HSRE007, HSRE008, HSRE009, HSRE010, HSRE011, HSRE012, HSRE013, and HSRE014. In certain embodiments, introns are also used to further enhance GCase expression. Table 1 provides the IDs and nucleotide sequences of various promoters and regulatory elements used in this disclosure.

[0128] Table 1. Different regulatory elements used in this disclosure

[0129]

[0130]

[0131] Example 2: In vitro screening of GBA1 transgenic constructs

[0132] To enhance GCase expression, 18 codon-optimized human GBA1 constructs (PG002-021) with the K321N mutation were constructed under the control of the universal CRE001 promoter. PG129 (SEQ ID 95 or 96), which harbors the K321N mutation in the wild-type GBA1 sequence, and PG001 (SEQ ID 1 or 20), which harbors the wild-type GBA1 sequence, were used as controls. All of these GBA1 constructs were transfected into Huh-7 cells according to the previously described method to screen for candidates exhibiting enhanced GCase activity. The codon-optimized nucleotide sequences of human GBA1 are shown in Table 2. These codon-optimized human GBA1 nucleotide sequences encode the polypeptide sequences of human GBA1 (mGBA) harboring the K321N mutation. Specifically, the polypeptide sequence of the codon-optimized human GBA1 lacking the signal peptide but harboring the K321N mutation is shown in SEQ ID NO: 39. The codon-optimized polypeptide sequence of human GBA1 including the signal peptide portion and the K321N mutation is shown as SEQ ID NO:40.

[0133] Table 2. Nucleotide sequence of human GBA1

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144] The polypeptide sequence of codon-optimized human GBA1 without the signal peptide portion but with the K321N mutation (SEQ ID NO: 39)

[0145] ARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDFLAPANATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSIHTYLWRRQ*

[0146] Polypeptide sequence of codon-optimized human GBA1 with a signal peptide portion and having a K321N mutation (SEQ ID NO: 40)

[0147] MEFSSPSREECPKPLSRVSIMAGSLTGLLLLQAVSWASGARPCIPKSFGYSSVVCVCNA

[0148] TYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVK

[0149] GFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTP

[0150] DDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSL

[0151] KGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPE

[0152] HQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVH

[0153] WYLDFLAPANATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSII

[0154] TNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSK

[0155] FIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSIHTYLWRRQ*

[0156] Compared with PG129, the GCase activity of PG011, PG006, PG005, PG002, PG003, PG007, PG004, PG010, PG018, PG014, PG019, PG009, PG016, PG021, PG013, and PG017 was significantly increased ( Figure 1 All these constructs were packaged into AAV9 vectors for further in vivo bioefficacy assays as described below.

[0157] Codon-optimized K321N constructs (PG002-PG007, PG009-PG011, PG013-PG015, PG017-PG021) and the control construct PG129 were packaged into rAAV9 and purified as described above. The bioefficacy of these rAAV9 candidates was evaluated by in vitro transduction assay in Huh-7 cells. All codon-optimized K321N constructs showed higher enzyme activity compared to PG129 ( Figure 2 ).

[0158] Example 3: In vivo therapeutic effect of codon-optimized constructs in Gaucher disease mice

[0159] To evaluate its therapeutic potential in Gaucher disease, a codon-optimized construct of PG011 was packaged with AAV9 and used in a therapeutic study in Gaucher disease mice along with a wild-type GBA1 construct of PG001. The constructs of PG011 and PG001 were injected at a dose of 2E12 vg / kg. The results showed that GCase was expressed in serum, liver and spleen of Gaucher disease mice ( Figure 3-5 ).

[0160] Compared with the buffer control group, all AAV injection groups showed higher GCase expression, and PG011 showed better expression than PG001. The results of glucose sphingosine accumulation were consistent with the GCase activity in the serum and tissues of the corresponding groups mentioned above ( Figure 5 All AAV-injected groups showed significantly reduced glucosphingosine accumulation in the liver, lungs, and spleen compared to the buffer control group. Notably, the PG011 construct significantly outperformed PG001 in both assays. In the liver and spleen, glucosphingosine accumulation was reduced to the same level in the PG011 group at a dose of 2E12 vg / kg. mice at a comparable level.

