Modified α-galactosidase A (α-GAL A) peptide and its functional variant, and related methods for treating Fabry disease

Abstract

Described herein are novel modified peptides, viral vectors, and their pharmaceutically acceptable compositions, as well as their use for increasing α-GAL A expression and for the treatment of α-GAL A deficiency-related conditions such as Fabry disease.

Description

Technical Field

[0001] Priority Claim

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 499,243, filed Apr. 30, 2023, and U.S. Provisional Application No. 63 / 515,558, filed Jul. 25, 2023, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing

[0002] This application includes a sequence listing compliant with ST.26, which is submitted simultaneously in xml format, the entire contents of which are incorporated herein by reference. A copy of the xml was created on Apr. 26, 2024, named 149731 - 8001WO00 Sequence Listing.xml, and is 62,000 bytes in size.

Background Art

[0003]

[0003] Fabry disease is an X - linked lysosomal storage disorder characterized by a deficiency of α - galactosidase A (α - GAL A). This disease can lead to a progressive and systemic accumulation of sphingolipids, such as globotriaosylceramide (GB3) and globotriaosylsphingosine (LYSO - GB3), in lysosomes of vascular endothelial cells, renal epithelial cells and endothelial cells, cardiomyocytes, etc. The deposition of sphingolipids can cause skin, heart, kidney, cerebrovascular, and other diseases, and may ultimately lead to premature death.

[0004]

[0004] Treatment options for Fabry disease are limited, including enzyme replacement therapy (ERT) that delivers recombinant human α-GAL A intravenously (J Med Gene 2015 May;52(5):353-8). While ERT is particularly effective in the early stages of the disease, the relatively short half-life of α-GAL A often necessitates regular infusions for patients (N Engl J Med. 2001 Jul 5;345(1):9-16), which can be a lifelong burden. Recently, the oral pharmacological chaperone Migalastat® has been approved as a treatment for Fabry disease, but it is only effective for specific α-GAL A gene mutations. Furthermore, three recombinant adeno-associated virus (rAAV) drugs using different rAAV serotypes, unique promoters, and other mechanisms are in clinical trials to promote α-GAL A expression (NCT04046224 / NCT05039886, NCT04519749, and NCT04040049). However, rAAV-mediated α-GAL A expression remains limited, as does the clearance of disease substrates Gb3 and lyso-Gb3.

[0005]

[0005] Despite these treatments, there is a need for novel, safe, and effective therapies for Fabry disease that have a sustained effect. [Overview of the project]

[0006]

[0006] The object of this technology is to provide novel compositions for increasing α-GAL A expression, and related methods for treating Fabry disease and / or symptoms of Fabry disease using such novel compositions.

[0007]

[0007] Unless otherwise specified, α-GAL A refers to the mature amino acid sequence after the wild-type signal peptide (wt sp) of α-GAL A has been cleaved, or its functional variant. Precursor or full-length α-GAL A refers to α-GAL A fused to one or more signal peptides, where the signal peptide is wt sp or heterologous sp (e.g., sp1, sp2, etc., as listed in Table 2). ..., sp22, etc. hGLA refers to a nucleotide sequence encoding α-GAL A, e.g., wild-type human α-galactosidase A (wt hGLA) and an optimized / modified nucleotide sequence encoding α-GAL A or a functional variant thereof, e.g., hGLA co v1 disclosed herein. Lowercase sp refers to a signal peptide or a functional variant thereof, and uppercase SP refers to a nucleotide sequence encoding a signal peptide or a functional variant thereof. The signal peptide referred to by sp may be wt sp or one of heterologous sp, and the nucleotide sequence SP encoding the signal peptide may be a nucleotide sequence SP encoding one of wt sp or one of heterologous sp.

[0008]

[0008] In some embodiments, the composition comprises a modified peptide comprising α-GAL A or a functional variant thereof, a signal peptide of tissue plasminogen activator (sp21) or a functional variant thereof, and optionally a cell-permeable peptide or a functional variant thereof. In some embodiments, the composition comprises a viral vector comprising an expression cassette comprising a first nucleotide sequence encoding α-GAL A or a functional variant thereof, a second nucleotide sequence encoding sp21 or a functional variant thereof, optionally a third nucleotide sequence encoding a cell-permeable peptide or a functional variant thereof, and a promoter.

[0009]

[0009] In some embodiments, the composition comprises a modified peptide comprising α-GAL A or a functional variant thereof, a wt signal peptide (wt sp) of α-GAL A or a functional variant thereof, and optionally a cell-permeable peptide or a functional variant thereof. In some embodiments, the composition comprises a viral vector comprising an expression cassette comprising a first nucleotide sequence encoding α-GAL A or a functional variant thereof, a fourth nucleotide sequence encoding wt sp or a functional variant thereof, optionally a third nucleotide sequence encoding a cell-permeable peptide or a functional variant thereof, and a promoter.

[0010]

[0010] Another object of this technology is to provide a method for treating Fabry disease or alleviating one or more symptoms of Fabry disease in a subject by administering the composition to that subject.

[0011]

[0011] Another objective of this technology is to provide a method for increasing the expression of α-GAL A in one or more target tissues by administering this composition to the subject.

[0012]

[0012] Another object of this technology is to provide a method for reducing the accumulation of sphingolipids in one or more target tissues of a subject by administering the composition to the subject.

[0013]

[0013] These and other objectives will become apparent in the following detailed description and accompanying drawings, but are achieved by the present technology comprising modified peptides comprising or consisting of a portion of α-GAL A or a functional variant thereof and at least a portion of a heterologous signal peptide, as well as a viral vector encoding it, which yield unexpectedly beneficial therapeutic activity through increased expression, secretion, localization, and activity for treating, mitigating, or otherwise improving Fabry disease.

[0014]

[0014] In some embodiments, the modified peptide comprises α-GAL A or a functional variant thereof and a heterologous signal peptide (e.g., sp21) or a functional variant thereof. Other examples of heterologous signal peptides include, but are not limited to, sp1-sp20 and sp22. In certain embodiments, the heterologous signal peptide is selected from the group consisting of sp1, sp3, sp4, sp18, sp20, and sp22, as disclosed herein. α-GAL A or a functional variant thereof may or may not include the wild-type (wt) signal peptide of α-GAL A.

[0015]

[0015] In some embodiments, the modified peptide further comprises a cell-permeable peptide or a functional variant thereof. Examples of cell-permeable peptides, but are not limited to, include TAT p47-57, TAT p48-60, penetratin p43-58, hCT p9-32, polyarginine (R7-R25), pVEC, Pep-1, Transportan, and MAP.

[0016]

[0016] In some embodiments, α-GAL A or a functional variant thereof comprises a peptide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 27.

[0017]

[0017] In some embodiments, sp1, sp3, sp4, sp18, sp20, sp21 and sp22 or their functional variants contain peptide sequences having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NOs. 29, 31, 32, 46, 48, 49, and 50, respectively.

[0018]

[0018] In some embodiments, the signal peptide or its functional variant comprises a peptide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NOs: 29, 31, 32, 46, 48, 49, and 50.

[0019]

[0019] In some embodiments, the cell-permeable peptide or its functional variant includes a peptide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with a peptide sequence selected from the group consisting of TAT p47-57 (SEQ ID NO: 55), TAT p48-60 (SEQ ID NO: 56), penetratin p43-58 (SEQ ID NO: 57), hCT p9-32 (SEQ ID NO: 58), polyarginine (SEQ ID NO: 59), pVEC (SEQ ID NO: 60), Pep-1 (SEQ ID NO: 61), transportan (SEQ ID NO: 62), and MAP (SEQ ID NO: 63).

[0020]

[0020] In some embodiments, a heterologous signal peptide (e.g., sp21) or a functional variant thereof is fused to the N-terminus of a portion of α-GAL A or its functional variant. In some embodiments, a portion of a heterologous signal peptide (e.g., sp21) or a functional variant thereof is fused to the C-terminus of a portion of α-GAL A or its functional variant. In some embodiments, a portion of a heterologous signal peptide (e.g., sp21) or a functional variant thereof is fused within a portion of α-GAL A or its functional variant.

[0021]

[0021] In some embodiments, the cell-permeable peptide or its functional variant is fused to a portion of α-GAL A or the C-terminus of its functional variant. In some embodiments, the cell-permeable peptide or its functional variant is fused to a portion of α-GAL A or the N-terminus of its functional variant. In some embodiments, the cell-permeable peptide or its functional variant is fused within a portion of α-GAL A or its functional variant.

[0022]

[0022] In some embodiments, the technology further comprises a viral vector having a nucleotide sequence encoding a modified peptide.

[0023]

[0023] In some embodiments, the viral vector further includes a promoter.

[0024]

[0024] In some embodiments, the composition of the present technology comprises a viral vector comprising an expression cassette comprising or consisting of a first nucleotide sequence encoding a part of α-GAL A or a functional variant thereof and a second nucleotide sequence encoding a part of a heterologous signal peptide (e.g., sp21) or a functional variant thereof. In some embodiments, the composition of the present technology comprises a viral vector comprising an expression cassette comprising or consisting of a first nucleotide sequence encoding a part of α-GAL A or a functional variant thereof, a fourth nucleotide sequence encoding a part of wt sp or a functional variant thereof, optionally a third nucleotide sequence encoding a part of a cell-penetrating peptide or a functional variant thereof, and a promoter.

[0025]

[0025] In some aspects, the second or fourth nucleotide sequence is 5' terminal relative to the first nucleotide, and / or the third nucleotide sequence is 3' terminal relative to the first nucleotide sequence.

[0026]

[0026] Another aspect described herein is a viral vector in which a part of α-GAL A or a functional variant thereof has a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to SEQ ID NO: 25 or 26.

[0027]

[0027] In some aspects, the nucleotide sequence encoding a part of sp21 or a functional variant thereof has a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to SEQ ID NO: 22.

[0028]

[0028] In some embodiments, the nucleotide sequence encoding a part of wt sp or its functional variant has a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to SEQ ID NO: 1.

[0029]

[0029] In some embodiments, the nucleotide sequence encoding a part of the heterologous signal peptide or its functional variant has a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 64, 65 and 66.

[0030]

[0030] In some embodiments, the nucleotide sequence encoding a part of the heterologous signal peptide or its functional variant has a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 19, 21, 22 and 23.

[0031]

[0031] In some embodiments, the nucleotide sequence encoding a part of the cell-penetrating peptide or its functional variant has a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to SEQ ID NO: 24.

[0032]

[0032] In some embodiments, the nucleotide sequence encoding a part of the cell-penetrating peptide or its functional variant has a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to SEQ ID NOs: 24, 52, 53 and 54.

[0033]

[0033] In some embodiments, the promoter is a tissue-specific promoter.

[0034]

[0034] In some embodiments, the tissue-specific promoter is a liver-specific promoter, a heart-specific promoter, or a kidney-specific promoter.

[0035]

[0035] In some embodiments, the liver-specific promoter is the TTR promoter.

[0036]

[0036] In some embodiments, the viral vector further comprises at least one reverse terminal repeat (ITR) nucleotide sequence, an intron, and a polyadenylated (poly-A) nucleotide sequence.

[0037]

[0037] In some embodiments, the viral vector is a modified recombinant adeno-associated virus (rAAV) vector.

[0038]

[0038] In some embodiments, the technology further includes a method for treating Fabry disease in a subject or alleviating one or more symptoms of Fabry disease by administering the composition to the subject.

[0039]

[0039] In some embodiments, the technology further includes a method of increasing the expression of α-GAL A in one or more target tissues of a subject by administering the composition to the subject.

[0040]

[0040] In some embodiments, the technology further includes a method for reducing sphingolipid accumulation in one or more target tissues of a subject by administering the composition to the subject.

[0041]

[0041] In some embodiments, the subject is suffering from Fabry disease or has symptoms of Fabry disease.

[0042]

[0042] In some embodiments, one or more target tissues are selected from the group consisting of liver tissue, heart tissue, or kidney tissue.

[0043]

[0043] In some embodiments, the method further comprises administering a composition of the present technology to a subject.

[0044]

[0044] In some embodiments, the composition of the technology is administered to the subject once.

[0045]

[0045] In some embodiments, the compositions of the present technology are administered to a subject by systemic administration.

[0046]

[0046] In some embodiments, the compositions of the present technology are administered to a subject by topical administration.

[0047]

[0047] In some embodiments, the composition of the technology is administered to a subject by tissue-specific administration.

