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WO2025111316A1 - Lentiviral gene therapy of mucopolysaccharidosis iva - Google Patents

Lentiviral gene therapy of mucopolysaccharidosis iva Download PDF

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WO2025111316A1
WO2025111316A1 PCT/US2024/056623 US2024056623W WO2025111316A1 WO 2025111316 A1 WO2025111316 A1 WO 2025111316A1 US 2024056623 W US2024056623 W US 2024056623W WO 2025111316 A1 WO2025111316 A1 WO 2025111316A1
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hgalns
galns
cells
cbh
promoter
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Shunji Tomatsu
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Nemours Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/06—Sulfuric ester hydrolases (3.1.6)
    • C12Y301/06004—N-Acetylgalactosamine-6-sulfatase (3.1.6.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the field relates to the treatment of mucopolysaccharidosis type IVA (MPS IVA).
  • MPS IVA mucopolysaccharidosis type IVA
  • Provided herein are methods and compositions for treatment of MPS IVA involving novel lentiviral vectors (LVs) gene therapy to produce and secrete active N-acetylgalactosamine-6- sulfate sulfatase (GALNS).
  • LVs novel lentiviral vectors
  • Mucopolysaccharidosis type IVA (MPS IVA; Morquio A Syndrome) is an autosomal recessive lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-6- sulfate sulfatase (GALNS) (Khan, et al., Mol Genet Metab., 2017; 120(l-2):78-95).
  • GAGs glycosaminoglycans
  • C6S chondroitin 6-sulfate
  • KS keratan sulfate
  • Other clinical manifestations of the disease can include hearing loss, heart valve involvement, and corneal opacity. Over 200 different mutations have been identified in patients, and the prevalence in the United States is approximately 1 in 250,000.
  • ERT Enzyme replacement therapy
  • HSCT hematopoietic stem cell transplantation
  • various surgical interventions are currently available as supportive therapy for patients with MPS IVA in clinical practice.
  • ERT Enzyme replacement therapy
  • HSCT hematopoietic stem cell transplantation
  • various surgical interventions are currently available as supportive therapy for patients with MPS IVA in clinical practice.
  • ERT elosulfase- alpha
  • ERT the current standard of care, results in partial improvement in soft tissue pathology and activity of daily living (ADL) of patients with MPS IVA; however, these therapies provide very limited impact in bone and cartilage due to the avascular character of these lesions.
  • ERT Current limitations of ERT include: i) weekly injections for 5-6 hours are required, ii) drug is rapidly cleared from the circulation, iii) the treatment cost is very expensive ($500,000 per year per patient), and v) the drug shows limited penetration to bone (Algahim and Almassi, Ther Clin Risk Manag., 2013;9:45-53;
  • HSCT may provide a better impact than ERT on bone
  • this cellbased therapy may not be applicable to all patients because of limited matched donors, the agelimit for effective treatment, a lack of well-trained facilities, the mortality risk of the procedure such as graft-versus-host disease (GVHD), infection, and other complications (Tomatsu et al., Drug Des Devel Then, 2015; 9: 1937-1953).
  • GVHD graft-versus-host disease
  • a novel drug for MPS IVA particularly a novel drug for treating skeletal dysplasia in patients with MPS IVA, is urgently required.
  • ERT enzyme replacement therapy
  • Vimizin® elosulfase alfa
  • HSCT hematopoietic stem cell transplantation
  • ERT and HSCT pharmacological chaperones
  • GT gene therapy
  • AAV adeno- associated viral vectors
  • CRISPR/Cas9 clustered regularly interspaced palindromic repeats
  • nanoparticles Sawamoto and Tomatsu, Int J Mol Sci 20 (2019) 4139; Almeciga-Diaz, et al., J Med Chem 62 (2019) 6175-6189; Leal, et al., Int J Mol Sci 24 (2023) 16148; Sawamoto, et al., Mol Ther Methods Clin Dev 18 (2020) 50-61; Leal, et al.. Scientific Rpts 2022 12: 1 1-15).
  • AAV gene therapy is an option for many genetic disorders; however, the episomal characteristic of AAV vectors has been affected by dilution factors over time.
  • Lentiviral (LV) mediated hematopoietic stem cell gene therapy has been attempted in other lysosomal storage diseases (LSDs) and blood-related disorders.
  • LSDs lysosomal storage diseases
  • off-target effects and potential immune reactions against infused LVs have been observed when delivered via in vivo direct infusion.
  • the present invention involves the use of novel lentiviral vectors (LVs) HSC-GT to produce and secrete the active GALNS enzyme supraphysiologic levels in cells of different organs of a subject diagnosed with MPS IVA.
  • MPS IVA mucopolysaccharidosis type IVA
  • rLVs recombinant lentiviral vectors
  • hGALNS human N-acetylgalactosamine-6-sulfate sulfatase
  • rLVs that can be used in the gene therapy methods, methods of making such rLVs, as well as polynucleotides, plasmids, and cells that can be used for making such rLVs.
  • a recombinant lentivirus has a human N- acetylgalactosamine-6-sulfate sulfatase (hGALNS) gene stably integrated in the LV genome (LV-hGALNS).
  • hGALNS human N- acetylgalactosamine-6-sulfate sulfatase
  • a hematopoietic stem cell transduced with LV- hGALNS.
  • a pharmaceutical composition comprising an LV-hGALNS provided herein and a pharmaceutically acceptable carrier.
  • a recombinant lentivirus (LV) comprising a recombinant LV genome comprising a human N-acetylgalactosamine-6-sulfate sulfatase (hGALNS) expression cassette wherein said hGALNS expression cassette comprises a promoter operably linked to a nucleotide sequence encoding the hGALNS protein.
  • LV lentivirus
  • the LV -hGALNS wherein the promoter of the hGALNS expression cassette is a tissue-specific promoter.
  • the LV -hGALNS wherein the tissue-specific promoteris a collagen-specific promoter; or comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90% identical, at least 95%, at least 98%, or at least 100% identical to SEQ ID NO: 6.
  • the LV -hGALNS wherein the expression cassette comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90% identical, at least 95%, at least 98%, or at least 100% identical to SEQ ID NO: 10.
  • the LV -hGALNS wherein the promoter of the hGALNS expression cassette is a ubiquitous promoter.
  • the ubiquitous promoter comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90% identical, at least 95%, at least 98%, or at least 100% identical to SEQ ID NO: 5.
  • the LV -hGALNS wherein the expression cassette comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90% identical, at least 95%, at least 98%, or at least 100% identical to SEQ ID NO: 8.
  • the LV -hGALNS wherein the nucleotide sequence encoding hGALNS or the nucleotide sequence encoding the fusion protein is codon-optimized.
  • a pharmaceutical composition comprising the hematopoietic stem cell transduced with a LV -hGALNS.
  • a method for treating a human subject diagnosed with mucopolysaccharidosis type IVA comprising administering to the human subject the LV-hGALNS or the hematopoietic stem cell transduced with LV-hGALNS or pharmaceutical composition comprising the hematopoietic stem cell transduced with LV-hGALNS.
  • a method for treating a human subject diagnosed with MPS IVA comprising delivering to the bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle, and/or heart valve of said human subject a therapeutically effective amount of a fusion protein that is hGALNS by administering to the human subject the LV- hGALNS or the hematopoietic stem cell transduced with LV-hGALNS or pharmaceutical composition comprising the hematopoietic stem cell transduced with LV-hGALNS.
  • FIG. 1 Schematic diagram of in vitro experiments. AB+/-: With or without Geneticin antibiotic.
  • FIG. 2 Schematic diagram of the experiment to confirm the uptake of secreted GALNS enzyme by MPS IVA patient’s fibroblasts.
  • FIGS. 3A and 3B Bar graphs depicting enzyme activity in fibroblasts derived from the MPS IVA patient.
  • A. Media enzyme activity. The GALNS enzyme activities of each vector were compared to the wild-type level through two-way ANOVA analysis. The inventors have shown the statistical significance of LV-COL2Al-hGALNS over time, but other vectors showed statistical significance as well at variations (p ⁇ 0.05), especially on day 8 (data not shown).