[0161] We used H&E staining ( Figure 6 ) and CD68 immunostaining ( Figure 7 ), the number of Gaucher cells in liver and lung sections of each group was determined. Treatment with the PG011 construct at doses of 2E12 and 2E13 vg / kg reduced the number of Gaucher cells to levels comparable to those in the liver and lungs of wild-type control mice ( Figure 6 and Figure 7 ).

[0162] In conclusion, PG011 showed significant therapeutic effects in Gaucher disease mice and will become a promising candidate for the treatment of Gaucher disease.

[0163] Example 4: In vitro screening of chimeric liver-specific promoters

[0164] To restrict the expression of GCase to the liver rather than other organs, a series of chimeric hepatic specific regulatory elements (CHSREs) were designed, containing a core promoter and 1, 2, 3 or more regulatory elements. The expression efficiency of GCase in HepG2 cells was compared in vitro or in vivo. mGBA-C110, a codon-optimized version of GBA1 with the K321N mutation, was expressed under the control of various CHSREs. Core promoter candidates were screened from four liver-specific promoters, HSRE002, HSRE005, HSRE004 and HSRE003. Figure 8 A, B, C and D).

[0165] CHSRE consists of 1, 2, 3 or more copies of an enhancer selected from HSRE010, HSRE014, HSRE012, HSRE006, and HSRE009, which are then combined with four promoters, HSRE002, HSRE004, HSRE003 or HSRE005, to drive the expression of the GBA1 gene. The resulting constructs, namely 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 GCase in HepG2 cells. The information of these constructs is shown in Table 3.

[0166] Table 3. Construct information

[0167]

[0168]

[0169] CHSREs with different combinations of liver regulatory elements and promoters showed similar or improved GCase expression compared to the promoter-only version. Constructs with chimeric HSREs, namely PG023-CHSRE002, PG026-CHSRE005, PG034-CHSRE013, PG041-CHSRE020, PG044-CHSRE023, PG048-CHSRE027, and PG051-CHSRE030, showed higher GCase activity compared to the other CHSREs ( Figure 8 A, B, C, D). Further increase in the copy number of HSRE010 (PG028 compared to PG025) or HSRE007 (PG029 compared to PG026) did not affect GCase expression ( Figure 8 A).

[0170] The effect of the position of HSRE012 relative to the HSRE002 promoter on GCase expression was tested, and the results showed that GCase expression was significantly reduced when HSRE012 was moved from the upstream (PG025-CHSRE004 and PG026-CHSRE005) to the downstream (PG053-CHSRE032 and PG054-CHSRE033) of the HSRE002 promoter. Figure 9A). Increasing the copy number of HSRE012 in the chimeric HSRE as shown in PG055-CHSRE034, PG056-CHSRE035, and PG057-CHSRE036 did not affect the expression level of GCase compared to the corresponding constructs with a single copy of HSRE012 (PG025-CHSRE004, PG026-CHSRE005, PG027-CHSRE006) ( Figure 9 B).

[0171] Example 5: In vitro screening of different introns

[0172] To further enhance GCase expression, endogenous introns selected from human GBA1, Int001 (inserted between 27-28 bp of SEQ ID NO: 30), Int002 (inserted between 115-116 bp of SEQ ID NO: 30), Int003 (inserted between 307-308 bp of SEQ ID NO: 30), Int004 (inserted between 454-455 bp of SEQ ID NO: 30), Int005 (inserted between 588-589 bp of SEQ ID NO: 30), Int006 (inserted between 761-762 bp of SEQ ID NO: 30), Int007 (inserted between 999-1000 bp of SEQ ID NO: 30), Int008 (inserted between 1224-1225 bp of SEQ ID NO: 30), Int009 (inserted between 1334-1336 bp of SEQ ID NO: 30), Int010 (inserted between 1335-1337 bp of SEQ ID NO: 30), Int011 (inserted between 1336-1337 bp of SEQ ID NO: 30), Int012 (inserted between 1337-1338 bp of SEQ ID NO: 30), Int013 (inserted between 1338-1339 bp of SEQ ID NO: 30), Int014 (inserted between 1339-1339 bp of SEQ ID NO: 30), Int015 (inserted between 1339-1339 bp of SEQ ID NO: SEQ ID NO: 30) and Int0010 (inserted between bp 1388-1389 of SEQ ID NO: 30) were cloned together with mGBA-C110 under the control of the HSRE002 promoter to generate constructs PG059, PG060, PG061, PG062, PG063, PG064, PG065, PG066, PG067, and PG068. The construct without any introns is PG058. These constructs, as well as PG058 and PG059, were transfected into Huh7 cells and used to study GCase activity in the cell culture supernatant.