[0048]

[0048] In some embodiments, the viral vector of a composition containing the viral vector of the present technology is administered to a subject in one or more doses ranging from 1E+11vg / kg to 5E+14vg / kg. [Brief explanation of the drawing]

[0049] [Figure 1A-1B]

[0049] Embodiments of the engineered recombinant adeno-associated virus (rAAV) vector of the present technology are shown. The rAAV vector comprises an expression construct having a human α-galactosidase A (hGLA) nucleotide sequence and a nucleotide sequence encoding a signal peptide (sp). The hGLA nucleotide sequence may be an embodiment of a wild-type hGLA (hGLA wt) or an optimized nucleotide sequence encoding α-GAL A (hGLAc.o.v1). The signal peptide nucleotide sequence may be a wild-type nucleotide sequence encoding wild-type sp of α-GAL A (wt SP), an optimized nucleotide sequence encoding wild-type signal peptide of α-GAL A (wtSP co), or a nucleotide sequence encoding a heterologous sp, for example, sp1 to sp22 (e.g., SP1 to SP22, respectively) as disclosed herein. Embodiments of rAAV vectors containing a nucleotide sequence encoding wt sp (wtSP, wtSP co-1, wtSP co-2, wtSP co-3, or wtSP co) or a nucleotide sequence encoding sp21 (SP21) are shown in Figures 1A and 1B. 1A shows two embodiments of a single-stranded rAAV vector (ss-rAAV): ss-wtspGLA contains wt SP and hGLA wt, and ss-sp21GLA contains SP21 and hGLA wt. 1B shows multiple embodiments of a self-complementary vector (sc-rAAV). FD802-1 contains wt SP and hGLA wt, FD802-2 contains SP21 and hGLA wt, FD802-3 contains wtSP co and hGLA co v1, FD802-4 contains SP21 and hGLA co v1, and FD802-5 contains wtSP co, hGLA co v1 and a nucleotide sequence (tat) which is a wild-type nucleotide sequence encoding TAT (e.g., TAT sequence (p47-57), TAT sequence (p48-60)). [Figure 2A-2B]

[0050] This shows that α-GAL A expression (Figure 2A) and activity levels (Figure 2B) in the culture medium removed from the HepG2 cell culture vehicle of HepG2 cells transfected with plasmids expressing α-GAL A+ heterologous sp(sp1, sp2, ..., and sp22) are increased compared to cells transfected with plasmids expressing α-GAL A+ wt sp. [Figure 3A-3B]

[0051] Figure 3A shows the genomic distribution of the ss-rAAV vector (Figure 3B) and hGLA mRNA expression (Figure 3B) in the livers of Fabry mice injected with low-dose (2E+12vg / kg), medium-dose (5E+12vg / kg), or high-dose (5E+13vg / kg) embodiments of ss-rAAV expressing α-GAL A+sp21 (ss-sp21GLA) or α-GAL A+wt sp (ss-wtspGLA), or untreated Fabry mice (0vg / kg dose). [Figure 4]

[0052] This study shows that α-GAL A activity in the plasma of Fabry mice injected with low-dose (2E+12vg / kg), medium-dose (5E+12vg / kg), or high-dose (5E+13vg / kg) embodiments of ss-rAAV expressing α-GAL A+sp21 (ss-sp21GLA) or embodiments of ss-rAAV expressing α-GAL A+wt sp (ss-wtspGLA) was increased compared to untreated Fabry mice and untreated wild-type mice. [Figures 5A-5D]

[0053] This study shows that α-GAL A activity in the liver (Figure 5A), heart (Figure 5B), kidney (Figure 5C), and spleen (Figure 5D) of Fabry mice injected with low-dose (2E+12vg / kg), medium-dose (5E+12vg / kg), or high-dose (5E+13vg / kg) embodiments of ss-rAAV expressing α-GAL A+sp21 (ss-sp21GLA) was increased compared to Fabry mice injected with low-dose (2E+12vg / kg), medium-dose (5E+12vg / kg), or high-dose (5E+13vg / kg) embodiments of ss-rAAV expressing α-GAL A+wt sp (ss-wtspGLA), untreated Fabry mice (untreated), and untreated wild-type mice (wild-type). [Figure 6A-6B]

[0054] Figure 6A shows that α-GAL A expression in the livers of Fabry mice 12 weeks after injection with low-dose (2E+12vg / kg), medium-dose (5E+12vg / kg), and high-dose (5E+13vg / kg) embodiments of ss-rAAV expressing α-GAL A+sp21 (ss-sp21GLA) was increased compared to mice injected with embodiments of ss-rAAV expressing α-GAL A+wt sp (ss-wtspGLA) (Figure 6A). Furthermore, the glycosylation level of α-GAL A under high-dose (5E+13vg / kg) α-GAL A+sp21-expressing embodiments of ss-rAAV (ss-sp21GLA) was higher than that of mice injected with high-dose (5E+13vg / kg) α-GAL A+wt sp. Figure 6B shows an increase compared to mice injected with an embodiment of ss-rAAV expressing sp (ss-wtspGLA). Recombinant α-GAL A was used as a positive control for the in vitro glycosylation test (Rec.hAGA). [Figure 7]

[0055] This study shows that α-GAL A expression in the liver, heart, and kidneys of Fabry mice 12 weeks after injection with a high-dose (5E+13vg / kg) embodiment of ss-rAAV expressing α-GAL A+sp21 (ss-sp21GLA) was increased compared to Fabry mice injected with a high-dose (5E+13vg / kg) embodiment of ss-rAAV expressing α-GAL A+wt sp (ss-wtspGLA) or untreated Fabry mice. [Figures 8A-8B]

[0056] This study shows that plasma lyso-Gb3 levels in Fabry mice injected with low-dose (2E+12vg / kg), medium-dose (5E+12vg / kg), or high-dose (5E+13vg / kg) embodiments of ss-rAAV expressing α-GAL A+sp21 (ss-sp21GLA) were reduced at both 12 weeks (Figure 8A) and 26 weeks (Figure 8B) post-injection compared to Fabry mice injected with low-dose (2E+12vg / kg), medium-dose (5E+12vg / kg), or high-dose (5E+13vg / kg) embodiments of ss-rAAV expressing α-GAL A+wt sp (ss-wtspGLA) and untreated Fabry mice. [Figure 9]

[0057] The body weight records of Fabry mice after injection of either an embodiment of ss-rAAV expressing α-GAL A+sp21 (ss-sp21GLA) or an embodiment of ss-rAAV expressing α-GAL A+wt sp (ss-wtspGLA) in addition to untreated Fabry mice (untreated) and untreated wild-type mice (wild-type), are shown. [Figure 10]

[0058] The images show liver tissue from Fabry mice, untreated Fabry mice, and wild-type mice without Fabry disease, after injection of either an embodiment of ss-rAAV expressing α-GAL A+sp21 (ss-sp21GLA) or an embodiment of ss-rAAV expressing α-GAL A+wt sp (ss-wtspGLA) at a high dose (5E+13vg / kg). No significant histopathological changes were observed in the liver after high-dose administration of ss-wtspGLA and ss-sp21GLA, indicating that this therapeutic administration has a certain degree of safety. [Figure 11]

[0059] This study shows that α-GAL A expression in HepG2 protein lysates after transfection with a plasmid expressing α-GAL A encoded by the functional variant hGLA nucleotide sequence (hGLA co v1, SEQ ID NO: 26) is increased compared to cells transfected with a plasmid expressing α-GAL A encoded by the wt hGLA nucleotide sequence (hGLA wt, SEQ ID NO: 25) and untransfected cells as a negative control (NC). GAPDH serves as a loading control in Western blotting. [Figure 12]

[0060] The α-GAL A activity in the plasma of Fabry mice after injection (FD802-3 and FD802-4) of an embodiment of the sc-rAAV vector expressing α-GAL A encoded by the functional variant hGLA nucleotide sequence hGLA cov1 at a low dose (2E+12vg / kg) or medium dose (5E+12vg / kg) was compared to the α-GAL A activity in the plasma of Fabry mice after injection (FD802-3 and FD802-4) of an embodiment of the sc-rAAV vector expressing α-GAL A encoded by the wt hGLA nucleotide sequence at a low dose (2E+12vg / kg) or medium dose (5E+12vg / kg), or the α-GAL A activity in the plasma of Fabry mice after injection (FD802-1 and FD802-2), or after injection of an embodiment of the sc-rAAV vector expressing α-GAL A encoded by the wt hGLA nucleotide sequence at a low dose (2E+12vg / kg) or medium dose (5E+12vg / kg). This shows an increase in Fabry mice injected with an embodiment of ss-rAAV expressing A+sp21(ss-sp21GLA) compared to untreated Fabry mice (untreated). [Figure 13]

[0061] This study shows that α-GAL A activity in the liver, heart, kidneys, and spleen of Fabry mice injected with moderate doses (5E+12vg / kg) of FD802-1, FD802-2, FD802-3, and FD802-4 was increased compared to Fabry mice injected with moderate doses (5E+12vg / kg) of ss-rAAV (ss-sp21GLA) expressing α-GAL A+sp21 and untreated Fabry mice (untreated). [Figure 14]

[0062] This study shows that the detection of α-GAL A in liver samples from Fabry mice injected with moderate doses (5E+12vg / kg) of FD802-1, FD802-2, FD802-3, and FD802-4 was increased compared to Fabry mice injected with moderate doses (5E+12vg / kg) of ss-rAAV (ss-sp21GLA) expressing α-GAL A+sp21, and untreated Fabry mice (untreated). [Figure 15]

[0063] This study shows that the detection of α-GAL A in the livers of Fabry mice injected with moderate doses (5E+12vg / kg) of FD802-1, FD802-2, FD802-3, and FD802-4 was increased compared to Fabry mice injected with moderate doses (5E+12vg / kg) of ss-rAAV expressing α-GAL A+sp21 (ss-sp21GLA) and untreated Fabry mice. [Figure 16]

[0064] This study shows that the levels of glycosylated α-GAL A in the liver of Fabry mice injected with moderate doses (5E+12vg / kg) of FD802-3 and FD802-4 were increased compared to Fabry mice injected with moderate doses (5E+12vg / kg) of FD802-1 or FD802-2. Recombinant α-GAL A was used as a positive control for glycosylation in vitro (Rec.hAGA). [Figure 17]

[0065] This study shows that lyso-Gb3 in the plasma of Fabry mice injected with moderate doses (5E+12vg / kg) of FD802-1, FD802-2, FD802-3, and FD802-4 was decreased at both 6 weeks (Figure 17A) and 14 weeks (Figure 17B) after injection, compared to Fabry mice injected with moderate doses (5E+12vg / kg) of ss-rAAV (ss-sp21GLA) expressing α-GAL A+sp21 and untreated Fabry mice. [Figure 18]

[0066] The histopathological staining of the livers of Fabry mice injected with medium doses (5E+12vg / kg) of FD802-1, FD802-2, FD802-3, and FD802-4 is shown, compared to Fabry mice injected with medium doses (5E+12vg / kg) of ss-rAAV expressing α-GAL A+sp21 (ss-sp21GLA) and untreated Fabry mice (untreated). No significant histopathological changes were observed in the liver after high-dose administration, indicating that this therapeutic administration has a certain degree of safety. [Figures 19A-19B]

[0067] Figure 19A shows the correlation between vector serotypes (rAAV1, rAAv2, ..., rAAV10) and α-GAL A expression in HepG2 cells (lysate) and secretion from HepG2 cells (culture medium) after transduction of various serotypes of sc-rAAV using the FD802-4 genome containing the functional variant hGLA nucleotide sequence (hGLA co v1, SEQ ID NO.) and SP21 (SEQ ID NO. 22). Figure 19B shows the activity analysis of secreted α-GAL A. NC: Negative control, protein sample from HepG2 cells without AAV transduction. PC: Positive control, protein sample containing α-GAL A. [Figures 20A-20B]

[0068] This figure shows the inclusion of the nucleotide sequence encoding TAT p47-57 in α-GAL A expression plasmids and its effect on α-GAL A activity in cell culture. Figure 20A shows that FD802-5 (Ctat-GLAco) produced lower levels of α-GAL A activity in culture medium compared to FD802-3 (GLAco). Figure 20B shows that the α-GAL A proteins expressed from both plasmids had equivalent enzymatic activity. [Figures 21A-21B]

[0069] Compared to untreated mice, Fabry mice injected with low-dose (2E+12vg / kg) or medium-dose (5E+12vg / kg) FD802-5 showed increased plasma α-GAL A activity, but there was no change in activity compared to equivalent doses of FD802-3 (Figure 21A). Activity levels were also shown in the liver, heart, kidney, and spleen tissues of Fabry mice injected with medium-dose (5E+12vg / kg) FD802-5 compared to both mice injected with medium-dose (5E+12vg / kg) FD802-3 and untreated mice (untreated) (Figure 21B). [Figure 22]

[0070] This study shows that α-GAL A detection in the liver, heart, and kidneys of Fabry mice injected with a moderate dose (5E+12vg / kg) of FD802-5 was increased compared to mice injected with an equivalent dose of FD802-3 and untreated mice. [Figures 23A-23B]

[0071] Figure 23A and Figure 23B show α-GAL A expression (Figure 23A) and glycosylation (Figure 23B) in the livers of Fabry mice 14 weeks after injection of medium doses (5E+12vg / kg) of FD802-3 and FD802-5. Recombinant α-GAL A was used as a positive control in the in vitro glycosylation test (Rec.hAGA). [Figures 24A-24B]

[0072] At 6 weeks (FIGURE 24A) and 14 weeks (FIGURE 24B) after administration of low-dose (2E+12vg / kg) or medium-dose (5E+12vg / kg) FD802-3 and FD802-5, Fabry mice showed a decrease in plasma lyso-Gb3 compared to untreated mice, demonstrating that FD802-5 has a dose-dependent effect. [Figure 25]

[0073] This study demonstrates that there are no visible adverse liver effects after injection of a moderate dose of FD802-5 compared to untreated Fabry mice and Fabry mice treated with a moderate dose of FD802-3. [Modes for carrying out the invention]

[0050]

[0074] This specification provides compositions useful for treating Fabry disease and / or alleviating the symptoms of Fabry disease.

[0051]

[0075] Regular infusions of recombinant α-GAL A, known as enzyme replacement therapy (ERT), are currently the primary treatment option for Fabry disease patients with non-adaptive mutations, although patients with adaptive mutations can benefit from both ERT and small molecule chaperone therapy. However, the efficacy of ERT may be limited due to the low physical stability, short circulating half-life, and inconsistent uptake by disease-related tissues of recombinant α-GAL A. The compositions provided herein deliver nucleic acid sequences encoding recombinant α-GAL A that are effective for gene therapy and enable longer-lasting circulating enzyme activity before α-GAL A is taken up by target tissues.

[0052]

[0076] In certain embodiments, the compositions and methods described herein include nucleic acid sequences, expression cassettes, vectors, recombinant viruses, peptide sequences, modified peptides, and other compositions and methods for the expression of functional α-GAL A. In certain embodiments, the compositions and methods described herein include nucleic acid sequences, expression cassettes, vectors, recombinant viruses, peptide sequences, modified peptides, host cells, other compositions, and methods for the production of a composition comprising any of the nucleic acid sequences encoding functional α-GAL A. In yet another embodiment, the compositions and methods described herein include nucleic acid sequences, expression cassettes, vectors, recombinant viruses, peptide sequences, modified peptides, and other compositions and methods for the delivery of nucleic acid sequences encoding functional α-GAL A to a subject for the treatment of Fabry disease. In one embodiment, the compositions and methods described herein are useful for providing therapeutic levels of functional α-GAL A in peripheral tissues such as the blood, liver, kidneys, and / or peripheral nervous system of a subject. In certain embodiments, the adeno-associated virus (AAV) vector-based methods described herein provide a novel therapeutic option that helps restore the desired function of α-GAL A and alleviate symptoms associated with α-GAL A deficiency and / or dysfunction (Fabry disease) by providing functional α-GAL A expression in subjects requiring it.