  • FIGS. 4A-4C Graphs depicting enzyme activity in media of HepG2 cultures treated under different MOIs of LVs.
  • FIGS. 5A-5C Bar graphs depicting intracellular enzyme activity of HepG2 cells treated under different MOIs of LVs.
  • FIGS. 6A-6E Bar graphs depicting enzyme activity in media of HEK293T cells treated under different MOIs with LVs.
  • FIGS. 7A-7E Bar graphs depicting intracellular enzyme activity of HEK293T cells treated under different MOIs with LVs.
  • FIG. 8 Bar graph depicting KS levels of MPS IVA fibroblasts after administering LVs. Comparison between untreated and treated groups was made according to Tukey multiple comparison test and two-way ANOVA (*: ⁇ 0.05, ***: ⁇ 0.001). Furthermore, each treatment was compared with wild-type, in which no statistical difference was found, while the inventors compared the cell collection days (8 and 30) via Tukey multiple comparison test and two-way ANOVA. Each treatment significantly reduced KS levels on days 8 and 30 (p ⁇ 0.0001).
  • FIGS. 9A-9C Bar graphs depicting vector copy numbers in MPS IVA fibroblasts.
  • A MPS IVA fibroblasts (MOI 20). Copy numbers were compared to untreated levels and each other using one-way ANOVA and Tukey multiple comparison test. Since the VCN is the only factor affected by the treatments, the inventors used one-way ANOVA.
  • B HEK293T (MOI 5,10,15, respectively)
  • C HepG2 cells (MOI 5,10,15, respectively).
  • Statistical methods used in Fig. 9B and C were Tukey multiple comparison and two-way ANOVA tests since the VCNs were affected by both LV treatments and MOIs.
  • FIGS. 10A-10B Lysosomal mass in MPS IVA fibroblasts treated with LVs; LV- CDl lb-hGALNS, LV-CBh-hGALNS, LV-CBh-hGALNSco, and LV-COL2Al-hGALNS at MOI 15.
  • A. A representative histogram from wild-type, untreated, and treated MPS IVA patient’s skin fibroblasts.
  • FIGS. 11A-11B Graphical depiction of Enzyme activity levels following uptake experiments.
  • A Media enzyme activities before and after adding into culture media in MPS IVA fibroblasts.
  • FIGS. 12A-12B Immunohistochemistry analysis of GALNS enzyme expression.
  • A IHC pictures from each experimental group.
  • FIG. 13 Bar graph depicting statistical analysis of KS GAG-positive areas in immunohistochemistry. Tukey multiple test and one-way ANOVA were used (**: ⁇ 0.005, ****:
  • FIG. 14A-14C Schematic of general workflow of Ex vivo gene therapy procedures. Isolation and modifications of HSCs removed from MPS IVA mice (A). Lentiviral vector constructs contained either the CBh or COL2A1 promoter driving native human GALNS (B). Isolation and transplantation of wild-type HSCs from healthy donors (C). WT: Wild-type, VSVG: Vesicular stomatitis virus G protein.
  • FIG. 15A-15H Graphs depicting GALNS enzyme activity in MPS IVA patients’ fibroblasts and media (A-B) and the percentage of cell survival 8 days LV-post-transduction (C), the GALNS enzyme activity in MPS IVA mouse HSCs 24h and 15 days and LVs-transduced wild-type HSCs (overexpression experiments) 15 days post-transduction and (D), number of CFU colonies 7 days post-transduced HSCs (E), VCNs; Gains'" HSCs 24h vs 15 days posttransduction, wild-type HSCs 15 days post-transduction, and MPS IVA fibroblasts 8 days posttransduction (F), and the GALNS enzyme activity of pooled CFU colonies (G), VCN of pooled CFU colonies originated from Gains'" HSCs and wild-type HSCs (H).
  • One-way ANOVA with Tukey’s post-hoc test *: ⁇ 0.05, **: ⁇ 0.005, ***: ⁇
  • FIGS. 16A-16I Graphs depictingeEnzyme activities in tissues after LV-modified HSCs transplantation and wild-type allo-HSCT (A-G); brain (A), trachea (B), lung (C), heart (D), liver (E), spleen (F) and bone (G) at 16 weeks old, and in plasma and WBCs over 16 weeks (H-I)
  • A-G brain
  • A trachea
  • B lung
  • C heart
  • D liver
  • E spleen
  • G bone
  • H-I One-way ANOVA with Tukey’s post-hoc test for plasma and WBCs week-by-week
  • *: LV-CBh-hGALNS vs. wild-type *: ⁇ 0.05, **: ⁇ 0.005, ***: ⁇ 0.001, ⁇ 0.0001 and #:
  • FIGS. 17A-17D Bar graphs depicting mono-sulfated keratan sulfate (KS) concentrations in plasma (A), WBCs (B), and bone (humerus) (C) at 16 weeks. VCN in the liver 16 weeks post-transplantation (D).
  • KS mono-sulfated keratan sulfate
  • FIGS. 18B-18C Bar graphs depicting Bone pathology of lentiviral vector (LV-CBh- hGALNS or LV-COL2Al-hGALNS)-treated and control groups. Chondrocyte vacuolization in the tibia growth plate (B) and chondrocyte column structure in the tibia growth plate (C).
  • FIGS. 19A-19X Bar graphs depicting bone morphometric analysis. Trabecular bone morphology (A-L) and cortical bone morphology (M-X). One-way ANOVA with Tukey’s post- hoc test; *: ⁇ 0.05, **: ⁇ 0.005, ***: ⁇ 0.001.
  • FIG. 20 Schematic of workflow of in vivo experiments
  • FIGS. 21A-21D Bar graphs depicting GALNS enzyme activity in (A) plasma, (B) liver, (C) heart, and (D) bone of treated and untreated mice at 16 weeks old.
  • FIGS. 22A-22C Bar graphs depicting plasma KS levels in plasma of (A) low-dose treated 4-week-old, (B) low-dose treated newborn, and (C) high-dose treated newborn mice.
  • FIGS. 23A-23C GAG level (KS) in liver (A), muscle (B) and bone (humerus) (C) samples treated with a dose of IxlO 11 TU/kg.
  • FIG. 24 Bar graph depicting VCN in liver samples.
  • FIGS. 25A-25B Bar graph depicting liver toxicity levels (A) AST and (B) ALT levels of mice treated with a dose of IxlO 11 TU/kg
  • FIG 26 Construct map for
  • FIG 27 Construct map for
  • FIG. 28 Construct map fo
  • FIG. 29. Construct map fo
  • FIG. 30 Construct map fo
  • MPS IVA is one of the lysosomal storage disorders with no effective cure to date, which affects multiple tissues, including bone and cartilage (Khan,S., et al. Mol Genet Metab 2017, 120, 78-95; Sawamoto, K., et al., IntJMol Set 2020, 21; Melbouci, M. et al., Mol Genet Metab 2018, 124, 1-10).
  • ERT and HSCT have little impact on bone, these treatments cannot fully recover hard-to-reach tissues.
  • AAV gene therapy is a highlighted option for many genetic disorders; however, the episomal characteristic of AAV vectors has been affected by dilution factors over time.
  • LV gene therapy provides stable and permanent transgene expression, a one-time treatment.
  • recombinant LVs for the treatment of MPS IVA The LV-hGALNS disclosed herein show effectiveness in increasing GALNS enzyme activity in a variety of cell types.
  • Lentiviral (LV) gene therapy provides stable and permanent transgene expression in a one-time treatment. Described herein is LV-mediated human stem cell gene therapy (HSC-GT) in the treatment of MPS IVA.
  • HSC-GT human stem cell gene therapy
  • Viral vectors have been widely explored in vivo and ex vivo.
  • the first ex vivo gene therapy with a recombinant retroviral vector (LGSN) was tested in the human MPS IVA fibroblasts, increasing GALNS enzyme activity for up to 6 days (Toietta, G., et al., Hum Gene Ther. 12(16):2007-2016, 2001).
  • the effect of CMV and EFla promoters driving the GALNS gene was tested with the co-expression of SUMF1 in HEK293 cells.