[0173] The PG059 construct with the endogenous Int001 inserted demonstrated significantly higher GCase expression compared to the intron-free control ( Figure 10A). Simultaneously, exogenous introns selected from Int012, Int013, Int014, Int015, Int016, Int017, Int018, Int019, and Int011 were cloned together with mGBA-C110 under the control of liver-specific promoters HSRE001, HSRE015, and HSRE016, respectively, to generate construct PG074-101. These constructs with exogenous introns inserted upstream of the GBA1 coding sequence were transfected into HepG2 cells and HEK293T cells to investigate GCase activity in the cell culture supernatant. The results showed that all constructs efficiently expressed GCase in HepG2 cells but not in HEK293T cells ( Figure 10 B and 10C).

[0174] The above results indicate that these expression cassettes constructed using the liver-specific promoter described herein in combination with endogenous or exogenous introns will express GBA1 specifically in liver cells.

[0175] Table 4. Sequences of introns

[0176]

[0177]

[0178]

[0179] Table 5. Construct information

[0180] Construct ID Regulatory element ID Intron ID Intron position description PG058 HSRE002 / / PG059 HSRE002 Int001 Inserted between 27-28 bp of SEQ ID NO: 30 PG060 HSRE002 Int002 Inserted between 115-116 bp of SEQ ID NO: 30 PG061 HSRE002 Int003 Inserted between 307-308 bp of SEQ ID NO: 30 PG062 HSRE002 Int004 Inserted between 454-455 bp of SEQ ID NO: 30 PG063 HSRE002 Int005 Inserted between 588-589 bp of SEQ ID NO: 30 PG064 HSRE002 Int006 Inserted between 761-762 bp of SEQ ID NO: 30 PG065 HSRE002 Int007 Inserted between 999-1000 bp of SEQ ID NO: 30 PG066 HSRE002 Int008 Inserted between 1224-1225 bp of SEQ ID NO: 30 PG067 HSRE002 Int009 Inserted between 1388-1389 bp of SEQ ID NO: 30 PG068 HSRE002 Int010 Inserted between 1505-1506 bp of SEQ ID NO: 30 PG074 HSRE015 / / PG075 HSRE015 Int012 Inserted between the promoter and GBA1 coding sequence PG076 HSRE015 Int013 Inserted between the promoter and GBA1 coding sequence PG077 HSRE015 Int014 Inserted between the promoter and GBA1 coding sequence PG078 HSRE015 Int015 Inserted between the promoter and GBA1 coding sequence PG079 HSRE015 Int016 Inserted between the promoter and GBA1 coding sequence PG080 HSRE015 Int017 Inserted between the promoter and GBA1 coding sequence PG081 HSRE015 Int011 Inserted between the promoter and GBA1 coding sequence PG082 HSRE016 / / PG083 HSRE016 Int012 Inserted between the promoter and GBA1 coding sequence PG084 HSRE016 Int013 Inserted between the promoter and GBA1 coding sequence PG085 HSRE016 Int014 Inserted between the promoter and GBA1 coding sequence PG086 HSRE016 Int015 Inserted between the promoter and GBA1 coding sequence PG087 HSRE016 Int016 Inserted between the promoter and GBA1 coding sequence PG088 HSRE016 Int017 Inserted between the promoter and GBA1 coding sequence PG089 HSRE016 Int018 Inserted between the promoter and GBA1 coding sequence PG090 HSRE016 Int019 Inserted between the promoter and GBA1 coding sequence PG091 HSRE016 Int011 Inserted between the promoter and GBA1 coding sequence PG092 HSRE001 / / PG093 HSRE001 Int012 Inserted between the promoter and GBA1 coding sequence PG094 HSRE001 Int013 Inserted between the promoter and GBA1 coding sequence PG095 HSRE001 Int014 Inserted between the promoter and GBA1 coding sequence PG096 HSRE001 Int015 Inserted between the promoter and GBA1 coding sequence PG097 HSRE001 Int016 Inserted between the promoter and GBA1 coding sequence PG098 HSRE001 Int017 Inserted between the promoter and GBA1 coding sequence PG099 HSRE001 Int018 Inserted between the promoter and GBA1 coding sequence PG100 HSRE001 Int019 Inserted between the promoter and GBA1 coding sequence PG101 HSRE001 Int011 Inserted between the promoter and GBA1 coding sequence