[0053]

[0077] As shown in the Examples section, several heterologous signal peptides (sp1-sp22) were screened in vitro for equivalent or superior extracellular secretion of α-GAL A, and sp1, 3, 4, 18, and 20-22 showed improved extracellular secretion of α-GAL A compared to wt sp (Example 1, Figures 2A and 2B). Human-derived sp21 was selected as an example of heterologous sp, and rAAV vectors consisting of nucleotide sequences encoding α-GAL A fused with the heterologous sp (Figures 1A and 1B, vector ss-sp21GLA, FD802-2, and FD802-4) were constructed and compared with rAAV vectors expressing α-GAL A fused with wt sp (Figures 1A and 1B, vector ss-wtspGLA, FD802-1, and FD802-3). Fabry mice were treated with rAAV vectors, and α-GAL A expression and activity, as well as lyso-Gb3 clearance, were compared in plasma, various organs, and / or tissues. The data showed that heterologous sps, such as sp21, were superior to wt sps in α-GAL A expression and secretion, and lyso-Gb3 clearance, without disrupting tissue structure, in various cases. In Examples 1-3, rAAV vectors containing nucleotide sequences encoding wt hGLA and sp (wt sp or sp21) were tested in Fabry mice. In the sp21 group, α-GAL A expression and activity were improved in plasma, liver, heart, kidney, and spleen, lyso-Gb3 clearance in plasma was improved, and weight gain was suppressed. In Examples 4-5, rAAV vectors containing optimized hGLA nucleotide sequences (hGLA cov1) and sp nucleotide sequences (wt SP, wtSP co, or SP21) were tested in Fabry mice. The hGLA cov1 group showed improved α-GAL A expression and activity in plasma (Figure 12), liver, heart, kidney, and spleen (Figure 13), and improved plasma lyso-Gb3 clearance (Figures 17A and 17B) compared to the corresponding wt hGLA group. Example 6 demonstrates that α-GAL A expression using the FD802-4 vector containing hGLA cov1 and SP21 was successful in various rAAV serotypes other than rAAV8 (Figures 19A and 19B).In Examples 7 and 8, rAAV vectors were tested with and without the presence of nucleotide sequences encoding hGLA cov1 and the wt SP nucleotide functional variant (wtSP co) and TAT p47-57 (tat). When administered to Fabry mice at 5E+12vg / kg, the rAAV vector containing hGLA cov1 and wtSP co and tat (FD802-5) showed higher α-GAL A activity in the heart and spleen, and lower α-GAL A activity in the liver, kidney (Figure 21B), and plasma (Figure 21A) compared with the rAAV vector containing hGLA cov1 and wtSP co but without tat (FD802-3). Improvements in α-GAL A protein levels were observed in the hearts of Fabry mice injected with FD802-5 compared with Fabry mice injected with FD802-3 (Figure 22). The fusion of TAT p47-57 and α-GAL A did not appear to alter the expression or glycosylation of α-GAL A in the liver of Fabry mice (Figure 23A-B). A vector expressing α-GAL A together with TAT p47-57 (FD802-5) also showed an effective reduction in lyso-Gb3 (Figure 24A-B), and no significant adverse effects on the liver tissue of Fabry mice were observed (Figure 25).

[0054] definition

[0078] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in which this art pertains. For the purposes of this art, the following terms are defined as follows:

[0055]

[0079] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, "an element" means one or more elements. Similarly, a singular reference may include multiple embodiments, and a reference to multiple components may include singular embodiments.

[0056]

[0080] The term "approximately" means a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that varies to a level acceptable in the art. In some embodiments, such variation may be approximately 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length. When the term "approximately" is used with a numerical range, the range is modified by extending the boundary above and below the numerical value.

[0057]

[0081] As used herein, the terms “therapeutic level” or “therapeutically effective” of functional α-GAL A mean at least about 5%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, more than 100%, about 2x, about 3x, or about 5x of the enzyme activity of a healthy control. Appropriate assays for measuring α-GAL A enzyme activity are known to those skilled in the art. In some embodiments, such therapeutic levels of α-GAL A may result in the alleviation of Fabry disease-related symptoms, improvement of Fabry disease-related biomarkers (e.g., a decrease in Gb3 levels in serum, urine, and / or other biological samples), enhancement of other treatments for Fabry disease (e.g., enzyme replacement therapy or chaperone therapy), prevention of neurocognitive decline, reversal of certain Fabry disease-related symptoms and / or prevention of progression of Fabry disease-related symptoms, or any combination thereof.

[0058]

[0082] As used herein, “healthy control” means a subject or a biological sample thereof that does not have Fabry disease or α-GAL A deficiency and / or functional impairment. A healthy control may be from a single subject. In another embodiment, a healthy control is a pooled sample from multiple subjects.

[0059]

[0083] As used herein, the term “biological sample” refers to any cell, biological fluid, or tissue. Suitable samples for use in the present invention may include, but are not limited to, whole blood, leukocytes, fibroblasts, serum, urine, plasma, saliva, bone marrow, cerebrospinal fluid, amniotic fluid, and skin cells. Such samples may be further diluted with saline, buffer, or a physiologically acceptable diluent. Alternatively, such samples may be concentrated by conventional methods.

[0060]

[0084] Each of the vectors and other compositions described herein is intended to be useful in other embodiments. Furthermore, each of the compositions described as useful in this method is also intended to be an embodiment of the present invention.

[0061]

[0085] As used herein, “disease,” “disorder,” and “condition” refer to Fabry disease and / or α-GAL A deficiency and / or dysfunction in the subject.

[0062]

[0086] In this specification, “modified” means constructing a peptide or vector to contain a specific peptide or genetic element, such as a specific nucleotide sequence, in order to produce a specific protein product. This also includes manipulating both endogenous and exogenous genetic or peptide elements to elicit a desired product, nucleic acid or protein sequence, and / or structure.

[0063]

[0087] As used herein, “part” means a nucleotide or amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of a complete gene element or peptide. For example, a part of an hGLA nucleotide sequence includes a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of a complete hGLA nucleotide sequence. Another example is that a part of α-GAL A includes an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of a complete α-GAL A.

[0064]

[0088] As used herein, “wild-type,” “wt,” and “endogenous” are used interchangeably to refer to naturally occurring or innate genes, proteins, nucleotide sequences, or peptide sequences.

[0065]

[0089] As used herein, “heterogeneous” is used to refer to a gene, protein, nucleotide sequence, or peptide sequence that is not naturally occurring or is exogenous.

[0066]

[0090] As used herein, “nucleic acid” refers to polymers of nucleotides, including RNA, mRNA, cDNA, genomic DNA, peptide nucleic acids (PNA), and the synthetic and mixed polymers described above. A nucleotide refers to a ribonucleotide, a deoxynucleotide, or a modified form of any type of nucleotide (e.g., a peptide nucleic acid oligomer). The term also includes single-stranded and double-stranded DNA. Those skilled in the art will understand that functional variants of these nucleic acid molecules are described herein. A functional variant is a nucleic acid sequence that, when directly translated using a standard genetic code, provides the same amino acid sequence as that translated from the parent nucleic acid molecule.

[0067]

[0091] In certain embodiments, nucleic acid molecules encoding functional α-GAL A, and other constructs described herein, are useful for generating expression cassettes and vector genomes and can be modified for expression in mammalian cells such as yeast cells, insect cells, or human cells. Methods are known and have been previously described, for example, in WIPO Patent Application Publication No. 1996 / 09378, which are incorporated herein by reference in their entirety. A nucleotide sequence is considered modified if, compared to the wt sequence, at least one non-preferred codon is replaced with a more preferred codon. Here, a non-preferred codon is a codon that is less frequently used in an organism than other codons encoding the same amino acid, and a more preferred codon is a codon that is more frequently used in an organism than a non-preferred codon. Codon frequencies for a particular organism can be found in codon frequency tables such as www.kazusa.jp / codon. Preferably, several non-preferred codons, preferably almost all or all non-preferred codons, are replaced with more preferred codons. Preferably, the most frequently used codon in the organism is used in the modified nucleotide sequence. Substitutions with preferred codons generally result in high expression. It will also be understood by those skilled in the art that, as a result of gene coding degeneracy, numerous different nucleic acid molecules may code for the same polypeptide. It will also be understood that, using routine techniques, nucleotide substitutions can be made that do not affect the amino acid sequence coded by the nucleic acid molecule, reflecting the codon usage of the specific host organism in which the polypeptide is expressed. Therefore, unless otherwise specified, “nucleic acid sequence encoding an amino acid sequence” and “nucleotide sequence encoding an amino acid sequence” include all nucleotide sequences that are degenerate versions of each other and code for the same amino acid sequence. Nucleic acid sequences can be cloned using routine molecular biological techniques or generated de novo by DNA synthesis. This can be done as a routine procedure by service companies operating in the field of DNA synthesis and / or molecular cloning (e.g., GeneArt, GenScript, Life Technologies, Eurofins).

[0068]

[0092] In certain embodiments, the nucleic acids, expression cassettes, and vector genomes described herein include a modified nucleotide sequence, which is an α-GAL A coding nucleotide sequence. In certain embodiments, the modified sequence is useful for improving production, transcription, expression, or safety in a subject. In certain embodiments, the modified sequence is useful for enhancing the efficacy of the resulting therapeutic composition or treatment. In further embodiments, the modified nucleotide sequence is useful for increasing the efficacy of the expressed functional α-GAL A and may also enable lower doses of therapeutic reagents encoding functional α-GAL A. In certain embodiments, the modified hGLA nucleotide coding sequence is characterized by an improved translation rate compared to the wild-type hGLA nucleotide coding sequence. "Modified" means that the nucleic acid sequence encoding the functional α-GAL A described herein is assembled, for example, to generate a non-viral delivery system (e.g., an RNA-based system, naked DNA, etc.), or to generate a viral vector within a packaging host cell and / or for delivery to a target host cell, and is placed on any suitable gene element, such as naked DNA, a phage, a transposon, a cosmid, or an episome, on which the hGLA nucleotide sequence is delivered to the host cell. In certain embodiments, the gene element is a vector. In one embodiment, the gene element is a plasmid. Methods used to construct such modified constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0069]

[0093] In the context of nucleic acid sequences, the terms “identity percentage (%)”, “sequence identity”, “sequence identity percentage”, or “percent identical” refer to residues in two nucleotide sequences that are identical when aligned for correspondence. The length of the sequence identity comparison may range from the full length of the construct, the full length of the gene-coding nucleotide sequence, or a fragment of at least about 500–1,000 nucleotides. However, identity between smaller fragments, for example, at least about 9 nucleotides, typically at least about 20–24 nucleotides, at least about 28–32 nucleotides, or at least about 36 nucleotides or more, may also be desired.

[0070]

[0094] The percentage of identity can be readily determined for amino acid sequences over the full length of a protein, polypeptide, approximately 100 amino acids, approximately 300 amino acids, or their peptide fragments, or the corresponding nucleic acid sequence coding sequence. A suitable amino acid fragment can be at least approximately 8 amino acids long and up to approximately 50 amino acids long. Generally, when referring to “identity,” “homology,” or “similarity” between two different amino acid sequences, “identity,” “homology,” or “similarity” is determined by referring to an “aligned” amino acid sequence. An “aligned” amino acid sequence, or “alignment,” is a set of nucleic acid sequences or protein (amino acid) sequences that often include corrections such as deletions or additions of bases or amino acids compared to a reference amino acid sequence.

[0071]

[0095] Identity can be determined by preparing an amino acid sequence alignment and using various algorithms and / or computer programs known in the art or commercially available (e.g., BLAST, ExPASy; Clustal Omega; FASTA; e.g., using the Needleman-Wunsch algorithm, Smith-Waterman algorithm). Alignment is performed using one of the various publicly available or commercially available Multiple Sequence Alignment Programs. Examples of amino acid sequence alignment programs include "Clustal Omega," "Clustal X," "MAP," "PIMA," "MSA," "BLOCKMAKER," "MEME," and "Match-Box" programs. Generally, these programs are used with their default settings, but those skilled in the art can change these settings as needed. Alternatively, those skilled in the art can utilize other algorithms or computer programs that provide at least the same degree of identity or consistency as those provided by the referenced algorithms and programs. For example, see JD Thomson et al, Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments," 27(13):2682-2690(1999).

[0072]

[0096] As used herein, the terms “human GLA,” “hGLA,” and “GLA” are used interchangeably to refer to therapeutic genes encoding mature α-GAL A or functional variants thereof. The Greek letter “alpha” and the symbol “α” will be understood to be used interchangeably throughout this specification. Examples of hGLA include, but are not limited to, natural (wild-type) hGLA encoding mature α-GAL A, functional variants of wt hGLA, and their optimized nucleotide sequences (e.g., hGLA co v1). In some embodiments, hGLA comprises or consists of SEQ ID NO: 25. In some embodiments, hGLA comprises or consists of a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 25. In certain embodiments, hGLA comprises or consists of a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 25. In certain embodiments, hGLA comprises or consists of a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 25. In some embodiments, hGLA comprises or consists of SEQ ID NO: 26. In some embodiments, hGLA comprises or consists of a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 26. In certain embodiments, hGLA comprises or consists of a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 26.

[0073]

[0097] As used herein, the term "α-GAL A" is used to refer to human α-galactosidase A protein or therapeutic peptide. Other names for alpha-galactosidase A include agalsidase alpha, alpha-D-galactosidase A, alpha-galactosidase A protein, alpha-galactosidase A enzyme, alpha-D-galactoside galactohydrolase, alpha-galactosidase, alpha-galactosidase A, ceramide trihexosidase, GALA, α-GAL A, galactosidase, alpha-galactosidase, and melibiase. It will be understood that the Greek letter "alpha" and the symbol "α" are used interchangeably throughout this specification. Examples of α-GAL A include, but are not limited to, natural (wild-type) α-GAL A, in particular variants of α-GAL A expressed from nucleic acid sequences provided herein, or functional fragments thereof, which, when delivered in or by the methods provided herein, restore desired function, alleviate symptoms, improve symptoms associated with Fabry disease-related biomarkers (e.g., serum α-GAL A), and / or promote other treatments for Fabry disease.