  • HEK293 cells transfected with EFla-pIRES- GALNS had a normal GALNS enzyme activity by 8 days, while co-transfection with SUMF1 plasmid increased the GALNS enzyme activity nearly 2.6-fold.
  • the study showed that the EFla promoter stably drove the GALNS gene (C.J. Almeciga-Diaz, M.A, et al., Mol Biol Rep 36 (2009) 1863-1870). Nevertheless, 6- or 8- days post-transduction is too short of a period to precisely state whether viral vectors stably and permanently expressed the proteins.
  • the LV-hGALNS viral vectors disclosed herein showed long-term, stable expression of hGALNS in transformed cells.
  • Exemplary LV-hGALNS viral vectors of the present disclosure are represented in the construct maps of FIG. 26, 27, 28, 29 and 30
  • LV-hGALNSs for use in the treatment of MPS IVA in a human subject in need of treatment.
  • These LV-hGALNS comprise a recombinant LV genome encoding for hGALNS.
  • the LV-hGALNS can be administered to an MPS IVA patient, resulting in the synthesis of hGALNS and the delivery of hGALNS to the affected tissues, such as bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle, and/or heart valve, thereby improving pathology, and preventing the progression of the disease.
  • polynucleotides comprising an hGALNS expression cassette as described herein.
  • plasmids and cells comprising a polynucleotide provided herein for making the LV-hGALNS for use with the methods and compositions provided herein.
  • methods for treating a human subject diagnosed with mucopolysaccharidosis type IVA MPS IVA.
  • the method comprises administering an LV-hGALNS described herein to the human subject.
  • the method comprises delivering hGALNS to the affected tissue(s) in the human subject.
  • the method comprises delivering hGALNS through the delivery of transduced cells to the human subject.
  • compositions and kits comprising LV- hGALNS and/or transduced cells described herein.
  • the manufacture, composition, and method of use of the LV-hGALNS can be modified such that it still results in the delivery of the hGALNS enzyme to the bone, cartilage, ligament, meniscus, and/or heart valve of a human subject as a treatment for MPS IVA.
  • LVs useful for the treatment of MPS IVA in a human subject in need thereof which LVs comprise an hGALNS expression cassette (LV-hGALNS).
  • LV-hGALNS hGALNS expression cassette
  • the LVs of the present invention may be used in HST-GT for patients with MPS-IVA.
  • LV-hGALNS resulting in stable long-term GALNS enzyme activity in transfected hematopoietic stem cells.
  • an LV-hGALNS comprising an hGALNS expression cassette, said hGALNS expression cassette comprising a nucleotide sequence encoding a hGALNS transgene.
  • the transgene encodes a fusion protein that is hGALNS fused to an acidic oligopeptide.
  • the hGALNS expression cassette may further comprise a nucleotide sequence encoding a tissue-specific promoter, wherein the nucleotide sequence encoding the tissue-specific promoter is operably linked to the nucleotide sequence encoding the hGALNS protein.
  • the nucleotide sequence encoding hGALNS or the hGALNS portion of the fusion protein comprises the sequence of SEQ ID NO: 1 or 2. In certain embodiments, the nucleotide sequence encoding hGALNS or the hGALNS portion of the fusion protein is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence set forth in SEQ ID NO: 1 or 2.
  • the nucleotide sequence encoding the fusion protein comprises the sequence of SEQ ID NO: 1 or 2. In certain embodiments, the nucleotide sequence encoding the fusion protein is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence set forth in SEQ ID NO: 1 or 2.
  • the nucleotide sequence encoding hGALNS or the hGALNS portion of the fusion protein comprises the cDNA sequence of hGALNS.
  • the nucleotide sequence encoding hGALNS or the hGALNS portion of the fusion protein is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the cDNA sequence of hGALNS.
  • the nucleotide sequence encoding the fusion protein comprises the cDNA sequence of the fusion protein. In certain embodiments, the nucleotide sequence encoding the fusion protein is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the cDNA sequence of the fusion protein.
  • the nucleotide sequence encoding hGALNS or the nucleotide sequence encoding the fusion protein is codon-optimized, for example, via any codonoptimization technique known to one of skill in the art (see, e.g., review by Quax et al., 2015, Mol Cell 59: 149-161).
  • CpG sites are depleted in the nucleotide sequence encoding hGALNS or the nucleotide sequence encoding the fusion protein.
  • Acidic oligopeptides have high binding affinities for hydroxyapatite, a major component of bones and cartilage.
  • the term “acid oligopeptide,” as used herein, refers to an oligopeptide with a repeating amino acid sequence of glutamic acid (E) and/or aspartic acid (D) residues.
  • the number of amino acid residues in an acidic oligopeptide may be, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In specific embodiments, the number of amino acid residues in an acidic oligopeptide is 4-8. In specific embodiments, the number of amino acid residues in an acidic oligopeptide is 6-8. In a specific embodiment, the number of amino acid residues in an acidic oligopeptide is 6. In another specific embodiment, the number of amino acid residues in an acidic oligopeptide is 8.
  • the acidic oligopeptide is D8 (i.e., an oligopeptide with an amino acid sequence of eight aspartic acid residues.
  • the acidic oligopeptide is E6 i.e., an oligopeptide with an amino acid sequence of six glutamic acid residues. The E6 sequence is described in Tomatsu et al., 2010, Molecular Therapy, 18(6): 11094-1102, which is incorporated by reference herein in its entirety.
  • the acidic oligopeptide is fused to the N-terminus of hGALNS. In another embodiment, the acidic oligopeptide is fused to the C-terminus of hGALNS. [0080] In a specific embodiment, the acidic oligopeptide is fused directly to hGALNS, with no intervening amino acid sequence. In another specific embodiment, the acidic oligopeptide is fused to hGALNS via a linker amino acid sequence e.g., an amino acid sequence that is 1-10, 2- 8, or 4-6 amino acid residues in length).
  • a linker amino acid sequence e.g., an amino acid sequence that is 1-10, 2- 8, or 4-6 amino acid residues in length).
  • the hGALNS enzyme can be delivered to the lysosomes in the bone and cartilage area to improve bone and cartilage pathology.
  • the hGALNS expression cassette described herein comprises components that modulate gene delivery or gene expression (e.g., “expression control elements”). In certain embodiments, the hGALNS expression cassette described herein comprises components that modulate gene expression. In certain embodiments, the hGALNS expression cassette described herein comprises components that influence binding or targeting cells. In certain embodiments, the hGALNS expression cassette described herein comprises components that influence the localization of the hGALNS within the cell after uptake. In certain embodiments, the hGALNS expression cassette described herein comprises components that can be used as detectable or selectable markers, e.g., to detect or select cells that have taken up the hGALNS expression cassette.
  • the hGALNS expression cassette described herein comprises nucleotide sequence(s) encoding one or more promoters, at least one of which is operably linked to the nucleotide sequence encoding hGALNS or the fusion protein that is hGALNS fused to an acidic oligopeptide.
  • the promoter can be a constitutive promoter. In alternate embodiments, the promoter can be an inducible promoter. [0083] In certain embodiments, the promoters are selected from known housekeeping promoters, including PGK, CMV, EFla, MND, MCU3, SFFV, and CBh.
  • the promoters are selected from tissue-specific promoters, including CDl lb, ALB, TBG, MHC, MLC2v, and cTnT.
  • the promoter is the COL2A1 promotion, a collagen type II specific promoter expressed in connective tissues and cartilage.
  • the promoter is the CBh promoter, which comprises CMV early enhancer fused to a modified chicken [Lactin promoter.
  • the promoter is the CD1 lb promotion, a myeloid cell-specific promoter from the alpha chain of Mac-1 integrin.
  • the promoter is a ubiquitous promoter, such as CBh.
  • the promoter is a collagen type II promoter, such as COL2A1, to provide for expression in cartilage and other connective tissues.
  • the promoter is a hematopoietic promoter, such as CD1 lb, which can provide expression in hematopoietic stem cells.
  • the promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5. In certain embodiments, the promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6.
  • the promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:7.
  • the liver-specific promoter is SEQ ID NO:5.