[0181] Table 6. Codon-optimized GBA1 nucleotide sequence with introns

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192] Example 6: In vitro detection of chimeric HSREs with introns

[0193] Based on the above results, constructs containing the endogenous GBA1 intron 1 (Int001, i1) and a 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 composed of CHSRE and mGBA-C110. Constructs containing Int001 inserted between bp 27 and 28 of SEQ ID NO: 30 of GBA1 are PG103, PG104, PG105, PG106, PG168, PG107, and PG108. Constructs containing an exogenous chimeric intron inserted between the chimeric HSRE and the GBA1 coding sequence are PG110, PG111, PG112, PG113, PG169, PG114, and PG115. HepG2 and HEK293T cells were transfected, and the culture supernatants were harvested for measuring GCase activity. The results showed that all of these constructs with a combination of chimeric HSRE with both endogenous intron 1 and exogenous chimeric introns efficiently expressed GCase in HepG2 cells ( Figure 11 A). Compared with the PG011 construct driven by the universal expression control promoter CRE001 to drive mGBA-C110, all these constructs showed very weak GCase activity in HEK293T cells ( Figure 11 B).

[0194] Table 7. Construct information

[0195] Construct ID Regulatory element ID Intron ID Intron position PG102 HSRE002 Int001 Inserted between 27-28 bp of SEQ ID NO: 30 PG103 CHSRE005 Int001 Inserted between 27-28 bp of SEQ ID NO: 30 PG104 CHSRE002 Int001 Inserted between 27-28 bp of SEQ ID NO: 30 PG105 CHSRE030 Int001 Inserted between 27-28 bp of SEQ ID NO: 30 PG106 CHSRE027 Int001 Inserted between 27-28 bp of SEQ ID NO: 30 PG107 CHSRE016 Int001 Inserted between 27-28 bp of SEQ ID NO: 30 PG108 CHSRE013 Int001 Inserted between 27-28 bp of SEQ ID NO: 30 PG109 HSRE002 Int011 Inserted between the promoter and GBA1 coding sequence PG110 CHSRE005 Int011 Inserted between the promoter and GBA1 coding sequence PG111 CHSRE002 Int011 Inserted between the promoter and GBA1 coding sequence PG112 CHSRE030 Int011 Inserted between the promoter and GBA1 coding sequence PG113 CHSRE027 Int011 Inserted between the promoter and GBA1 coding sequence PG114 CHSRE016 Int011 Inserted between the promoter and GBA1 coding sequence PG115 CHSRE013 Int011 Inserted between the promoter and GBA1 coding sequence PG168 HSRE005 Int001 Inserted between 27-28 bp of SEQ ID NO: 30 PG169 HSRE005 Int011 Inserted between the promoter and GBA1 coding sequence

[0196] Example 7: In vivo studies of constructs with chimeric HSREs and introns using AAV8 vectors in wild-type mice

[0197] Based on the above results, constructs PG102, PG026, PG103, PG104, PG037, PG107, PG108, PG051, PG105, and PG106 showed enhanced GCase expression in vitro and were selected for packaging with AAV8 and for further study in wild-type mice. These AAV8 constructs and the reference AAV product PG127 constructed by combining the sequences 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 12 ). A buffer control group and an enzyme replacement therapy (imiglucerase) group were also included in this study. The results showed that all of these AAV8 products effectively increased GCase activity in serum, liver, spleen, and lung. The enzyme activity results from serum were highly consistent with those from different tissue lysates. Most of the AAV8 products PG102, PG026, PG103, PG104, PG037, PG107, PG108, PG051, PG105, and PG106 performed better than the imiglucerase group or at least comparable to the imiglucerase group.

[0198] In conclusion, constructs harboring the HSRE or chimeric HSRE described herein in combination with Int001 packaged with AAV8 delivered GBA1 specifically to the liver and systemically increased GCase activity in serum and other target tissues.