[0074]

[0098] Unless otherwise specified, “α-GAL A” refers to the mature protein after the signal peptide has been cleaved, the variant protein described herein, or a functional fragment. As used herein, the term “functional α-GAL A” refers to the enzyme having the full-length amino acid sequence of the natural (wild-type) protein (indicated by UniProtKB accession number: P06280-1), its variants (including those described herein having specific amino acid substitutions), its variants having conserved amino acid substitutions, its fragments, the full-length or fragments of any combination of variants and variants having conserved amino acid substitutions, and functional α-GAL A provides at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, or nearly the same, or greater than 100% of the biological activity level of natural (wild-type) α-GAL A. In some embodiments, α-GAL A is encoded by a nucleotide sequence containing or consisting of SEQ ID NO: 25. In some embodiments, α-GAL A is encoded by a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 25. In certain embodiments, α-GAL A is encoded by a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 25. In certain embodiments, α-GAL A is encoded by a nucleotide sequence having about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 25. In some embodiments, α-GAL A is encoded by a nucleotide sequence comprising or consisting of SEQ ID NO: 26. In some embodiments, α-GAL A is encoded by a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 26.In certain embodiments, α-GAL A is encoded by a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 26.

[0075]

[0099] As used herein, the term “functional variant” of a peptide or nucleotide sequence means a protein or nucleotide variant of a peptide or nucleotide sequence (including those described herein having certain one or more amino acids and substitutions), a variant thereof having a conserved amino acid or nucleotide substitution, a fragment thereof, the full length or fragment thereof of any combination of a variant having a conserved amino acid or nucleotide substitution and a variant thereof, and the functional variant provides at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, or nearly the same, or more than 100% of the biological activity level or expression capacity of the peptide (e.g., wt α-GAL A) or nucleotide sequence (e.g., wt hGLA).

[0076]

[0100] As used herein, “signal peptide” or “sp” refers to a peptide sequence that is fused to or within a therapeutic peptide and affects the function or localization of the therapeutic peptide. A signal peptide (sp) may be fused to or near the N-terminus of a therapeutic peptide, to or near the C-terminus of a therapeutic peptide, to or within a therapeutic peptide, or any combination thereof. sp may be wild-type or heterologous. Lowercase sp refers to a signal peptide or a functional variant thereof, and uppercase SP refers to a nucleotide sequence that codes for a signal peptide or a functional variant thereof. A nucleotide sequence that codes for an SP may be called SP. The signal peptide referred to by sp may be one of the wt sp or heterologous sp disclosed herein, and the nucleotide sequence SP that codes for a signal peptide may be a nucleotide sequence SP that codes for one of the wt sp or heterologous sp.

[0077]

[0101] As used herein, “tissue-specific promoter” refers to a predefined tissue or a genetic element that stimulates the expression of a peptide sequence in a tissue. A tissue-specific promoter can stimulate the expression of a peptide sequence in one or more tissues.

[0078]

[0102] As used herein, “vector” refers to a nucleotide sequence that introduces an exogenous or modified nucleotide sequence into a cell. A vector may be a linear or round element. A vector may be, for example, a plasmid, virus, phage, or cosmid. A vector may consist of one or more expression cassettes. A vector may be engineered.

[0079]

[0103] As used herein, “viral vector” refers to the use of a viral genome to deliver genetic material to a cell. A viral genome may be modified to include or exclude genetic elements. Viral vectors may be designed or synthesized in vivo or in vitro. Viral vectors may be retroviruses, lentiviruses, poxviruses, adenoviruses, and adeno-associated viruses. Viral vectors may also be single-stranded or self-complementary, such as ss-rAAV or sc-rAAV, respectively.

[0080]

[0104] As used herein, “serotype” refers to the biologically distinguishable features and / or differences of particles, such as surface antigens. Serotypes may be selected from at least one of rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rAAV13, and their derivatives (e.g., modified using directed evolution).

[0081]

[0105] As used herein, “cell-permeable peptide” means a peptide, protein, or protein fragment that can traverse a cell membrane or organelle membrane, or has the ability to transport cargo across a cell membrane or organelle membrane. A cell-permeable peptide may be fused to or near the N-terminus of a therapeutic peptide, to or near the C-terminus of a therapeutic peptide, to be fused within a therapeutic peptide, or any combination thereof.

[0082]

[0106] As used herein, “encapsulated” and “encapsulation” refer to the packaging, immobilization, or encapsulation of a vector by structural proteins and / or lipid molecules.

[0083]

[0107] As used herein, “Gb3” refers to the sphingolipid, glycolipid globotriaosylceramide. Gb3 may also be known as “Gls” or “GL3”. Gb3 may be a free sphingolipid or may be bound to another molecule or cell membrane. Gb3 may also refer to the acetylated version of Gb3 or acetylated Lyso-Gb3.

[0084]

[0108] As used herein, “lyso-Gb3” refers to the deacetylated version of Gb3. Lyso-Gb3 also refers to a plasma biomarker for Fabry disease. Lyso-Gb3 may also be known as “lyso-Gls” or “lyso-GL3”.

[0085]

[0109] As used herein, “patient” or “subject” refers to a male or female mammal, such as a human, a dog, or an animal model used in clinical research. In certain embodiments, the subjects of these methods and compositions are humans diagnosed with Fabry disease. In further embodiments, the human subjects of these methods and compositions are prenatal, neonatal, infant, toddler, preschool child, primary school child, teenager, young adult, or adult.

[0086]

[0110] As used herein, “sphingolipid accumulation” refers to processes including the anabolism and catabolism of sphingolipids, and the distribution of sphingolipids in leukocytes and tissues including but not limited to bone marrow, skin, and muscle. Sphingolipid accumulation may also refer to α-GAL A activity. Sphingolipid storage disease refers to α-GAL A deficiency or dysfunction, or a lack of enzymes involved in sphingolipid metabolism.

[0087]

[0111] As used herein, the terms “Fabry disease,” “Fabry-related symptoms,” or “symptoms” refer to the symptoms found in patients with Fabry disease and in animal models of Fabry disease. Such symptoms include, but are not limited to, keratotic hemangiomas, acrosensory dyspareunia, hypohidrosis / anhidrosis, corneal opacity, lens opacity, cardiac disorders, pain, and renal dysfunction. In addition, common cardiac signs and symptoms of Fabry disease include left ventricular hypertrophy, valvular heart disease (particularly mitral valve prolapse and / or regurgitation), early coronary artery disease, angina pectoris, myocardial infarction, conduction abnormalities, arrhythmias, and congestive heart failure. Fabry disease is also known by other names such as alpha-galactosidase A deficiency, Anderson-Fabry disease, and diffuse keratotic hemangioma.

[0088]

[0112] As used herein, “Fabry mouse” refers to an α-GAL A-deficient mouse model that can be used as a model of Fabry disease. See, for example, doi:10.1073 / pnas.94.6.2540 and Proc Natl Acad Sci US A. 1997 Mar 18;94(6):Proc Natl Acad Sci US A. 1997 Mar 18;94(6):2540-42540-4.

[0089]

[0113] As used herein, “administer,” “dosage,” and “administer” refer to the delivery of the therapy or composition of this technology to a subject by either local or systemic administration. Administration may be intratracheal, nasal, dermal, transdermal, oral, or parenteral administration. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, or intracranial (e.g., intrathecal or intraventricular) administration.

[0090]

[0114] As used herein, “therapeutic effective dose” refers to the amount that produces the desired effect on a subject for an indication, condition, disease, or disorder. In certain embodiments, the therapeutic effective dose is the amount that produces the greatest therapeutic effect. In other embodiments, the therapeutic effective dose produces a therapeutic effect less than the greatest therapeutic effect. For example, the therapeutic effective dose is the amount that produces a therapeutic effect while avoiding one or more side effects associated with the dose that produces the greatest therapeutic effect. The therapeutic effective dose of a particular composition will vary based on a variety of factors, including but not limited to the properties of the therapeutic composition (e.g., activity, pharmacokinetics, pharmacodynamics, and bioavailability); the physiological state of the subject (e.g., age, weight, sex, type and stage of disease, medical history, general condition, responsiveness to a given dose, and other current medications); the properties of pharmaceutically acceptable carriers, additives, and preservatives in the composition; and the route of administration. Those skilled in the art of clinical and pharmacological fields may determine the therapeutic effective dose by routine experimentation, i.e., by monitoring the subject's response to administration of the therapeutic composition and adjusting the dose accordingly.

[0091]

[0115] As used herein, “clinically effective dose,” “clinically effective concentration,” or “clinically effective dose” refers to the concentration or dose of a peptide, composition, or pharmaceutical composition that has been shown to be effective in a clinical trial or is predicted to be effective based on early-stage or preclinical studies. In some embodiments, the “clinically effective dose” is the same as the “therapeutic effective dose.” In some embodiments, the “clinically effective dose” is greater than or less than the “therapeutic effective dose.” For further guidance, see Remington: The Science and Practice of Pharmacy, 21st Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.

[0092] Modified peptide

[0116] This specification describes modified peptides designed to deliver α-GAL A to cells or subjects in need. α-GAL A catalyzes the hydrolysis of sphingoglycolipids and is involved in the degradation of sphingoglycolipids in lysosomes. The absence or dysfunction of α-GAL A can lead to the accumulation of sphingoglycolipids and their byproducts to harmful levels. This accumulation and α-GAL A deficiency are commonly observed in subjects with Fabry disease. Treatment for Fabry disease requires therapies that promote increased α-GAL A activity. One possible therapy is the administration of α-GAL A to subjects via modified peptides. Restoring α-GAL A levels using modified peptides may promote metabolic homeostasis, particularly sphingolipid metabolism. Modified peptides containing α-GAL A may be further modified to enhance peptide efficiency, targeting, and stability. Modifications may include alterations to the wt α-GAL A amino acid sequence to generate functional variants, which may be more effective in restoring α-GAL A function than the wt α-GAL A amino acid sequence.

[0093]

[0117] In some embodiments, α-GAL A is human α-GAL A. In some embodiments, the modified peptide comprises α-GAL A or a functional variant thereof. In some embodiments, the modified peptide further comprises one or more signal peptides or functional variants thereof, and optionally one or more cell-permeable peptides or functional variants thereof. One or more signal peptides or functional variants thereof, and one or more cell-permeable peptides or functional variants thereof, may be fused to the C-terminus and / or N-terminus or vicinity thereof of α-GAL A or its functional variant, and / or internally fused within the sequence of α-GAL A or its functional variant. In certain embodiments, the modified peptide comprises a spacer sequence between α-GAL A or its functional variant and one or more signal peptides or functional variants. In certain embodiments, the modified peptide comprises a spacer sequence between α-GAL A or its functional variant and one or more cell-permeable peptides or functional variants. In certain embodiments, the modified peptide comprises at least one signal peptide fused to or near the N-terminus of α-GAL A or a functional variant thereof. In certain embodiments, the modified peptide comprises multiple signal peptides. The multiple signal peptides may be sequentially fused at the N-terminus. In certain embodiments, the modified peptide comprises multiple cell-permeable peptides. The multiple cell-permeable peptides may be sequentially fused at any site on the protein.

[0094]

[0118] In certain embodiments, human α-GAL A is encoded by wt hGLA (SEQ ID NO: 25). In certain embodiments, human α-GAL A is encoded by a functional variant of the wt nucleotide sequence. In certain embodiments, the functional variant is hGLA co v1 (SEQ ID NO: 26).

[0095]

[0119] In some embodiments, the modified peptide comprises α-GAL A or a functional variant thereof containing a peptide sequence having at least about 60% to 100% identity with SEQ ID NO: 27.

[0096]

[0120] In some embodiments, the modified peptide comprises α-GAL A or a functional variant thereof containing a peptide sequence having at least 60% to 100% identity with SEQ ID NO: 27.

[0097]

[0121] In some embodiments, the modified peptide comprises α-GAL A or a functional variant thereof containing a peptide sequence having approximately 60% to 100% identity with SEQ ID NO: 27. Table 1: Amino acid sequence of α-GAL A and embodiments of the nucleotide sequence encoding α-GAL A. TIFF2026515280000002.tif254170TIFF2026515280000003.tif254170TIFF2026515280000004.tif45170

[0098] Signal peptide

[0122] Modified peptides containing α-GAL A may be further modified to enhance the peptide's efficiency, targeting, and stability. Modifications may include alterations to the signal peptide sequence, which may be more effective in restoring α-GAL A function than the wt amino acid sequence. Signal peptides are peptides that facilitate, target, and localize the transport of therapeutic peptides, and provide other signals. A signal peptide (sp) can be a signal sequence, targeting signal, localization signal, localization sequence, transport peptide, leader sequence, or leader peptide. Modified peptides may contain endogenous sps or modified sps. In some cases, modified peptides may have multiple sps. Inclusion, deletion, alteration, substitution, or editing of sps may alter protein function. The position of the sp peptide may also affect activity. Furthermore, sp sequences may be modified or synthesized to improve protein function, abundance, and activity. This technology allows for the incorporation of different sps into modified peptide sequences to enhance the delivery, secretion, and / or overall potency of α-GAL A from the modified peptide.

[0099]

[0123] In some embodiments, the modified peptide comprises a portion of the signal peptide or a functional variant thereof. In some embodiments, a portion of sp or its functional variant is fused to the N-terminus or vicinity of a portion of α-GAL A or its functional variant. In some embodiments, a portion of sp or its functional variant is fused to the C-terminus or vicinity of a portion of α-GAL A or its functional variant. In some embodiments, a portion of sp or its functional variant is internally fused within a portion of α-GAL A or its functional variant.