  • the liver-specific promoter is SEQ ID NO:6.
  • the liver-specific promoter is SEQ ID NO:7.
  • the promoter comprises one or more elements that enhance the expression of hGALNS or the fusion protein. In certain embodiments, the promoter comprises a TATA box.
  • one or more promoter elements can be inverted or moved relative to one another.
  • the elements of the promoter can be positioned to function cooperatively.
  • the elements of the promoter can be positioned to function independently.
  • the hGALNS expression cassette described herein comprises one or more ubiquitous promoters.
  • the hGALNS expression cassette provided herein comprises one or more tissue-specific promoters.
  • the hGALNS expression cassette comprises a fusion protein that is hGALNS fused to an acidic oligopeptide (preferably D8).
  • the hGALNS expression cassette described herein comprises one or more untranslated regions (UTRs), e.g., 3’ and/or 5’ UTRs.
  • the UTRs are optimized for the desired level of protein expression.
  • the UTRs are optimized for the mRNA half-life of the hGALNS.
  • the UTRs are optimized for the stability of the mRNA of the hGALNS.
  • the UTRs are optimized for the secondary structure of the mRNA of the hGALNS.
  • compositions comprising an LV-hGALNS provided herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be prepared as individual, single unit dosage forms.
  • the pharmaceutical compositions provided herein can be formulated for, for example, parenteral, subcutaneous, intramuscular, intravenous, intraperitoneal, intranasal, intrathecal, or transdermal administration.
  • the pharmaceutical composition is formulated for intravenous administration.
  • a suitable pharmaceutically acceptable carrier e.g., for intravenous administration and transduction in liver cells
  • kits comprising a pharmaceutical composition described herein, contained in one or more containers.
  • the containers that the pharmaceutical composition can be packaged in can include but are not limited to, bottles, packets, ampoules, tubes, inhalers, bags, vials, and containers.
  • the kit comprises instructions for administering the pharmaceutical administration.
  • the kit comprises devices that can be used to administer the pharmaceutical composition, including, but not limited to, syringes, needle-less injectors, drip bags, patches, and inhalers.
  • hematopoietic stem cells transduced with an LV containing a hGALNS expression cassette as described herein.
  • hematopoietic stem cells comprising an hGALNS expression cassette.
  • said hGALNS expression cassette comprises a nucleotide sequence encoding a transgene, such as the transgene encoding a fusion protein that is hGALNS.
  • the hGALNS expression cassette may further comprise a nucleotide sequence encoding a collagenspecific promoter (for example, a COL2A1 promoter), wherein the nucleotide sequence encoding the collagen-specific promoter is operably linked to the nucleotide sequence encoding the fusion protein.
  • the collagen-specific promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6.
  • the expression cassette comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 10.
  • said hGALNS expression cassette comprises a nucleotide sequence encoding a transgene, such as the transgene encoding a fusion protein that is hGALNS.
  • the hGALNS expression cassette may further comprise a nucleotide sequence encoding a ubiquitous promoter (for example, a COL2A1 promoter), wherein the nucleotide sequence encoding the ubiquitous promoter is operably linked to the nucleotide sequence encoding the fusion protein.
  • the ubiquitous promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:5.
  • the expression cassette comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 8.
  • lentivirus packaging of one or more genes of interest may be performed through vector packaging vendors.
  • the LV-hGALNS provided herein were prepared through the lentivirus packaging service, Vector Builder.
  • cell culture assays can be used to measure hGALNS expression from a cell transduced with the LV-hGALNS described herein, thus indicating, e.g., the potency of the LV-hGALNS.
  • Cells utilized for the assay can include, but are not limited to, A549, WEHI, 10T1/2, BHK, MDCK, C0S1, COS7, BSC 1 , BSC 40, BMT 10, VERO, W138, HeLa, HEK293, HEK293-T, HuH7, Saos, C2C12, L, HT1080, HepG2, primary fibroblast, hepatocyte, and myoblast cells.
  • the cells utilized in the cell culture assay comprise HuH7 cells.
  • cells transfected with the LV-hGALNS can be analyzed for hGALNS enzyme activity.
  • mice may also be used to assess the expression of hGALNS from an LV- hGALNS described herein and its efficacy.
  • Mouse models for MPS IVA have been described (see, e.g., Tomatsu etal., 2003, Hum Mol Genet 12(24):3349-3358).
  • the mouse model for MPS IVA has a targeted disruption of Exon 2 of mouse GALNS. These mice have no detectable GALNS enzyme activity, and increased levels of GAGs are detected in the urine. At 2 months old, increased storage of GAGs is seen in the reticuloendothelial cells, Kupffer cells, and the sinusoidal cells which line the spleen.
  • vacuolar change is observed in the visceral epithelial cells of glomeruli and cells at the base of heart valves, but it is not present in parenchymal cells such as hepatocytes and renal tubular epithelial cells. Lysosomal storage of GAGs is seen in hippocampal and neocortical neurons and meningeal cells. Keratan sulfate (KS) and chondroitin-6-sulfate (C6S) is increased in the corneal epithelial cells of this mouse model compared to wild type; however, no skeletal indications become evident in the mouse model.
  • KS Keratan sulfate
  • C6S chondroitin-6-sulfate
  • the methods include gene therapy vectors, e.g., the combination of regulatory elements and transgenes that provide increased expression of a functional hGALNS protein.
  • expression may be measured 1) by several proteins (hGALNS) determination assays known to the skilled person, not limited to sandwich ELISA, Western Blot, histological staining, and liquid chromatography tandem mass spectrometry (LC- MS/MS); 2) by several protein activity assays, such as enzymatic assays or functional assays; and/or 3) by several substrate detection assays, not limited to keratan sulfate (KS), glycosaminoglycans (CAG), and/or chondroitin-6-sulfate (C6S) detection, and be determined as efficacious and suitable for human treatment (Hintze, J.P.
  • KS keratan sulfate
  • CAG glycosaminoglycans
  • C6S chondroitin-6-sulfate
  • the invention thus provides methods and gene therapy vectors that increase intracellular hGALNS enzyme activity in tissue cells, e.g. including hepatic, muscle, white blood cells, kidney, lung, spleen cardiac, bone, or cartilage cells of the subject to levels compared to wild-type levels, or that increase intracellular hGALNS enzyme activity to about 2-fold wildtype hGALNS activity levels, or about 5-fold wild-type hGALNS activity levels, about 10-fold wild-type hGALNS activity levels, about 25-fold wild-type hGALNS activity levels, about 40- fold wild-type hGALNS activity levels, about 50-fold wild-type hGALNS activity levels, about 60-fold wild-type hGALNS activity levels, about 70-fold wild-type hGALNS activity levels, about 75-fold wild-type hGALNS activity levels, about 80-fold wild-type hGALNS activity levels, about 85-fold wild-type hGALNS activity levels, about 90-fold wild
  • gene therapy provides a method of increasing hGALNS activity levels in the subject two weeks after administration of the gene therapy as compared to the levels prior administration or the average levels in the untreated subjects. In some embodiments, gene therapy provides a method of increasing hGALNS activity levels in the subject two weeks after administration of the gene therapy. In some embodiments, gene therapy provides a method of increasing hGALNS activity levels in blood or tissues, for example, liver, muscle, kidney, lung, spleen, heart, bone, or cartilage of the subject two weeks after administration of the gene therapy. In some embodiments, the increase in hGALNS activity levels in the subject is measured ten weeks after administration of the gene therapy.
  • the invention also provides methods and gene therapy vectors that reduce blood (e.g., plasma or serum) levels or tissue levels of KS in the subject to levels compared to the levels of KS in untreated wild-type subjects or that reduce KS levels to about 1.1 -fold wild-type KS levels, or about 1.2-fold wild-type KS levels, about 1.3 -fold wild-type KS levels, about 1.4-fold wild-type KS levels, about 1.5-fold wild-type KS levels, about 1.6-fold wild-type KS levels, about 1.7-fold wild-type KS levels, about 1.8-fold wild-type KS levels, about 1.9-fold wild-type KS levels, about 2-fold wild-type KS levels, about 2.5-fold wild-type KS levels, about 3-fold wild-type KS levels, about 3.5-fold wild-type KS levels, or about 4-fold wild-type KS levels, as measured by a KS assay, e.g., using an assay format as described in the Examples herein, or
  • gene therapy provides a method of reducing KS levels in the subject two weeks after administration of the gene therapy.