[0199] Example 8: In vivo study of the therapeutic potential of AAV8 candidates for Gaucher disease

[0200] The above constructs PG011, PG103, PG104, PG107, and PG105 all carry ampicillin resistance genes, which were replaced with kanamycin resistance genes and renamed PG117, PG118, PG119, PG120, PG121, and PG122. To study the long-term therapeutic effects of these AAV8 candidates for Gaucher disease gene therapy, constructs PG119, PG120, PG121, and PG122 using CHSRE-driven mGBAi1-C110, construct PG118 using CHSRE-driven GBAi1-C110, and construct PG117 using CRE001-driven mGBA-C110 were packaged into AAV8. These AAV8 candidates, along with the wild-type GBA1 control PG001 and the reference product PG127, were administered to Gaucher disease mice via tail vein injection at a dose of 2E12 vg / kg for a 12-week therapeutic efficacy study. Buffer control and enzyme replacement therapy (imiglucerase) groups. Serum enzyme activity and glucosphingosine accumulation were monitored at different time points after injection (weeks 1, 2, 4, 6, 8, and 12). The results showed that all AAV8 injection groups showed high and stable GCase activity in serum during the study time frame. Compared with the buffer control group, imiglucerase group, and PG127 reference product, the AAV8 candidates PG117, PG118, PG119, PG120, PG121, and PG122 showed higher GCase activity ( Figure 13 A). Serum glucosphingosine levels decreased rapidly after injection of AAV8 candidates PG117, PG118, PG119, PG120, PG121, and PG122; 8 weeks after injection, glucosphingosine levels in these groups decreased to levels comparable to those in the control group. Comparable levels in wild-type mice ( Figure 13 B).

[0201] Tissue samples were collected at the end of the study for enzyme activity and glucosphingosine analysis. GCase enzyme activity was elevated in the liver, lung, and spleen of animals in all AAV-injected groups. Consistent with the results of the serum studies of GCase activity and glucosphingosine accumulation described above, the AAV8 product groups of PG117, PG118, PG119, PG120, PG121, and PG122 showed higher GCase activity and lower glucosphingosine accumulation in all tissues tested compared to the values ​​of the imiglucerase group and the PG127 reference product group ( Figure 14 and Figure 15 ).

[0202] In conclusion, the AAV8 products of PG117, PG118, PG119, PG120, PG121, and PG122 were demonstrated to be ideal candidates for the treatment of Gaucher disease and outperformed existing imiglucerase therapy.

[0203] Table 8. Construct information

[0204]

[0205]

[0206] Example 9: In vivo studies of constructs containing introns using AAV9 vectors in wild-type mice

[0207] In our study, we aimed to enhance the therapeutic potential of AAV9 gene therapy products expressing GCase for the treatment of Gaucher disease, Parkinson's disease, and Alzheimer's disease. We achieved this by inserting endogenous GBA1 introns (Int001, Int002, Int005, or a combination of Int001 and Int005) into the mGBA-C110 expression cassette under the control of the universal promoter CRE001. This resulted in four new constructs: mGBAi1-C110 (PG123), mGBAi2-C110 (PG124), mGBAi5-C110 (PG125), and mGBAi1i5-C110 (PG126).

[0208] These AAV9 products were injected into wild-type mice at a dose of 2E12 vg / kg via tail vein injection along with a reference product of PG128 constructed using the sequence combination of SEQ ID NO: 1 (149 bp-3806 bp) as described in patent US10837028B2. A buffer control group and an enzyme replacement therapy (imiglucerase) group were also included in this study. Compared with the buffer control, PG001 and PG128 reference product groups, the AAV9 products of PG123, PG124, PG125 and PG126 showed higher GCase activity in both serum and tissue lysates. Notably, the GCase activity levels were consistent in the serum and different tissue lysates of all groups ( Figure 16 ).

[0209] These findings demonstrate that our novel AAV9 products have the potential to increase GCase activity in both serum and tissues, indicating their superior therapeutic potential for Gaucher disease, Parkinson's disease, and Alzheimer's disease. AAV9's ability to cross the blood-brain barrier and efficiently deliver the GBA1 gene to the central nervous system (CNS) has the potential to alleviate neurological symptoms and benefit individuals with Gaucher disease types II and III, Parkinson's disease, and Alzheimer's disease.

Claims

1. A polynucleotide encoding human GBA1, wherein the polynucleotide is codon-optimized for expression in humans and comprises the coding sequence of the human GBA1 gene, wherein the coding sequence is as shown in SEQ ID NO:

11.