[0100]

[0124] In some embodiments, the modified peptide comprises endogenous human α-GAL A wt sp (SEQ ID NO: 28) or a functional variant thereof.

[0101]

[0125] In some embodiments, the modified peptide comprises a heterologous signal peptide or a functional variant thereof.

[0102]

[0126] In some embodiments, the modified peptide comprises a wt sp or a functional variant thereof and a heterologous sp or a functional variant thereof. In some embodiments, the modified peptide comprises two or more heterologous sps or functional variants thereof.

[0103]

[0127] In certain embodiments, one or more heterologous signal peptides are selected from the group consisting of sp1, sp2, ..., sp22, and their amino acid sequences are provided in Table 2. In some embodiments, one or more heterologous signal peptides are selected from the group consisting of sp1, sp3, sp4, sp18, sp20, sp21, and sp22.

[0104]

[0128] In certain embodiments, the wt sp or its functional variant is encoded by a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 1 or 51. In certain embodiments, the wt sp or its functional variant is encoded by a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 1 or 51. In certain embodiments, the wt sp or its functional variant is encoded by a nucleotide sequence having about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 1 or 51.

[0105]

[0129] In certain embodiments, one or more heterologous signal peptides or their functional variants are encoded by one or more nucleotide sequences that are at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 2, 3, 4, ..., 23, 64, 65, and 66 as listed in Table 2. In certain embodiments, one or more heterologous signal peptides or functional variants thereof are encoded by one or more nucleotide sequences that are approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 2, 3, ..., 23, 64, 65, and 66, as listed in Table 2. Table 2: Embodiments of endogenous human α-GAL A wt sp, heterologous sp, and embodiments of nucleotide sequences encoding wt sp and heterologous sp. TIFF2026515280000005.tif254170TIFF2026515280000006.tif209170

[0106]

[0130] In some embodiments, the nucleotide sequence encoding sp includes or consists of SEQ ID NO: 1 or 51. In some embodiments, the nucleotide sequence encoding sp includes or consists of a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 1 or 51. In certain embodiments, the nucleotide sequence encoding sp includes or consists of a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 1 or 51. In certain embodiments, the nucleotide sequence encoding sp includes or consists of a sequence having about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 1 or 51. In some embodiments, sp is sp21. In some embodiments, the nucleotide sequence encoding sp includes or consists of SEQ ID NO: 22. In some embodiments, the nucleotide sequence encoding sp includes or consists of a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 22. In certain embodiments, the nucleotide sequence encoding sp includes or consists of a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 22. In certain embodiments, the nucleotide sequence encoding sp includes or consists of a sequence having about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 22.

[0107] Cell-permeable peptides

[0131] As previously mentioned, modified peptides containing α-GAL A may be further modified to enhance the peptide's efficiency, targeting, and stability. Modifications may include the inclusion or alteration of cell-permeable peptide sequences. Cell-permeable peptides can assist in the delivery of molecules and the transport of peptides across the cell membrane or organelle membrane. By incorporating cell-permeable peptide sequences or functional variants thereof into modified peptide sequences, peptide delivery and / or secretion can be promoted. The position of the cell-permeable peptide within the peptide sequence may also affect peptide delivery and / or secretion. Cell-permeable peptides may also be incorporated into modified peptides. This specification also describes the incorporation of TAT sequences into modified peptides. TAT sequences may include arginine-rich sequences that directly permeate the cell membrane or organelle membrane. In this technology, incorporating TAT or other cell-permeable peptides into modified peptide sequences can enhance the delivery, secretion, and / or overall potency of α-GAL A from the modified peptide. Examples of cell-permeable peptides include, but are not limited to, TAT p47-57, TAT p48-60, penetratin p43-58, hCT p-9-32, polyarginine, pVEC, Pep-1, transportan, and MAP.

[0108]

[0132] Several embodiments of this technology describe the use of a modified peptide comprising α-GAL A and a cell-permeable peptide, the modified peptide may be incorporated into the modified peptide sequence to modulate different peptide properties, including but not limited to enhancing peptide secretion and delivery to target cells or tissues. In some embodiments, the modified peptide comprises the cell-permeable peptide or a functional variant thereof. In some embodiments, the cell-permeable peptide comprises or consists of a portion of TAT p47-57 (SEQ ID NO: 55) or a functional variant thereof. In certain embodiments, the cell-permeable peptide is encoded by a nucleotide sequence having at least about 60-100% identity to SEQ ID NO: 24. In certain embodiments, the cell-permeable peptide is encoded by a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 24.

[0109]

[0133] In certain embodiments, the cell-permeable peptide is encoded by a nucleotide sequence having at least 60-100% identity with SEQ ID NO: 24.

[0110]

[0134] In certain embodiments, the cell-permeable peptide is encoded by a nucleotide sequence having approximately 60–100% identity to SEQ ID NO: 24. In certain embodiments, the cell-permeable peptide is encoded by a nucleotide sequence having approximately 80%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 24.

[0111]

[0135] In some embodiments, the cell-permeable peptide comprises or consists of at least a portion of peptides selected from the group consisting of TAT p47-57 (SEQ ID NO: 55), TAT p48-60 (SEQ ID NO: 56), penetratin p43-58 (SEQ ID NO: 57), hCT p9-32 (SEQ ID NO: 58), polyarginine (SEQ ID NO: 59), pVEC (SEQ ID NO: 60), Pep-1 (SEQ ID NO: 61), transportan (SEQ ID NO: 62), and MAP (SEQ ID NO: 63), or functional variants thereof. In certain embodiments, the cell-permeable peptide is encoded by a nucleotide sequence having at least about 60-100% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24, 52, 53, and 54. In certain embodiments, the cell-permeable peptide is encoded by a nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24, 52, 53, and 54.

[0112]

[0136] In certain embodiments, the cell-permeable peptide is encoded by a nucleotide sequence having at least 60-100% identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24, 52, 53, and 54.

[0113]

[0137] In certain embodiments, the cell-permeable peptide is encoded by a nucleotide sequence having approximately 60-100% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24, 52, 53, and 54. In certain embodiments, the cell-permeable peptide is encoded by a nucleotide sequence having approximately 80%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, 99%, or 100% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24, 52, 53, and 54.

[0114]

[0138] In some embodiments, the cell-permeable peptide is fused to the C-terminus of α-GAL A. In some embodiments, the cell-permeable peptide is fused to the N-terminus of α-GAL A. In certain embodiments, the cell-permeable peptide is internally fused to the interior of α-GAL A.

[0115]

[0139] In certain embodiments, the modified peptide comprises multiple cell-permeable peptides or functional variants thereof. In some embodiments, the modified peptide comprises a first cell-permeable peptide or functional variant fused to or near the N-terminus of α-GAL A, and a second cell-permeable peptide or functional variant fused to or near the C-terminus of α-GAL A. In some embodiments, the modified peptide comprises a first cell-permeable peptide or functional variant fused to or near the N-terminus of α-GAL A, and a second cell-permeable peptide or functional variant internally fused within α-GAL A. In some embodiments, the modified peptide comprises a first cell-permeable peptide or functional variant fused to or near the C-terminus of α-GAL A, and a second cell-permeable peptide or functional variant internally fused within α-GAL A.

[0116]

[0140] In some embodiments, the modified peptide comprises a first cell-permeable peptide or a functional variant thereof fused to the N-terminus or vicinity of α-GAL A, a second cell-permeable peptide or a functional variant thereof fused to the C-terminus or vicinity of α-GAL A, and a third cell-permeable peptide or a functional variant thereof internally fused within α-GAL A. Table 3: Examples of cell-permeable peptides and nucleotide sequences encoding cell-permeable peptides TIFF2026515280000007.tif128170

[0117] Viral vector

[0141] Treatment for Fabry disease requires therapies that promote the enhancement of α-GAL A activity. One possible therapy involves administering α-GAL A to the target via a modified peptide expressed from a viral vector. The viral vector may be constructed to deliver genetic material and express the modified peptide in target cells or tissues that require it. The viral genome may be modified to include or exclude genetic elements. One of these genetic elements is an expression cassette containing a nucleic acid sequence that is expressed as a protein. The viral vector may be designed or synthesized in vivo or in vitro and may be a retrovirus, lentivirus, poxvirus, adenovirus, or adeno-associated virus. Embodiments of this technology include an rAAV vector containing an rAAV genome, the rAAV genome containing a nucleic acid sequence encoding a modified peptide containing α-GAL A. The viral vector may also be single-stranded or self-complementary, referred to as ss-rAAV or sc-rAAV, respectively. Furthermore, the viral vector may be modified by encapsulation or serotype selection, each modifying the efficacy of the viral vector. This specification describes the use of vectors to increase the expression and delivery of α-GAL A.

[0118]

[0142] In some embodiments, the viral vector is encapsulated. In some embodiments, the viral vector includes a serotype. In certain embodiments, the serotype is rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, or rAAV13. In certain embodiments, the serotype may be a derivative of rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, or rAAV13.

[0119]

[0143] In some embodiments, the viral vector further comprises a promoter. In some embodiments, the viral vector is a tissue-specific promoter. In some embodiments, the viral vector is a liver, heart, kidney, or spleen-specific promoter. In certain embodiments, the liver-specific promoter is a TTR, HBV, ALB, human α-1 antitrypsin (hAAT) promoter, or phosphoenolpyruvate carboxykinase (PEPCK) gene promoter. In certain embodiments, the liver-specific promoter is a modified liver-specific promoter.

[0120]

[0144] In certain embodiments, the viral vector includes at least one ITR nucleotide sequence. In certain embodiments, the viral vector includes a 5'ITR and a 3'ITR. In certain embodiments, the viral vector does not include an intron. In certain embodiments, the viral vector includes at least one intron. In certain embodiments, the vector includes a polyadenylated (poly-A) nucleotide sequence. In certain embodiments, the viral vector includes at least one ITR nucleotide sequence, an intron, and / or a poly-A nucleotide sequence. Examples of ss-rAAV and sc-rAAV are shown in Figures 1A and 1B.

[0121]

[0145] In some embodiments, the viral vector includes a 5'ITR, a 3'ITR, a tissue-specific promoter, an intron, a stop codon, a poly(A) nucleotide sequence, an hGLA nucleotide sequence, and a nucleotide sequence encoding sp. In certain embodiments, the hGLA nucleotide sequence is wt hGLA (SEQ ID NO: 25) or hGLA cov1 (SEQ ID NO: 26). In certain embodiments, sp is wt sp or sp21. In certain embodiments, the sp nucleotide sequence is one of the sp nucleotide sequences listed in Table 2. In certain embodiments, the tissue-specific promoter is a TTR. In certain embodiments, the viral vector is an ss-rAAV vector or an sc-rAAV vector.

[0122]

[0146] In some embodiments, the viral vector is rAAV8. In some embodiments, the sc-rAAV vector is ss-wtspGLA or ss-sp21GLA. In some embodiments, the ss-rAAV vector is FD802-1, FD802-2, FD802-3, FD802-4 or FD802-5.

[0123]

[0147] In some embodiments, the hGLA sequence is expressed from a vector. In certain embodiments, the vectors containing hGLA are ss-rAAV expressing α-GAL A+wt sp, ss-rAAV expressing α-GAL A+sp21, FD802-1, or FD802-2. In certain embodiments, the vectors containing hGLA cov1 (sequence number 26) are FD802-3, FD802-4, or FD802-5.

[0124]

[0148] In some embodiments, the signal peptide is expressed from a vector. In some embodiments, the vector expressing the wt sp peptide is ss-rAAV, FD802-1, FD802-3, or FD802-5 expressing α-GAL A+wt sp. In some embodiments, the vector expressing the sp21 peptide is ss-rAAV, FD802-2, or FD802-4 expressing α-GAL A+sp21.

[0125]

[0149] In some embodiments, the cell-permeable peptide is expressed from a vector. In some embodiments, the vector for expressing the cell-permeable peptide is FD802-5.

[0126] Pharmaceutical composition

[0150] The modified peptides and viral vectors described herein may be formulated into a pharmaceutical composition containing, essentially, or comprising an effective amount of one or more of the modified peptides or viral vectors described herein. The pharmaceutical composition may be modified for different routes of administration or to enhance the stability of the composition. Furthermore, the pharmaceutical composition may be modified to deliver different doses of the active ingredient, such as the modified peptides, viral vectors, and / or viral compositions. In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic agents, which are not the modified peptides or viral vectors disclosed herein. In some embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable carriers, additives, excipients, preservatives, or combinations thereof. Examples of acceptable carriers include physiologically acceptable solutions such as sterile saline and sterile buffered saline.

[0127]

[0151] In some embodiments, the modified peptide is present in the pharmaceutical composition. In some embodiments, the viral vector is present in the pharmaceutical composition. In some embodiments, the pharmaceutical composition includes an encapsulated vector.

[0128]

[0152] In some embodiments, the pharmaceutical composition further comprises additives. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or additives. In some embodiments, the pharmaceutically acceptable carrier and / or additives comprises water.

[0129]

[0153] In some embodiments, the pharmaceutical composition is formulated for intraperitoneal, intravenous, parenteral, subcutaneous, intramuscular, intraventricular, or oral administration. In some embodiments, a single pharmaceutical composition is formulated for subcutaneous administration.

[0130]

[0154] In some embodiments, the pharmaceutical composition is administered together with a second Fabry disease treatment. In certain embodiments, the second Fabry disease treatment comprises a modified peptide, vector, or viral vector.

[0131]

[0155] Viral vectors can be formulated into pharmaceutical or viral compositions to deliver genetic material to a target or therapeutic peptide in order to express that peptide. In some embodiments, the technology provides a viral composition for increasing α-GAL A expression in a target. In other embodiments, the technology provides a viral composition for treating Fabry disease or for alleviating the symptoms of Fabry disease in a target. The α-GAL A of the viral vector is expressed from an expression cassette further comprising at least one ITR nucleotide sequence, an intron, and / or a polyadenylated polyA nucleotide sequence. The viral composition may be modified to increase the expression of the peptide of interest, such as one containing α-GAL A. Other parameters of the viral composition may be modified to enhance the stability of the composition or the efficiency of delivery to target cells or tissues. This includes, but is not limited to, encapsulation of the viral vector to enhance delivery and stability, and selection of a serotype that allows binding to a specific cell surface protein of interest.