  • the gene therapy provides a method of reducing tissue levels of KS in the subject two weeks after administration of the gene therapy.
  • the KS assay comprises measurement of mono-sulfated KS in blood or tissue, and the gene therapy provides a method of reducing mono-sulfated KS levels in the subject two weeks after administration of the gene therapy.
  • the method comprises administering LV-hGALNS in conjunction with Hematopoietic Stem Cell Transplant (HSCT).
  • HSCs are collected from a subject’s donor cells (autologous) and are modified with LV-hGALNS in vitro. Patients are pre-conditioned with an applicable method (such as administration of busulfan or fludarabine) to eliminate existing blood cells before transplanting modified HSCs to engraft in the bone marrow.
  • an applicable method such as administration of busulfan or fludarabine
  • the method comprises administering to the human subject an LV- hGALNS described herein, or cells transduced with LV-hGALNS or a pharmaceutical composition described herein.
  • the method comprises delivering to the bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle, and/or heart valve (e.g., delivering to the bone and/or cartilage) of said human subject a therapeutically effective amount of a hGALNS, by administering to the human subject an HSC capable of expressing the hGALNS gene through incorporation of the LV-hGALNS expression casette provided herein.
  • the LV-hGALNS expression cassette comprises a nucleotide sequence encoding a tissue-specific promoter, wherein the nucleotide sequence encoding the tissuespecific promoter is operably linked to a nucleotide sequence encoding the fusion protein.
  • the tissue-specific promoter is a COL2A1 promoter.
  • the LV-hGALNS comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 10.
  • the tissue-specific promoter is a CD1 Ib-hGALNS promoter.
  • the tissue-specific promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 11.
  • the promoter is a ubiquitous promoter.
  • the ubiquitous promoter is a CBh promoter.
  • the ubiquitous promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 8 or to SEQ ID NO:9.
  • the method comprises delivering to the bone, cartilage, ligament, growth plate, meniscus, liver, spleen, lung, kidney, trachea, heart muscle, and/or heart valve (e.g, delivering to the bone and/or cartilage) of said human subject a therapeutically effective amount of a fusion protein that is hGALNS fused to an acidic oligopeptide (such as an acidic oligopeptide, for example, D8), wherein the fusion protein is produced from a genome in which the hGALNS gene cassette has been stably integrated from the LV-hGALNS.
  • the genome may comprise a portion of the hGALNS expression cassette.
  • the method comprises delivering to the bone, cartilage, ligament, growth plate, meniscus, liver, spleen, lung, kidney, trachea, heart muscle, and/or heart valve (e.g., delivering to the bone and/or cartilage) of said human subject a therapeutically effective amount of hGALNS protein through administration of LV-hGALNS.
  • the method comprises delivering to the bone, cartilage, ligament, growth plate, meniscus, liver, spleen, lung, kidney, trachea, heart muscle, and/or heart valve (e.g., delivering to the bone and/or cartilage) of said human subject a therapeutically effective amount of a fusion protein that is hGALNS fused to an acidic oligopeptide (such as an acidic oligopeptide, for example, D8), through administration of LV-hGALNS.
  • a fusion protein that is hGALNS fused to an acidic oligopeptide (such as an acidic oligopeptide, for example, D8), through administration of LV-hGALNS.
  • the LV-hGALNS may comprise an hGALNS expression cassette.
  • the LV-hGALNS expression cassette comprises a nucleotide sequence encoding a tissue-specific promoter, wherein the nucleotide sequence encoding the tissue-specific promoter is operably linked to a nucleotide sequence encoding the fusion protein.
  • the tissue-specific promoter is a COL2A1 promoter.
  • the LV-hGALNS comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 10.
  • the tissue-specific promoter is a CD1 lb promoter.
  • the tissue-specific promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 11.
  • the promoter is a ubiquitous promoter.
  • the ubiquitous promoter is a CBh promoter.
  • the ubiquitous promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 8 or to SEQ ID NOV.
  • the human subject or patient is an individual who has been diagnosed with MPS IVA (Morquio A syndrome).
  • the patient has one or more of the following symptoms of MPS IVA: abnormal heart valve morphology, carious teeth, cervical myelopathy, cervical subluxation, chondroitin sulfate excretion in urine, coarse facial features, constricted iliac wings, coxa valga, disproportionate short-trunk, short stature, epiphyseal deformities of tubular bones, flaring of rib cage, genu valgum, grayish enamel, hearing impairment, hepatomegaly, hyperlordosis, hypoplasia of the odontoid process, inguinal hernia, joint laxity, juvenile onset, keratin sulfate excretion in urine, kyphosis, large elbow, mandibular prognathism, metaphyseal widening, opacification of the corneal
  • the patient has been identified as responsive to treatment with hGALNS.
  • the patient has a severe and rapidly progressive, early-onset form of MPS IVA. In another specific embodiment, the patient has a slowly progressive, later- onset form of MPS IVA.
  • the patient is an adult (at least age 16). In another specific embodiment, the patient is an adolescent (age 12-15). In another specific embodiment, the patient is a child (under age 12).
  • the patient is under age 6.
  • the route of administration of an LV described herein and the amount of LV to be administered to the human patient can be determined based on the severity of the disease, condition of the human patient, and the knowledge of the treating physician.
  • the amount of LV-hGALNS administered to a human subject is sufficient to supply a therapeutically effective amount of hGALNS to the affected tissue (bone, cartilage, ligament, meniscus, and/or heart valve).
  • dosages are measured by the number of genome copies administered to the human subject via LV-hGALNS provided herein.
  • 1 x 10 10 to 1 x 10 16 genome copies are administered.
  • 1 x 10 10 to 1 x 10 11 genome copies are administered.
  • 1 x 10 11 to 1 x 10 12 genome copies are administered.
  • 1 x 10 12 to 1 x 10 13 genome copies are administered.
  • 1 x 10 13 to 1 x 10 14 genome copies are administered.
  • 1 x 10 14 to 1 x IO 15 genome copies are administered.
  • 1 x IO 13 to 1 x 10 16 genome copies are administered.
  • the LV-hGALNS can be present in a pharmaceutical composition in order to be administered to the human subject.
  • the LV-hGALNS can be administered, for example, by parenteral, subcutaneous, intramuscular, intravenous, intraperitoneal, intranasal, intrathecal, or transdermal administration.
  • the LV-hGALNS is administered by intravenous administration.
  • LV-hGALNS While the delivery of LV-hGALNS should minimize immune reactions, the clearest potential source of toxicity related to gene therapy is generating immunity against the expressed hGALNS protein in human subjects who are genetically deficient for hGALNS and, therefore, potentially not tolerant of the enzyme or the LV-hGALNS. Thus, in a certain embodiment, it is advisable to co-treat the patient with immune suppression therapy — especially when treating patients with severe disease who have close to zero levels of hGALNS. Immune suppression therapies involving a regimen of tacrolimus or rapamycin (sirolimus) in combination with mycophenolic acid or other immune suppression regimens used in tissue transplantation procedures can be employed.
  • immune suppression treatment may be administered during the course of gene therapy, and in certain embodiments, pre-treatment with immune suppression therapy may be preferred.
  • Immune suppression therapy can be continued subsequent to the gene therapy treatment, based on the judgment of the treating physician, and may thereafter be withdrawn when immune tolerance is induced; e.g., after 180 days.
  • the methods of treatment provided herein further comprise administering to the human patient an immune suppression regimen comprising prednisolone, mycophenolic acid, and tacrolimus. In certain embodiments, the methods of treatment provided herein further comprise administering to the human patient an immune suppression regimen comprising prednisolone, mycophenolic acid, and rapamycin (sirolimus). In certain embodiments, the methods of treatment provided herein further comprise administering to the human patient an immune suppression regimen that does not comprise tacrolimus.