2. The polynucleotide according to claim 1, further comprising a nucleotide sequence encoding a signal peptide, wherein the polynucleotide sequence is shown in SEQ ID NO:

30. The polynucleotide according to claim 1 , further comprising an untranslated intron region.

4. The polynucleotide according to claim 3, wherein the untranslated intron region is selected from at least one sequence in the group consisting of SEQ ID NOs: 41-59 and 94.

5. The polynucleotide according to claim 3, wherein the untranslated intron region comprises the first sequence shown in SEQ ID NO:

41.

6. The polynucleotide according to claim 3, wherein the untranslated intron region comprises a first sequence as shown in SEQ ID NO: 41 and a second sequence as shown in SEQ ID NO:

45. 7 . The polynucleotide according to claim 3 , wherein the untranslated intron region is operably linked to the 5′ end of the coding sequence of the human GBA1 gene.

8. The polynucleotide of claim 3, wherein the untranslated intron region is located between 27-28 bp, 115-116 bp, 307-308 bp, 454-455 bp, 588-589 bp, 761-762 bp, 999-1000 bp, 1224-1225 bp, 1388-1389 bp, or 1505-1506 bp of SEQ ID NO:

30. 9 . The polynucleotide according to claim 4 , wherein the polynucleotide encoding human GBA1 is represented by the nucleotide sequence of any one of SEQ ID NOs: 60-71.

10. An expression construct comprising the polynucleotide of any one of claims 1 to 9 and a transcriptional regulatory element operably linked to the polynucleotide, wherein the transcriptional regulatory element comprises a promoter and / or an enhancer. The expression construct according to claim 10 , wherein the enhancer is upstream of the promoter.

12. The expression construct according to claim 10 or 11, wherein the promoter is selected from the group consisting of SEQ ID NOs: 77-82 and 92-93.

13. The expression construct according to claim 10 or 11, wherein the enhancer is selected from the sequence of at least one of the group consisting of SEQ ID NOs: 83-91.

14. The expression construct of claim 13, wherein the enhancer is a single copy or multi-copy sequence.

15. The expression construct according to claim 10 or 11, wherein the transcriptional regulatory element comprises, in 5' to 3' direction: (a) enhancer 3, which is optional; (b) enhancer 2; (c) enhancer 1; and (d) a promoter; wherein The promoter is selected from the group consisting of SEQ ID NOs: 78-82; The enhancer 1 is selected from at least one sequence in the group consisting of SEQ ID NOs: 89 and 91, The enhancer 2 is selected from at least one sequence in the group consisting of SEQ ID NOs: 84-87 and 89, and The enhancer 3 is selected from at least one sequence in the group consisting of SEQ ID NOs: 84, 85, 86 and 87.

16. A vector comprising the polynucleotide according to any one of claims 1 to 9 or the expression construct according to any one of claims 10 to 15.

17. The vector according to claim 16, wherein the vector is a viral vector.

18. The vector of claim 16, wherein the vector is an AAV vector.

19. The vector of claim 16, wherein the vector further comprises two adeno-associated virus inverted terminal repeat (ITR) sequences flanking the expression construct.

20. The vector of claim 16, wherein the vector further comprises a poly A sequence.

21. An adeno-associated virus (AAV) comprising the vector according to any one of claims 16 to 20 and a capsid protein.

22. The AAV of claim 21, 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, AAVhu37, or any AAV serotype isolated from a human or non-human mammal, or a variant thereof.

23. A composition comprising: The polynucleotide of any one of claims 1-9, the expression construct of any one of claims 10-15, the vector of any one of claims 16-20, or the AAV of any one of claims 21-22, further comprising a pharmaceutically acceptable excipient.

24. Use of the polynucleotide of any one of claims 1-9, the expression construct of any one of claims 10-15, the vector of any one of claims 16-20, the AAV of any one of claims 21-22, or the composition of claim 23 in the preparation of a medicament for treating Gaucher disease or Parkinson's disease in a subject.

25. The use according to claim 24, wherein Treatment comprises administering to the subject an effective amount of a polynucleotide according to any one of claims 1-9, an expression construct according to any one of claims 10-15, a vector according to any one of claims 16-20, an AAV according to any one of claims 21-22, or a composition according to claim 23.

26. The use according to claim 24, wherein the Gaucher disease is type I, II or III Gaucher disease or neuropathic Gaucher disease.

Images