[0132]

[0156] In some embodiments, the viral composition includes a viral vector expressing α-GAL A. In some embodiments, the viral vector is encapsulated. In some embodiments, the viral vector is an rAAV vector. In certain embodiments, the viral vector is an sc-rAAV or ss-rAAV vector. In certain embodiments, the viral vector includes serotypes rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, or rAAV13.

[0133]

[0157] In some embodiments, the viral composition is formulated for intraperitoneal, intravenous, parenteral, subcutaneous, intramuscular, intraventricular, or oral administration. In some embodiments, a single pharmaceutical composition is formulated for subcutaneous administration.

[0134] Related methods

[0158] The modified peptides, viral vectors, and pharmaceutical compositions described herein are intended for use in methods of treating Fabry disease or reducing symptoms associated with Fabry disease. Symptoms associated with Fabry disease include, but are not limited to, keratotic hemangiomas, acrosensory dyspareunia, hypohidrosis / anhidrosis, corneal opacity, lens opacity, cardiac disorders, pain, renal dysfunction, or symptoms resulting from α-GAL A dysfunction or deficiency.

[0135]

[0159] In some embodiments, the technology provides a method for treating Fabry disease in a subject. In some embodiments, the technology provides a method for reducing one or more symptoms associated with Fabry disease in a subject. In some embodiments, the technology provides a method for increasing α-GAL A expression in a subject. In some embodiments, α-GAL expression and / or activity are increased in target tissue and / or plasma of the subject. In certain embodiments, the target tissue is the liver, heart, kidney, and / or spleen.

[0136]

[0160] In some embodiments, the technology provides a method for treating Fabry disease in a subject or reducing one or more symptoms of Fabry disease, comprising administering a modified peptide containing α-GAL A, a viral vector expressing α-GAL A, or a pharmaceutical composition containing a modified peptide containing α-GAL A or a viral vector expressing α-GAL A to a subject in need thereof.

[0137]

[0161] In some embodiments, the technology provides a method for increasing the expression of α-Gal A in a subject, comprising administering a modified peptide containing α-Gal A, a viral vector expressing α-Gal A, or a pharmaceutical composition containing a modified peptide containing α-Gal A or a viral vector expressing α-Gal A to a subject requiring such action.

[0138]

[0162] In some embodiments, the technology provides a method for reducing lyso-Gb3 in a target tissue and / or plasma. In some embodiments, the technology provides a method for increasing the glycosylation of α-GAL A in a target tissue and / or plasma. In some embodiments, the technology provides a method for increasing mature α-GAL A in a target tissue and / or plasma.

[0139]

[0163] In some embodiments, the technology provides methods for modulating sphingolipid accumulation in target tissues and / or plasma of a subject. In certain embodiments, modulating sphingolipid accumulation involves decreasing or increasing sphingolipids in target tissues and / or plasma of a subject. In some embodiments, the technology provides methods for reducing sphingolipid accumulation in one or more target tissues of a subject, comprising administering a modified peptide containing α-GAL A, a viral vector expressing α-GAL A, or a pharmaceutical composition containing a modified peptide containing α-GAL A or a viral vector expressing α-GAL A to a subject requiring such treatment.

[0140] Dosage

[0164] Depending on the indication, severity, and route of administration, an appropriate dosage may be selected. For example, in acute indications, a higher dose may be administered in fewer treatments, while in chronic indications requiring frequent and prolonged treatment, a lower dose may be administered. In tissues with high lyso-Gb3 levels or low α-GAL A expression, higher doses of modified peptides, viral vectors, and / or pharmaceutical compositions may be required. The dosage may be determined by the effective amount of modified peptides, viral vectors, and / or pharmaceutical compositions to improve symptoms or molecular outcomes associated with Fabry disease and reduced α-GAL A expression.

[0141]

[0165] The dosage of modified peptides, viral vectors, and / or pharmaceutical compositions expressing α-GAL A expression may be determined by an effective dose that increases α-GAL A expression and / or activity, decreases lyso-GB3, or reduces symptoms associated with Fabry disease. The modified peptides, viral vectors, and / or pharmaceutical compositions may be administered as a single or multiple dose to subjects requiring them.

[0142]

[0166] In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition are administered to the subject in a single dose.

[0143]

[0167] In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition are administered together with a second Fabry disease treatment.

[0144]

[0168] It is understood that a specific dose regimen for a given subject should be adjusted over time according to the severity of the disease or disease-related symptoms in the subject requiring it. For example, if sufficient therapeutic activity is not obtained at low doses, the dose of the modified peptide, viral vector, or pharmaceutical composition may be increased.

[0145]

[0169] In some embodiments, the dosage is determined by an effective amount of a modified peptide, viral vector, and / or pharmaceutical composition to reduce one or more symptoms associated with Fabry disease. In some embodiments, the dosage is determined by an effective amount of a modified peptide, viral vector, and / or pharmaceutical composition to increase the expression and / or activity of α-GAL A. In some embodiments, the dosage is determined by an effective amount of a modified peptide, viral vector, and / or pharmaceutical composition to lower plasma and / or tissue lyso-Gb3 levels.

[0146]

[0170] The dosage of a viral vector and / or a pharmaceutical composition containing a viral vector may be adjusted depending on the specific amount of viral genome that is to be delivered to the target that requires it.

[0147]

[0171] In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition are administered in doses of at least about 0.5E+10vg / kg to 14E+14vg / kg.

[0148]

[0172] In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition are administered in doses of at least about 0.5E+10vg / kg to 7E+10vg / kg. In some embodiments, the modified peptide, viral vector and / or pharmaceutical composition is administered in doses of at least about 0.5E+10vg / kg, at least about 1E+10vg / kg, at least about 1.5E+10vg / kg, at least about 2E+10vg / kg, at least about 2.5E+10vg / kg, at least about 3E+10vg / kg, at least about 3.5E+10vg / kg, at least about 4E+10vg / kg, at least about 4.5E+10vg / kg, at least about 5E+10vg / kg, at least about 5.5E+10vg / kg, at least about 6E+10vg / kg, at least about 6.5E+10vg / kg, or at least about 7E+10vg / kg.

[0149]

[0173] In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition is administered in doses of at least 0.5E+10vg / kg to 7E+10vg / kg. In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition is administered in doses of at least 0.5E+10vg / kg, at least 1E+10vg / kg, at least 1.5E+10vg / kg, at least 2E+10vg / kg, at least 2.5E+10vg / kg, at least 3E+10vg / kg, at least 3.5E+10vg / kg, at least about 4E+10vg / kg, at least 4.5E+10vg / kg, at least 5E+10vg / kg, at least 5.5E+10vg / kg, at least 6E+10vg / kg, at least 6.5E+10vg / kg, or at least 7E+10vg / kg.

[0150]

[0174] In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition is administered in doses of approximately 0.5E+10vg / kg to 7E+10vg / kg. In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition is administered in doses of approximately 0.5E+10vg / kg, approximately 1E+10vg / kg, approximately 1.5E+10vg / kg, approximately 2E+10vg / kg, approximately 2.5E+10vg / kg, approximately 3E+10vg / kg, approximately 3.5E+10vg / kg, approximately 4E+10vg / kg, approximately 4.5E+10vg / kg, approximately 5E+10vg / kg, approximately 5.5E+10vg / kg, approximately 6E+10vg / kg, approximately 6.5E+10vg / kg, and approximately 7E+10vg / kg.

[0151]

[0175] In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition are administered in doses of at least about 0.5E+11vg / kg to 7E+11vg / kg. In some embodiments, the modified peptide, viral vector and / or pharmaceutical composition is administered in doses of at least about 0.5E+11vg / kg, at least about 1E+11vg / kg, at least about 1.5E+11vg / kg, at least about 2E+11vg / kg, at least about 2.5E+11vg / kg, at least about 3E+11vg / kg, at least about 3.5E+11vg / kg, at least about 4E+11vg / kg, at least about 4.5E+11vg / kg, at least about 5E+11vg / kg, at least about 5.5E+11vg / kg, at least about 6E+11vg / kg, at least about 6.5E+11vg / kg, or at least about 7E+11vg / kg.

[0152]

[0176] In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition is administered in doses of at least 0.5E+11vg / kg to 7E+11vg / kg. In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition is administered in doses of at least 0.5E+11vg / kg, at least 1E+11vg / kg, at least 1.5E+11vg / kg, at least 2E+11vg / kg, at least 2.5E+11vg / kg, at least 3E+11vg / kg, at least 3.5E+11vg / kg, at least about 4E+11vg / kg, at least 4.5E+11vg / kg, at least 5E+11vg / kg, at least 5.5E+11vg / kg, at least 6E+11vg / kg, at least 6.5E+11vg / kg, or at least 7E+11vg / kg.

[0153]

[0177] In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition is administered in doses of approximately 0.5E+11vg / kg to 7E+11vg / kg. In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition is administered in doses of approximately 0.5E+11vg / kg, approximately 1E+11vg / kg, approximately 1.5E+11vg / kg, approximately 2E+11vg / kg, approximately 2.5E+11vg / kg, approximately 3E+11vg / kg, approximately 3.5E+11vg / kg, approximately 4E+11vg / kg, approximately 4.5E+11vg / kg, approximately 5E+11vg / kg, approximately 5.5E+11vg / kg, approximately 6E+11vg / kg, approximately 6.5E+11vg / kg, and approximately 7E+11vg / kg.

[0154]

[0178] In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition are administered in doses of at least about 0.5E+12vg / kg to 7E+12vg / kg. In some embodiments, the modified peptide, viral vector and / or pharmaceutical composition is administered in doses of at least about 0.5E+12vg / kg, at least about 1E+12vg / kg, at least about 1.5E+12vg / kg, at least about 2E+12vg / kg, at least about 2.5E+12vg / kg, at least about 3E+12vg / kg, at least about 3.5E+12vg / kg, at least about 4E+12vg / kg, at least about 4.5E+12vg / kg, at least about 5E+12vg / kg, at least about 5.5E+12vg / kg, at least about 6E+12vg / kg, at least about 6.5E+12vg / kg, or at least about 7E+12vg / kg.

[0155]

[0179] In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition is administered in doses of at least 0.5E+12vg / kg to 7E+12vg / kg. In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition is administered in doses of at least 0.5E+12vg / kg, at least 1E+12vg / kg, at least 1.5E+12vg / kg, at least 2E+12vg / kg, at least 2.5E+12vg / kg, at least 3E+12vg / kg, at least 3.5E+12vg / kg, at least 4E+12vg / kg, at least 4.5E+12vg / kg, at least 5E+12vg / kg, at least 5.5E+12vg / kg, at least 6E+12vg / kg, at least 6.5E+12vg / kg, or at least 7E+12vg / kg.

[0156]

[0180] In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition is administered in doses of approximately 0.5E+12vg / kg to 7E+12vg / kg. In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition is administered in doses of approximately 0.5E+12vg / kg, approximately 1E+12vg / kg, approximately 1.5E+12vg / kg, approximately 2E+12vg / kg, approximately 2.5E+12vg / kg, approximately 3E+12vg / kg, approximately 3.5E+12vg / kg, approximately 4E+12vg / kg, approximately 4.5E+12vg / kg, approximately 5E+12vg / kg, approximately 5.5E+12vg / kg, approximately 6E+12vg / kg, approximately 6.5E+12vg / kg, and approximately 7E+12vg / kg.

[0157]

[0181] In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition are administered in doses of at least about 0.5E+13vg / kg to 7E+13vg / kg. In some embodiments, the modified peptide, viral vector and / or pharmaceutical composition is administered in doses of at least about 0.5E+13vg / kg, at least about 1E+13vg / kg, at least about 1.5E+13vg / kg, at least about 2E+13vg / kg, at least about 2.5E+13vg / kg, at least about 3E+13vg / kg, at least about 3.5E+13vg / kg, at least about 4E+13vg / kg, at least about 4.5E+13vg / kg, at least about 5E+13vg / kg, at least about 5.5E+13vg / kg, at least about 6E+13vg / kg, at least about 6.5E+13vg / kg, or at least about 7E+13vg / kg.

[0158]

[0182] In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition is administered in doses of at least 0.5E+13vg / kg to 7E+13vg / kg. In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition is administered in doses of at least 0.5E+13vg / kg, at least 1E+13vg / kg, at least 1.5E+13vg / kg, at least 2E+13vg / kg, at least 2.5E+13vg / kg, at least 3E+13vg / kg, at least 3.5E+13vg / kg, at least 4E+13vg / kg, at least 4.5E+13vg / kg, at least 5E+13vg / kg, at least 5.5E+13vg / kg, at least 6E+13vg / kg, at least 6.5E+13vg / kg, or at least 7E+13vg / kg.

[0159]

[0183] In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition is administered in doses of approximately 0.5E+13vg / kg to 7E+13vg / kg. In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition is administered in doses of approximately 0.5E+13vg / kg, approximately 1E+13vg / kg, approximately 1.5E+13vg / kg, approximately 2E+13vg / kg, approximately 2.5E+13vg / kg, approximately 3E+13vg / kg, approximately 3.5E+13vg / kg, approximately 4E+13vg / kg, approximately 4.5E+13vg / kg, approximately 5E+13vg / kg, approximately 5.5E+13vg / kg, approximately 6E+13vg / kg, approximately 6.5E+13vg / kg, and approximately 7E+13vg / kg.

[0160]

[0184] In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition are administered in doses of at least about 0.5E+14vg / kg to 7E+14vg / kg. In some embodiments, the modified peptide, viral vector and / or pharmaceutical composition is administered in doses of at least about 0.5E+14vg / kg, at least about 1E+14vg / kg, at least about 1.5E+14vg / kg, at least about 2E+14vg / kg, at least about 2.5E+14vg / kg, at least about 3E+14vg / kg, at least about 3.5E+14vg / kg, at least about 4E+14vg / kg, at least about 4.5E+14vg / kg, at least about 5E+14vg / kg, at least about 5.5E+14vg / kg, at least about 6E+14vg / kg, at least about 6.5E+14vg / kg, or at least about 7E+14vg / kg.