  • the methods of treatment provided herein further comprise administering to the human patient an immune suppression regimen comprising one or more corticosteroids such as methylprednisolone and/or prednisolone, as well as tacrolimus and/or sirolimus.
  • the immune suppression therapy comprises administering a combination of (a) tacrolimus and mycophenolic acid, or (b) rapamycin and mycophenolic acid to said subject before or concurrently with the hGALNS treatment and continuing thereafter.
  • the immune suppression therapy is withdrawn after 180 days. In certain embodiments, the immune suppression therapy is withdrawn after 30, 60, 90, 120, 150, or 180 days.
  • Combination therapy involving the administration of the LV-hGALNS as described herein to the human subject accompanied by the administration of other available treatments are encompassed by the methods of the described embodiment.
  • the additional treatments may be administered before, concurrently, or after the gene therapy treatment.
  • Available treatments for MPS IVA that could be combined with the gene therapy of the invention include but are not limited to enzyme replacement therapy (ERT) and/or HSCT therapy.
  • efficacy of a treatment method as described herein may be monitored by measuring reductions in biomarkers of disease (such as GAG, KS, and C6S storage) and/or increase in hGALNS enzyme activity in bone, cartilage, ligament, meniscus, heart valve, urine, and/or serum. Signs of inflammation and other safety events may also be monitored.
  • biomarkers of disease such as GAG, KS, and C6S storage
  • hGALNS enzyme activity in bone, cartilage, ligament, meniscus, heart valve, urine, and/or serum. Signs of inflammation and other safety events may also be monitored.
  • the efficacy of a treatment method as described herein is monitored by measuring the level of a disease biomarker in the patient.
  • the level of the disease biomarker is measured in the serum of the patient.
  • the level of the disease biomarker is measured in the urine of the patient.
  • the disease biomarker is GAG.
  • the disease biomarker is KS.
  • the disease biomarker is C6S.
  • the disease biomarker is hGALNS enzyme activity.
  • the efficacy of a treatment method as described herein can be monitored by measuring physical characteristics associated with lysosomal storage deficiency in the patient.
  • the physical characteristics can be storage lesions.
  • the physical characteristic can be short neck and trunk.
  • the physical characteristic can be pectus carinatum.
  • the physical characteristic can be the laxity of joints.
  • the physical characteristic can be kyphoscoliosis.
  • the physical characteristic can be tracheal obstruction.
  • the physical characteristic can be spinal cord compression.
  • the physical characteristic can be hearing impairment.
  • the physical characteristic can be corneal opacity.
  • the physical characteristics can be bone and joint deformities.
  • the physical characteristic can be cardiac valve disease.
  • the physical characteri sitess can be restrictive/obstructive airway. Such physical characteristics may be measured by any method known to one of skill in the art.
  • LVs carrying the native GALNS encoding sequence were produced under three different promoters: CBh, COL2A1, and CDl lb. Moreover, the inventors designed LVs carrying the native GALNS cDNA under CD1 lb promoter tagged with D8 octapeptide and codon-optimized GALNS cDNA under CBh promoter, respectively. These LVs were transduced in MPS IVA patient fibroblasts, HEK293T, and HepG2 cells at an increasing multiplicity of infections. Transduced cells were cultured for 8 and 30 days, respectively, and media was collected every three days.
  • VCN Vector copy numbers
  • LVs were provided by VectorBuilder (Chicago, IL). The LVs were designed under different promoters: ubiquitous CBh, collagen targeting COL2A1, myeloid cell targeting CD1 lb, and D8 octapeptide tagged CD1 lb. Furthermore, LVs expressing the native hGALNS gene and codon-optimized hGALNS (hGALNSco) cDNA under the CBh promoter were also compared. The final constructs for the treatment of MPS IVA were:
  • MPS IVA fibroblasts, HEK293T, and HepG2 cells were analyzed. Fibroblasts derived from MPS IVA patients were cultured in complete Dulbecco’s modified Eagle’s medium nutrient mixture F-12 (DMEM/F12, Gibco#l 1320033, Grand Island, NY, USA) supplemented with 20% fetal bovine serum (FBS; Gibco#10082147), 1% streptomycin/penicillin.
  • HEK293T cells were cultured in chemically defined medium Pro293sTMCDM (Lonza#12-765Q, Rockland, ME) supplemented with 15% FBS and 1% streptomycin/penicillin.
  • HepG2 cells were cultured in Eagle’s minimum essential medium (EMEM; ATCC# 30-2003, Manassas, VA) supplemented with 15% FBS and 1% streptomycin/penicillin. Cultured cells were incubated at 37°C and 5% CO2.
  • EMEM Eagle’s minimum essential medium
  • Transduction of LVs The effect of LV gene therapy was first evaluated on MPS IVA fibroblasts, HEK293T, and HepG2 following transduction at different multiplication of infections (MOIs). HEK293T and HepG2 cells were transduced at MOI 5, 10, and 15, and MPS IVA fibroblasts at MOI 20. The transduction process is shown in FIG. 1. Briefly, previously cultured 90% confluent cells were harvested, and 3 x 10 5 cells per well of each cell line were seeded into 6-well culture plates (Day 0). After 24 h incubation, 6-well culture plates were transduced with LVs (Day 1).
  • Enzyme Assay with 4-Methylumbelliferone (4-MU) GALNS activity in cell homogenates was performed using a 4-methylumbelliferone (4-MU) assay (Melford Laboratories Ltd, Suffolk, UK). Briefly, cells were sonicated in 30-60 ul of homogenization buffer (25 mM Tris-HCl, pH 7.2, 1 mM PMSF) for 30 seconds on ice. The activity was expressed as nanomoles of 4-methylumbelliferone released per milligram of protein per hr. Protein concentrations were determined using PierceTM BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL).
  • GAGs Glycosaminoglycans assay with Liquid Chromatography Mass Spectrometry (LC-MS/MS): KS levels in untreated and treated cells were measured by LC-MS/MS (J.P. Hintze, et al., Biomark Insights 6 (2011) 69-78; S. Tomatsu, et al., J Inherit Metab Dis 33 Suppl 3 (2010). GAGs were extracted from the cell lysates (S.A. Khan, et al., Mol Genet Metab 125 (2016) 44-52; S.A. Khan, et al., Methods Mol Biol 2619 (2023) 3-24).
  • Vector Copy Numbers Viral genome and mRNA were extracted on day 30 from each cell line transduced by the LVs expressing hGALNS at different MOIs. Quantification of vectors was done by digital droplet PCR (ddPCR) using the primers specific to p24 capsid protein of LV, 5'-CGACTGGTGAGTACGCCAAA-3' and 5'-CCCGCTTAATACTGACGCTCTC-3' and produce an 82-bp PCR-product (Probe; AGCGGAGGCTAGAAGGAGAGATGGGT).
  • ddPCR digital droplet PCR
  • Lysosomal mass was determined using the pH-responsive broad LysoTrackerTM Deep Red (Thermo Fisher Scientific #L 12492, Carlsbad, CA) by flow cytometry on MPS IVA fibroblasts treated with LV-CBh-hGALNS, LV-CBh-hGALNSco, LV-CD11b- hGALNS, and LV-COL2A1 -hGALNS over 30 days. Briefly, MPS IVA fibroblasts in monolayer were stained with 50 pM LysoTrackerTM Deep Red in supplemented DMEM. After one hour of incubation, cells were washed three times with IX PBS and harvested by trypsinization.
  • Pelleted cells were washed twice and resuspended on 500 pL IX warmed Hank’s buffer salt solution (HBSS) for analysis.
  • HBSS buffer salt solution
  • a Novocyte 3000 Flow Cytometer (Exc/Em: 647/668 nm) was used to acquire at least 50,000 events following the protocol described previously [47], Propidium iodide (PI; Img/mL, Sigma-Aldrich#P4864, Saint Louis, MO) was used to identify viable cells.
  • the mean fluorescence intensity (MFI) from singlets was included for the analysis on FlowJo® software.
  • Immunohi stochemi stry To confirm enzyme expression in each cell, the inventors performed immunohistochemistry (IHC) for GALNS (anti-GALNS). Additionally, the inventors analyzed KS levels (anti-KS) following IHC. Harvested cells on day 30 were fixed in 10% formalin and sectioned with 5 pm-thickness for immunohistochemistry. The distribution and intensity patterns of GALNS and KS were investigated to determine any correlation with therapeutic effects. KS and GALNS were stained by anti-KS (1 :50, Santa Cruz Biotechnology#sc-73518, Santa Cruz, CA) and anti-GALNS antibodies (Leal, A. F. , et al., Mol Ther Methods Clin Dev 2023 , 31, 101153).
  • Enzyme Activity in MPS IVA Fibroblasts Normal skin fibroblasts (as wild-type) and MPS IVA patient’s skin fibroblasts (as treated or untreated) were used to confirm hGALNS enzyme activity and the effectiveness of LVs under CBh, CD1 lb, COL2A1, and CD1 lb*D8 at MOI 20. Following LV transduction, treated and control groups were cultured in two sets over 8 and 30 days. The secreted enzyme activity in the media of each group was analyzed 30 days post-transduction (FIG. 3A).
  • the enzyme activity was below the wild-type level.
  • the inventors found that the GALNS enzyme activity gradually increased over time, and the media enzyme activity treated with LV- COL2Al-hGALNS was at the highest level, nearly 12 nmol/h/ml in MPS IVA patient fibroblasts on day 29, which was significant compared to the wild-type control (1.271 ⁇ 0.1 nmol/h/ml) (p 0.0366).
  • Each LV, except the CD1 lb*D8-related LV was expressed at similar levels above wild-type; however, no significant difference was detected among these treatments on day 29.
  • the intracellular hGALNS enzyme activity in transduced cells was measured in individual experiments (FIG. 3B).
  • LV-COL2A1 -hGALNS vector had the highest expression level (138.9 nmol/h/mg on day 8 and 170.0 nmol/h/mg on day 30), although the LV-CD1 lb*D8-hGALNS vector was expressed at the lowest level (0.54 nmol/h/mg on day 8 and 0.06 nmol/h/mg on day 29). There were no statistical differences regarding intracellular enzyme activities between days 8 and 30 under COL2A1, CD1 lb, and CBh vectors.
  • the GALNS enzyme was stably expressed at the highest level in MPS IVA fibroblasts treated with LV-COL2A1 -hGALNS (FIG. 3A-3B).
  • Enzyme Activity in HepG2 To confirm if LV could be transduced and expressed in the cells originating from different organs, the inventors also used HEK293T and HepG2 cells originating from the kidney and liver, respectively. In these experiments, the inventors tested three different MOIs (5, 10, 15) and analyzed the cells at 8- and 30-days post-transduction. The secreted enzyme levels in the media of HepG2 cells treated with LVs illustrated a similar tendency among groups and MOIs (FIG. 4A-C). All three vectors were expressed in HepG2 cells, but LV-CD1 Ib-hGALNS lost the expression over time (FIG. 4C).
  • LV-COL2Al-hGALNS the hGALNS enzyme activity was found -60.98 and 22.95 nmol/h/ml at MOI 5, -78.97 and 39.17 nmol/h/ml at MOI 10, and -75.74 and 49.22 nmol/h/ml at MOI 15 on day 8 and day 30, respectively.
  • the media enzyme activity in the treatment with LV-CD1 Ib-hGALNS was -3.00 and 0.69 nmol/h/ml at MOI 5, -3.75 and 2.49 nmol/h/ml at MOI 10, and -11.99 and 3.9 nmol/h/ml at MOI 15 on day 8 and day 30, respectively.
  • the GALNS enzyme activity peaked on days 11 to 14, it was stabilized by the end of treatments.
  • LV-COL2A1 -hGALNS expression was the highest in all groups. At day 30 with MOI 15, this vector reached approximately 500 nmol/h/mg hGALNS enzyme expression (FIG. 5B). Increased MOIs also showed higher GALNS enzyme activity in HepG2 cells. Nevertheless, the inventors could not detect this phenomenon in all other vectors or MOIs. Depending on the promoters, the enzyme activity had variations among treatments.
  • HepG2 cells had the highest intracellular GALNS enzyme activity at MOI 15 under the COL2A1 promoter on day 30, while it was -164.0 nmol/h/mg under the CBh promoter and -39.53 nmol/h/mg under the CD1 lb promoter, respectively, compared to the enzyme activity in untreated HepG2 cells (9.25 ⁇ 11.7 nmol/h/mg).
  • the inventors did not find statistical differences among MOIs regarding intracellular enzyme activity in HepG2 cells.
  • the GALNS enzyme activity was found to be stable over time in these cells (FIG. 5A-C).
  • the GALNS enzyme activity at MOI 15 increased 47.4-fold by LV-COL2 Al -hGALNS, 17.7-fold by LV-CBh-hGALNS, and 4.27-fold by LV-CD1 Ib-hGALNS in HepG2 cells on day 30.
  • Enzyme Activity in HEK293 Cells In HEK293T cells, the secreted enzyme levels in media showed several variations among treatment groups and MOIs (FIG. 6A-C). All five vectors were expressed in HEK293T cells, but LV-CD1 Ib-hGALNS and LV-CD1 lb*D8- hGALNS had less expression over time (FIG. 6D-E).
  • LV-COL2A1 -hGALNS the hGALNS enzyme activity was found -0.15 and 2.23 nmol/h/ml at MOI 5, -0.33 and 0.75 nmol/h/ml at MOI 10, and -3.28 and 6.80 nmol/h/ml at MOI 15 on day 8 and day 29, respectively (FIG. 6B).
  • the media enzyme activity in the treatment with LV-CD1 Ib-hGALNS was -0.31 and 0.54 nmol/h/ml at MOI 5, -0.56 and 0.13 nmol/h/ml at MOI 10, and -1.80 and 0.79 nmol/h/ml at MOI 15 on day 8 and day
  • the media enzyme activity under the LV-CD1 lb*D8-hGALNS vector was -0.75 and 1.04 nmol/h/ml at MOI 5, -0.61 and 0.80 nmol/h/ml at MOI 10, and -0.58 and 0.93 nmol/h/ml at MOI 15 on day 8 and day 29, respectively while the LV-CBh-hGALNSco LV provided the enzyme activity in media by 0.53 and -5.63 nmol/h/ml at MOI 5, 0.51 and 7.94 nmol/h/ml at MOI 10, and -0.20 and 6.24 nmol/h/ml at MOI 15 on day 8 and day 29, respectively (FIG.
  • LVs under the CBh promoter with either native or codon-optimized hGALNS had relatively higher expression in HEK293T culture media than wild-type controls and other treatment groups.
  • the lowest GALNS enzyme activity was detected in HEK293T media under each treatment compared to MPS IVA fibroblasts and HepG2.
  • the intracellular GALNS enzyme activity of HEK293T cells was confirmed under each treatment with LVs.
  • the inventors detected untreated intracellular GALNS enzyme activity by 1.26 ⁇ 0.33 nmol/h/mg.
  • LV-CBh-hGALNS, LV-COL2A1 -hGALNS, and LV-CBh- hGALNSco LVs were expressed higher than CD 11 -related LVs (FIG. 7A-E).
  • the enzyme activity via LV-CBh-hGALNS was approximately the same under MOI 5 (-23.6 and -23.5 nmol/h/mg on day 8 and 30, respectively) and MOI 10 (-27.8 and 32.2 nmol/h/mg on day 8 and
  • LV-COL2A1 -hGALNS vector showed the highest expression (-77.90 nmol/h/mg) at MOI 5 among treated groups on day 30, while it was -32.17 nmol/h/mg on day 8.
  • the enzyme activity was -41.73 and 74.26 nmol/h/mg on days 8 and 30, respectively.
  • HEK293T cells treated with LV-CBh- hGALNSco the inventors detected the intracellular enzyme activity by -6.06 and 10.6 nmol/h/mg at MOI 5, -9.1, and 20.0 nmol/h/mg at MOI 10, and -14.8 and 23.06 nmol/h/mg at MOI 15 on days 8 and 30, respectively.