[0161]

[0185] In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition is administered in doses of at least 0.5E+14vg / kg to 7E+14vg / kg. In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition is administered in doses of at least 0.5E+14vg / kg, at least 1E+14vg / kg, at least 1.5E+14vg / kg, at least 2E+14vg / kg, at least 2.5E+14vg / kg, at least 3E+14vg / kg, at least 3.5E+14vg / kg, at least 4E+14vg / kg, at least 4.5E+14vg / kg, at least 5E+14vg / kg, at least 5.5E+14vg / kg, at least 6E+14vg / kg, at least 6.5E+14vg / kg, or at least 7E+14vg / kg.

[0162]

[0186] In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition is administered in doses of approximately 0.5E+14vg / kg to 7E+14vg / kg. In some embodiments, the modified peptide, viral vector, and / or pharmaceutical composition is administered in doses of approximately 0.5E+14vg / kg, approximately 1E+14vg / kg, approximately 1.5E+14vg / kg, approximately 2E+14vg / kg, approximately 2.5E+14vg / kg, approximately 3E+14vg / kg, approximately 3.5E+14vg / kg, approximately 4E+14vg / kg, approximately 4.5E+14vg / kg, approximately 5E+14vg / kg, approximately 5.5E+14vg / kg, approximately 6E+14vg / kg, approximately 6.5E+14vg / kg, and approximately 7E+14vg / kg.

[0163]

[0187] It will be apparent to those skilled in the art that various equivalents, modifications, and alterations can be made without departing from the scope of this technology, and it will be understood that embodiments of such equivalents are included herein.

[0164]

[0188] The following examples are intended to illustrate various embodiments of the present technology. Thus, the specific embodiments described are not intended to limit the scope of the present technology. [Examples]

[0165]

[0189] Decreased activity or expression of α-galactosidase A (α-GAL A) can cause Fabry disease.

[0166]

[0190] The function of α-GAL A can be restored using gene therapy approaches such as exogenous expression of α-GAL A using viral vectors. To determine whether vector-mediated delivery of α-GAL A is achievable, a series of viral vectors modified to express α-GAL A were synthesized (Figures 1A and 1B). Each vector contained an expression cassette having either the wt hGLA nucleotide sequence (SEQ ID NO: 25) or its functional variant, e.g., hGLA cov1 (SEQ ID NO: 26), and either the endogenous wt signal peptide (wt sp) nucleotide sequence of hGLA or a heterologous sp nucleotide sequence, e.g., sp1-sp22 shown in Table 2.

[0167]

[0191] Vector generation: mRNA was extracted from hEK293 cells and reverse transcribed into cDNA. The wt signal peptide of hGLA was substituted at the N-terminus with a heterologous sp sequence by cloning the PCR fragment into a restriction digest plasmid. The nucleotide sequence encoding α-GAL A, fused with either the wt sp or one of sp1 to sp22, was cloned into an expression construct containing a liver-specific TTR promoter and a polyadenylated sequence flanked by AAV2 reverse terminal repeats (ITRs). The vector was constructed using either a single-stranded rAAV8 vector (ss-rAAV; Figure 1A) or a self-complementary rAAV8 vector (sc-rAAV; Figure 1B). Sequences were validated by enzymatic digestion and Sanger sequencing.

[0168]

[0192] Unless otherwise specified, in the Examples section, low dose refers to 2E+12vg / kg, medium dose refers to 5E+12vg / kg, high dose refers to 5E+13vg / kg, wild-type mouse refers to a healthy mouse that is not Fabry disease and has been treated with PBS, untreated refers to a Fabry disease mouse treated with PBS, which is the same medium used for vector injections that do not contain vectors, and Rec.hAGA refers to recombinant human α-GAL A used as a positive control in the in vitro glycosylation test.

[0169] Example 1: rAAV expressing α-GAL A fused with several heterologous sps increased α-GAL A expression and activity in HepG2 cells and Fabry mice.

[0193] To determine whether the incorporation of heterologous sps into α-GAL A increases α-GAL A expression, HepG2 cells were transfected with a plasmid expressing α-GAL A fused to one sp selected from the group consisting of sp1, sp2, ..., and sp22. α-GAL A was detected in the culture medium using Western blotting (WB) technique. α-GAL A fused to sp1, sp3, sp4, sp18, and sp20-22 correlated with increased extracellular secretion of α-GAL A (Figure 2A) and α-GAL A activity (Figure 2B) compared to α-GAL A fused to wt sp. α-GAL A fused to heterologous sp demonstrated increased activity in HepG2 culture medium compared to α-GAL A fused to wt sp. This suggests that α-GAL A expression and secretion may be increased by substituting wt sp with heterologous sp.

[0170]

[0194] To evaluate the genomic expression and distribution of ss-sp21GLA after injection, rAAV genomic DNA (gDNA) and hGLA RNA were measured in the livers of Fabry mice 12 weeks after injection of either low, medium, or high doses of ss-wtspGLA or ss-sp21GLA. The rAAV8 genomic distribution in the liver, quantified at 12 weeks post-injection, showed a dose-dependent effect, with both vectors exhibiting higher levels at higher doses and higher transduction efficiency than those injected at lower doses of the corresponding vectors (Figure 3A). mRNA levels of modified hGLA in the liver were also analyzed at 12 weeks post-injection, and the same trend as the rAAV8 genomics was observed (Figure 3B). AAV vectors were transduced into the livers of mice, and the AAV genome was detected in liver genomic samples from both vector-treated groups. The transduced AAV vectors were transcribed because transgene mRNA was detected in liver mRNA samples from both vector-treated groups. Successful introduction and transcription of AAV are prerequisites for effective treatment.

[0171]

[0195] To determine whether α-GAL A fused to a heterologous sp increases α-GAL A activity in Fabry mice, Fabry mice were injected with rAAV vectors expressing α-GAL A fused to either wt sp (ss-wtspGLA) or heterologous sp21 (ss-sp21GLA) at doses of 2E+12vg / kg (low dose), 5E+12vg / kg (medium dose), or 5E+13vg / kg (high dose). α-GAL A activity was quantified from orbital blood-derived plasma at weeks 0, 2, 6, 8, and 12 post-injection. Compared to Fabry mouse controls, both vectors showed high levels of α-GAL A activity (Figure 4). In all three dose groups, mice administered with ss-sp21GLA showed higher levels of α-GAL A activity compared to mice injected with ss-wtspGLA (Figure 4). The highest activity was observed under high doses of ss-sp21GLA. Similarly, liver, heart, kidney, and spleen tissues were collected 12 weeks post-injection from mice treated under the aforementioned conditions. As a result, in mice injected with ss-sp21GLA, the overall α-GAL A activity levels in various organs showed a dose-dependent effect, while in mice treated with ss-wtspGLA, the overall α-GAL A activity levels in various organs showed little dose-dependent effect (Figures 5A-D for liver, heart, kidney, and spleen tissues, respectively).

[0172] Example 2: rAAV expressing α-GAL A fused with sp21 increased the expression of α-GAL A in the liver, heart, and kidneys in Fabry mice.

[0196] To evaluate whether ss-sp21GLA administration increases α-GAL A liver expression and produces the active form with post-translational modifications, liver samples from Fabry mice were examined 12 weeks after injection of low, medium, or high doses of ss-sp21GLA or ss-wtspGLA. Mice treated with any dose of ss-sp21GLA showed higher levels of α-GAL A in the liver, heart, and kidneys compared to mice treated with ss-wtspGLA, as shown in Figures 6A and 7. Furthermore, glycosylation analysis of liver proteins at 12 weeks post-injection showed that α-GAL A produced by both vectors was glycosylated and processed into the mature form (Figure 6B). This suggests that administration of ss-sp21GLA increased the presence of α-GAL A in the liver of Fabry mice compared to ss-wtspGLA, and that the α-GAL A produced by both vectors was glycosylated and processed into the mature form. Recombinant α-GAL A was used as a positive control in the in vitro glycosylation test (Rec.hAGA).

[0173] Example 3: rAAV expressing α-GAL A fused with sp21 can reduce lyso-Gb3 and delay weight gain without disrupting tissue structure.

[0197] α-GAL A deficiency can lead to progressive and systemic accumulation of sphingolipids such as globotriaosylceramide (Gb3) and its deacylated derivatives, and globotriaosylsphingosine (lyso-Gb3). Therefore, restoring α-GAL A may reduce levels of Gb3 and lyso-Gb3. To determine whether ss-sp21GLA is effective in reducing lyso-Gb3, mice were treated with low, medium, and high doses of ss-sp21GLA or ss-wtspGLA. At both 12 weeks (Figure 8A) and 26 weeks (Figure 8B) after injection, mice injected with ss-sp21GLA showed a greater reduction in lyso-Gb3 at all doses compared to mice injected with ss-wtspGLA. This suggests that ss-sp21GLA administration had a greater effect on reducing lyso-Gb3 compared to ss-wtspGLA administration.

[0174]

[0198] To determine whether ss-sp21GLA administration is effective for mouse growth, i.e., whether the treatment is safe, mice were treated with low, medium, or high doses of ss-sp21GLA or ss-wtspGLA. Body weight was recorded at weeks 0, 2, 4, 6, and 9. While the rate of body weight gain throughout the growth process was generally similar, the body weight of mice injected with medium or high doses of ss-sp21GLA decreased slightly at week 4 after injection, similar to that of mice injected with medium doses of ss-wtspGLA (Figure 9). This suggests that ss-sp21GLA administration may have a delayed effect on body weight gain depending on the dose.

[0175]

[0199] To determine whether α-GAL A expression induced by ss-sp21GLA administration adversely affects the liver, tissue samples were collected from wild-type mice, untreated Fabry mice, and Fabry mice treated with high doses of ss-wtspGLA or ss-sp21GLA and evaluated by hematoxylin-eosin staining (HE). No significant pathological changes were observed in the livers of Fabry mice after treatment. This suggests that high doses of ss-sp21GLA are not harmful to the liver.

[0176] Example 4: A functional variant of the hGLA nucleotide sequence encoding α-GAL A increased the expression of α-GAL A in HepG2 cells and mouse tissues.

[0200] To determine whether modification of the wt hGLA nucleotide sequence increases α-GAL A expression, plasmids containing a functional variant of wt SP (wt SP co) and a functional variant of wt hGLA, hGLA cov1 (sometimes referred to as "cohGLA"), were transfected into HepG2 cells, and the α-GAL A expression of the protein lysates was evaluated. As shown in Figure 11, transfection with the hGLA cov1 nucleotide sequence was associated with increased α-GAL A expression, suggesting that optimizing the wt hGLA nucleotide sequence to hGLA cov1 can increase intracellular α-GAL A expression.

[0177]

[0201] To determine whether sc-rAAV vectors containing the hGLA cov1 nucleotide sequence can modulate α-GAL A expression and activity in mice, Fabry mice were administered low or medium doses of ss-sp21GLA, FD802-1, FD802-2, FD802-3, or FD802-4. Plasma α-GAL A activity was measured at weeks 0, 2, 4, 6, and 8. Vectors FD802-1, 2, 3, and 4 containing the hGLA cov1 nucleotide sequence showed increased α-GAL A activity in all measurements compared to ss-sp21GLA, as shown in Figure 12. Similarly, vectors FD802-1, 2, 3, and 4 containing the hGLA cov1 nucleotide sequence correlated with increased α-GAL A activity in the liver, heart, kidney, and spleen compared to the ss-sp21GLA vector administered at the same dose, as shown in Figures 13 and 14. This suggests that the hGLA cov1 nucleotide sequence increased the expression and activity of α-GAL A compared to the wt hGLA nucleotide sequence.

[0178] Example 5: A functional variant of the hGLA nucleotide sequence encoding α-GAL A increased α-GAL A expression in mouse liver and decreased lyso-Gb3 without causing pathological changes in the liver.

[0202] To evaluate whether the hGLA cov1 nucleotide sequence increases α-GAL A expression in the liver compared to the wt hGLA nucleotide sequence, liver samples from Fabry mice injected with moderate doses of FD802-1, FD802-2, FD802-3, FD802-4, and ss-sp21GLA at 14 weeks were examined for α-GAL A expression (Figure 15). α-GAL A expression levels detected in the livers of mice injected with moderate doses of FD802-1, FD802-2, FD802-3, or FD802-4 were higher than those of mice injected with ss-sp21GLA, with the highest α-GAL A expression levels observed in mice treated with FD802-4 (Figure 15).

[0179]

[0203] Furthermore, glycosylation analysis of liver proteins 14 weeks after injection of FD802-1, 2, 3, and 4 vectors containing hGLA cov1 showed that α-GAL A produced by these vectors was glycosylated and processed into its mature form (Figure 6B). Recombinant α-GAL A was used as a positive control for the in vitro glycosylation test (Rec.hAGA).

[0180]

[0204] As previously mentioned, α-GAL A deficiency can lead to progressive and systemic accumulation of sphingolipids such as Gb3 and its deacylated derivatives, lyso-Gb3. Therefore, restoring α-GAL A may reduce the levels of Gb3 and lyso-Gb3. To determine whether FD802-1, 2, 3, and 4 vectors containing the hGLA cov1 nucleotide sequence are effective in reducing lyso-Gb3, mice were treated with low or medium doses of FD802-1, FD802-2, FD802-3, FD802-4, and ss-sp21GLA. Quantification of plasma lyso-Gb3 by LC-MS / MS at 6 weeks (Figure 17A) and 14 weeks (Figure 17B) after injection showed a dose-dependent effective reduction of the substrate by α-GAL A in vector-treated mice compared to untreated mice. FD802-1, FD802-2, FD802-3, and FD802-4 resulted in better Lyso-Gb3 removal compared to ss-sp21GLA. In mice injected with low or medium doses of the vector, residual substrate levels were less than 5% of untreated controls even as early as 6 weeks post-injection (Figure 17A), with mice treated with FD802-4 showing the best clearance. Mice injected with one of the four sc-AAV8 vectors (FD802-1, FD802-2, FD802-3, and FD802-4) showed higher α-GAL A activity levels.

[0181]

[0205] To evaluate whether administration of FD802-1, FD802-2, FD802-3, and FD802-4 vectors containing the hGLA cov1 nucleotide sequence alters the histological features of tissue, liver tissue was collected from untreated Fabry mice and Fabry mice treated with moderate doses of FD802-1, FD802-2, FD802-3, FD802-4, or ss-sp21GLA. Hematoxylin-eosin (HE) analysis by H&E staining demonstrated no pathological changes in the livers of Fabry mice treated with moderate doses of FD802-1, 2, 3, and 4, as shown in Figure 18. This suggests the safety of treatment in Fabry mice administered with moderate doses of FD802-1, FD802-2, FD802-3, and FD802-4 vectors containing the hGLA cov1 nucleotide sequence.

[0182] Example 6: Successful expression of α-GAL A using other vector serotypes.

[0206] To determine whether rAAV serotypes correspond to α-GAL A expression and secretion, HepG2 cells were transduced with FD802-4 containing serotypes rAAV1, rAAV2, ..., or rAAV10, and α-GAL A activity was measured. From the WB data, α-GAL A expression (lysate) and secretion (culture medium) from HepG2 cells delivered by rAAV encapsulated with various serotype capsids were confirmed (Figure 19A). Activity analysis of the secreted α-GAL A showed that the α-GAL A produced by these vectors was processed into the active form (Figure 19B), with serotype rAAV6 corresponding to the highest α-GAL A activity. The variability in expression and activity levels among these vectors is thought to be due to packaging efficiency. Various serotypes of AAV may be used to introduce α-GAL A expression into human cells for therapeutic purposes.

[0183] Example 7: α-GAL A fused with TAT p47-57 did not alter the activity of secreted α-GAL A.

[0207] To determine whether α-GAL A activity can be improved by incorporating the nucleotide sequence of the cell-permeable peptide TAT p47-57 into an rAAV vector containing the hGLA nucleotide sequence, HepG2 cells were transfected with plasmids containing the TAT nucleotide sequence (tat, wild-type nucleotide sequence expressing TAT p47-57), wtSP co, and hGLA cov1, as well as plasmids containing hGLA cov1 and wtSP co. The activity of the secreted α-GAL A was quantified. Quantification of α-GAL A activity using culture medium revealed that hGLA fused with TAT p47-57 (Ctat-GLAco) produced α-GAL A at a lower activity level compared to hGLA not fused with TAT p47-57 (GLAco) (Figure 20A). On the other hand, quantification of α-GAL A activity using equal amounts of α-GAL A produced by the two vectors showed no difference between the two vectors (Figure 20B). This indicates that the fusion of TAT p47-57 and hGLA reduced the expression or secretion of the transgene, but did not affect α-GAL A activity.

[0184] Example 8: α-GAL A fused with TAT p47-57 can increase the tissue permeability of α-GAL A without altering α-GAL A expression or glycosylation, or lyso-Gb3 clearance.

[0208] To determine whether α-GAL A fused with TAT p47-57 increases α-GAL A activity compared to α-GAL A not fused with TAT, Fabry mice were administered either low or medium doses of FD802-3 or FD802-5 (where FD802-5 contained the TAT nucleotide sequence), as shown in Figure 1B. Mice injected with FD802-3 or FD802-5 vectors expressing TAT p47-57 showed higher levels of α-GAL A activity compared to untreated Fabry mice, while the FD802-5 vector showed dose-dependent levels of α-GAL A activity, as shown in Figure 21A. Similarly, α-GAL A activity levels were measured in the liver, heart, kidneys, and spleen of Fabry mice 14 weeks after injection of low or medium doses of FD802-3 or FD802-5. Mice injected with a vector expressing TAT p47-57 (FD802-5) showed a slight decrease in α-GAL A activity in the liver, but a slight increase in α-GAL A activity levels in the heart and kidney (Figure 21B). Immunohistochemical data from tissue samples at 14 weeks post-injection confirmed α-GAL A expression in the liver of mice administered a moderate dose of the vector. In the hearts of Fabry mice injected with FD802-5, improved α-GAL A protein levels were observed compared to Fabry mice injected with FD802-3 (Figure 22). This suggests that administering a vector expressing α-GAL A fused with TAT p47-57 may improve the tissue permeability of α-GAL A depending on the tissue type.

[0185]

[0209] To determine whether the fusion of TAT p47-57 to α-GAL A alters α-GAL A expression or glycosylation in the liver, liver samples from Fabry mice injected with a moderate dose of FD802-3 or FD802-5 were evaluated for α-GAL A expression and glycosylation 14 weeks later. Mice injected with the FD802-3 or FD802-5 vector showed increased α-GAL A expression compared to untreated mice, as shown in Figure 23A. However, there was no noticeable difference in expression between vectors expressing the TAT peptide and those not expressing it. Glycosylation analysis of liver proteins 14 weeks post-injection showed that α-GAL A produced by both vectors was glycosylated and processed into its active form (Figure 23B). This suggests that the fusion of TAT p47-57 to α-GAL A did not inhibit α-GAL A expression or glycosylation.

[0186]

[0210] To determine whether the fusion of TAT p47-57 to α-GAL A is effective in reducing lyso-Gb3, mice were treated with low or medium doses of FD802-3 and FD802-5, and lyso-Gb3 levels were assessed. At both 6 weeks (Figure 24A) and 14 weeks (Figure 24B) post-injection, plasma lyso-Gb3 levels decreased with all administrations of FD802-3 and FD802-5, with FD802-5 showing a dose-dependent effect. Medium-dose FD802-5 injection resulted in similar lyso-Gb3 removal compared to FD802-3. In mice injected with low or medium dose vectors, residual substrate levels were less than 5% of PBS even at the early post-injection stage (6 weeks) (Figure 24A).

[0187]

[0211] Liver tissue from Fabry mice treated with medium doses of FD802-3 or FD802-5 was evaluated using H&E staining. As shown in Figure 25, no adverse effects on the liver were observed. This suggests the safety of the treatment in Fabry mice, as administration of α-GAL A fused with TAT p47-57 did not alter tissue integrity.

[0188] Example 9:

[0212] 1.Clinical dose

[0213] (1) Patients with Fabry disease were recruited for an investigator-initiated clinical trial (IIT). A single intravenous dose of rAAV expressing α-GAL A fused to sp21 at 1 × 10¹³ vg / kg was administered to adult male patients with classical Fabry disease. The plan was to enroll 1 to 3 patients in the treatment group, and to date, 2 patients have been enrolled. Enzyme activity tests were performed weekly from 1 to 8 weeks after administration, every two weeks from 8 to 12 weeks, every four weeks from 12 to 32 weeks, and once each at 40 and 52 weeks after administration. If patients entered the long-term follow-up period, tests were performed once each at 1.5, 2, 3, 4, and 5 years after drug administration. One week after administration, the plasma enzyme activity of the two subjects was above normal, with the peak value reaching 3.87 to 15.74 times the plasma enzyme activity of normal individuals, and remained stable. The substrate Gb3 was effectively removed from the plasma, and plasma Gb-3 concentrations decreased by 56.22% and 44.90% in two patients at 16 weeks and 8 weeks after administration, respectively.

[0189]

[0214] (2) For rAAV expressing α-GAL A fused to sp21, 1 to 3 subjects are to be enrolled in the 3 × 10¹³ vg / kg dose group, but no subjects have been enrolled yet.

Claims

1. A modified peptide comprising, or consisting of, a portion of human alpha-galactosidase A (α-GAL A) or a functional variant thereof, and a portion of a tissue plasminogen activator signal peptide (sp21, sp18, sp20, or sp22) or a functional variant thereof.

2. The modified peptide according to claim 1, further comprising a portion of a cell-permeable peptide or a functional variant thereof.

3. A modified peptide comprising, or consisting of, a portion of human alpha-galactosidase A (α-GAL A) or its functional variant, a portion of the signal peptide of α-GAL A (wt sp) or its functional variant, and a portion of the cell-permeable peptide or its functional variant.

4. A modified peptide according to any one of claims 1 to 3, wherein a portion of α-GAL A or a functional variant thereof contains a peptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:

25.

5. A modified peptide according to any one of claims 1 to 4, wherein a part of sp21, sp18, sp20, or sp22 or a functional variant thereof contains a peptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NOs: 22, 19, 21, or 23.

6. A modified peptide according to any one of claims 2 to 5, wherein a portion of the cell-permeable peptide or a functional variant thereof comprises a peptide sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NOs: 24, 52, 53, or 54.

7. A modified peptide according to any one of claims 1 to 6, wherein a portion of sp21 or a functional variant thereof is fused to the N-terminus of a portion of α-GAL A or a functional variant thereof.

8. A modified peptide according to any one of claims 2 to 7, wherein a portion of the cell-permeable peptide or a functional variant thereof is fused to the C-terminus of a portion of α-GAL A or a functional variant thereof.

9. A viral vector comprising a nucleotide sequence encoding the modified peptide described in any one of claims 1 to 8.

10. The viral vector according to claim 9, further comprising a promoter.

11. A viral vector comprising an expression cassette comprising a first nucleotide sequence encoding a portion of α-GAL A or a functional variant thereof, a second nucleotide sequence encoding a portion of sp21 or a functional variant thereof, a third nucleotide sequence optionally encoding a portion of a cell-permeable peptide or a functional variant thereof, and a promoter.

12. A viral vector comprising an expression cassette comprising a first nucleotide sequence encoding a portion of α-GAL A or a functional variant thereof, a fourth nucleotide sequence encoding a portion of wt sp or a functional variant thereof, a third nucleotide sequence optionally encoding a portion of a cell-permeable peptide or a functional variant thereof, and a promoter.

13. The viral vector according to claim 11, wherein the second nucleotide sequence is located at 5' of the first nucleotide sequence, and / or the third nucleotide sequence is located at 3' of the first nucleotide sequence.

14. The viral vector according to claim 12, wherein the fourth nucleotide sequence is located at 5' of the first nucleotide sequence, and / or the third nucleotide sequence is located at 3' of the first nucleotide sequence.

15. The viral vector according to any one of claims 9 to 14, wherein the nucleotide sequence encoding a part of α-GAL A or a functional variant thereof has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 25 or 26.

16. The viral vector according to any one of claims 9 to 15, wherein a nucleotide sequence encoding a part of sp21, sp18, sp20, or sp22 or a functional variant thereof has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs. 22, 19, 21, or 23.

17. The viral vector according to any one of claims 9 to 16, wherein a nucleotide sequence encoding a portion of wt sp or a functional variant thereof has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:

51.

18. The viral vector according to any one of claims 9 to 17, wherein a nucleotide sequence encoding a portion of a cell-permeable peptide or a functional variant thereof has at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NOs: 24, 52, 53, or 54.

19. The viral vector according to any one of claims 10 to 18, wherein the promoter is a tissue-specific promoter.

20. The viral vector according to claim 19, wherein the tissue-specific promoter is a liver-specific promoter, a heart-specific promoter, or a kidney-specific promoter.

21. The viral vector according to claim 20, wherein the liver-specific promoter is the TTR promoter.

22. A viral vector according to any one of claims 9 to 21, further comprising at least one reverse-terminal repeat (ITR) nucleotide sequence, an intron, and a polyadenylated (poly-A) nucleotide sequence.

23. A viral vector according to any one of claims 9 to 22, which is a modified recombinant adeno-associated virus (rAAV) vector.

24. A method for treating Fabry disease or reducing one or more symptoms of Fabry disease in a subject, comprising administering to the subject a modified peptide according to any one of claims 1 to 8, a viral vector according to any one of claims 9 to 23, or a pharmaceutical composition comprising a modified peptide according to any one of claims 1 to 8 or a viral vector according to any one of claims 9 to 23.

25. A method for increasing α-Gal A expression in one or more target tissues, comprising administering to the target a modified peptide according to any one of claims 1 to 8, a viral vector according to any one of claims 9 to 23, or a pharmaceutical composition comprising the modified peptide according to any one of claims 1 to 8 or the viral vector according to any one of claims 9 to 23.

26. A method for reducing sphingolipid accumulation in one or more target tissues, comprising administering to the target a modified peptide according to any one of claims 1 to 8, a viral vector according to any one of claims 9 to 23, or a pharmaceutical composition comprising a modified peptide according to any one of claims 1 to 8 or a viral vector according to any one of claims 9 to 23.

27. The method according to claim 24 or 25, wherein the subject is suffering from Fabry disease or has symptoms of Fabry disease.

28. The method according to claim 25 or 26, wherein one or more target tissues are selected from the group consisting of liver tissue, heart tissue, or kidney tissue.

29. The method according to any one of claims 24 to 28, wherein a modified peptide according to any one of claims 1 to 8, a viral vector according to any one of claims 9 to 23, or a pharmaceutical composition comprising the modified peptide according to any one of claims 1 to 8 or the viral vector according to any one of claims 9 to 23 is administered to a subject.

30. The method according to any one of claims 24 to 28, wherein a pharmaceutically acceptable composition comprising a modified peptide according to any one of claims 1 to 8, a viral vector according to any one of claims 9 to 23, or a modified peptide according to any one of claims 1 to 8 or a viral vector according to any one of claims 9 to 23 is administered to a subject once every 12 months.

31. The method according to any one of claims 24 to 28, wherein a modified peptide according to any one of claims 1 to 8, a viral vector according to any one of claims 9 to 23, or a pharmaceutical composition comprising the modified peptide according to any one of claims 1 to 8 or the viral vector according to any one of claims 9 to 23 is administered to a subject by systemic administration.

32. The method according to any one of claims 24 to 28, wherein a viral vector according to any one of claims 9 to 23 or a pharmaceutical composition comprising a viral vector according to any one of claims 9 to 23 is administered to a subject once or more times at a dose of 0.5E+10vg / kg to 7E+14vg / kg.

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