  • the enzyme activity by LV-CBh-hGALNS co was significant under different MOIs, especially MOI 10 (p ⁇ 0.0001) and 15 (p ⁇ 0.0001) on days 8 and 30.
  • GAG Levels of MPS IVA Fibroblasts Following Lentiviral Gene Therapy To confirm whether accumulated GAG levels decreased following the LV treatments, MPS IVA fibroblasts were harvested separately on days 8 and 30. After isolating GAGs from the cells according to the methods described above, LC-MS/MS was performed. KS is one of the clinical biomarkers of MPS IVA (J.P. Hintze, et al., Biomark Insights 6 (2011) 69-78; S. Tomatsu, et aL, J Inherit Metah Dis 33 Suppl 3 (2010). Thus, KS levels were analyzed and reported (FIG. 8).
  • the inventors also tested other GAGs, including heparan sulfate and dermatan sulfate (data not shown). The results indicated that LV gene therapy under CBh, COL2A1, and CD1 lb promoters reduced KS levels in MPS IVA fibroblasts compared to untreated (25.9 ⁇ 1.6 ng/mg) and wildtype control ones (8.25 ⁇ 1.35 ng/mg).
  • CBh-related LVs reduced KS on day 30 compared to day 8 (p ⁇ 0.0001).
  • KS levels were found to increase by 1.4-fold in CD1 lb (p ⁇ 0.0001) and 1.3- fold in COL2A1 -related promoters (p ⁇ 0.0001) on day 30 compared to day 8 (FIG. 8).
  • LV-specific p24 protein was tested via digital droplet PCR (ddPCR) in the LV-treated MPS IVA fibroblasts (MOI 20), HEK293T (MOI 5,10,15, respectively), and HepG2 cells (MOI 5,10,15, respectively) at day 30 (FIG. 9A-C).
  • Each lentiviral treatment had similar copy numbers in MPS IVA fibroblasts; 4.6 ⁇ 0.007, 3.8 ⁇ 0.007, 3.6 ⁇ 0.02, 3.6 ⁇ 0.02, and 3.5 ⁇ 0.01 copies per diploid cells by LV-CBh- hGALNS, LV-CBh-hGALNSco, LV-COL2Al-hGALNS, LV-CD1 Ib-hGALNS, and LV- CD1 lb*D8-hGALNS, respectively.
  • VCN was found significant in all treated groups compared to untreated (p ⁇ 0.0001) (FIG. 9A).
  • LV-CBh- GALNS and LV-CBh-hGALNSco vectors had the highest copies (p ⁇ 0.0001). The lowest copies were detected in HEK293T cells, varying from 0.2 to 1.8 copies per diploid cell (FIG. 9B), and the highest vector copies were detected in HepG2 cells ranging from 0.5 to 12.5 copies per diploid cell (FIG. 9C).
  • VCNs were found at 0.7 ⁇ 0.13, 0.2 ⁇ 0.04, 1.15 ⁇ 0.03, 0.8 ⁇ 0.02, and 0.9 ⁇ 0.08 copies per diploid cell at MOI 5, while 0.9 ⁇ 0.12, 0.4 ⁇ 0.007, 1.2 ⁇ 0.007, 1.1 ⁇ 0.007, and 1.8 ⁇ 0.02 copies per diploid cell at MOI 15 for LV-CBh-hGALNS, LV-CBh-hGALNSco, LV-COL2Al-hGALNS, LV-CD1 Ib-hGALNS, and LV-CD1 lb*D8-hGALNS, respectively (p ⁇ 0.05).
  • LV-CBh-hGALNS In HepG2 cells, the inventors detected VCN only for LV-CBh-hGALNS, LV-COL2Al-hGALNS, and LV-CD1 Ib-hGALNS, which was 0.3 ⁇ 0.0009, 3.05 ⁇ 0.006, and 3.3 ⁇ 0.005 copies per diploid cell at MOI 5 and 0.8 ⁇ 0.0005, 6.7 ⁇ 0.008, 12.3 ⁇ 0.03 copies per diploid cell at MOI 15, respectively (p ⁇ 0.05).
  • -CD1 Ib-hGALNS provided the highest level, approximately 12.5 copies per diploid cell, although nearly 1.1 copies per diploid cell in HEK293T cells (FIG. 9B-C).
  • Lysosomal Mass To confirm GAG reduction in lysosomes, flow cytometry analysis was performed on days 8 and 30. Following a 3.3-fold difference between wild-type and untreated MPS IVA fibroblasts, the inventors found that all LV treatments significantly reduced the lysosomal mass on day 30.
  • the inventors measured the enzyme activity in media before and after transferring into the untreated MPS IVA fibroblasts. Following 13-hour incubation in secreted hGALNS enzyme, the cells were harvested, and enzyme activity was measured. The media enzyme activities under each promoter were found at -0.75, 0.41, 1.92, and 1.34 nmol/h/ml before treatment, while at -0.17, 0.27, 1.38, and 0.85 nmol/h/ml after treatment with LV-CBh-hGALNS, LV-CBh-hGALNSco, LV-COL2A1 -hGALNS, and LV- CD1 Ib-hGALNS at MOI 20, respectively.
  • the intracellular enzyme activity was followed as 1.31 ⁇ 0.8, 0.94 ⁇ 0.5, 2.27 ⁇ 0.3, and 2.24 ⁇ 0.7 nmol/h/mg for LV-CBh-hGALNS, LV-CBh-hGALNSco, LV-COL2A1 -hGALNS, and LV-CD1 Ib-hGALNS, respectively.
  • the enzyme activity was elevated in MPS IVA fibroblasts incubated with culture media, including the secreted GALNS enzyme (p ⁇ 0.05) (FIG. 11B).
  • LV transduced-MPS IVA fibroblasts and HepG2 cells stably expressed the hGALNS transgene in the cells and secreted into the extracellular area better than HEK293T cells over time.
  • the GALNS enzyme activity in MPS IVA fibroblasts was elevated ⁇ 40-fold with LV-CBh-hGALNS, ⁇ 54-fold with LV-CBh-hGALNSco, ⁇ 170-fold with LV-COL2A1 -hGALNS, ⁇ 75-fold with LV-CD1 Ib-hGALNS and 0.7-fold with LV-CD1 lb*D8-hGALNS as compared to untreated fibroblasts.
  • promoters have been found to impact transgene expression in MPS IVA fibroblasts.
  • MOIs might have toxic effects on cellular GALNS expression.
  • the intracellular enzyme activity increased 15.8-fold at MOI 5 and 17.7-fold at MOI 15 under LV-CBh-hGALNS. The difference is too small, while MOI is three times higher in the same cell line.
  • LV-COL2A1 -hGALNS treated HepG2 cells showed a 30.6-fold and 47.4-fold increase at MOI 5 and MOI 15, respectively.
  • LV-CD1 Ib-hGALNS vector increased the GALNS enzyme activity 2.9-fold at MOI 5 and 4.3-fold at MOI 15 in HepG2 cells.
  • AAV vectors under CMV, EFl , and al -antitrypsin (AAT) promoters driving the GALNS gene were transduced into HEK293, human MPS IVA fibroblasts and murine MPS IVA chondrocytes.
  • the GALNS enzyme activity showed a 13-fold to 30-fold increase in HEK293 cells with AAV vectors under CMV, EFla, and AAT promoters.
  • the efficiency of the CMV promoter remained poor since HEK293 cells did not show any GALNS activity between 2 days to 10 days post-transduction.
  • the GALNS enzyme activity increased 1.8-fold, 3.5-fold, and 4.0- fold under CMV, AAT, and EFla promoters, respectively.
  • MPS IVA human fibroblasts transduced with AAV under CMV, AAT, and EFla showed 36.5%, 54.6%, and 15.3% increase in the GALNS enzyme activity, respectively, and the co-transduction with SUMF1 elevated the enzyme activity by 1.5-fold (C.J.
  • PGK, CMV, EFla, MND, MCU3, SFFV, and CBh have been used as housekeeping promoters in a variety of LSDs, whereas CD1 lb, ALB, TBG, MHC, MLC2v, cTnT as tissue-specific promoters (E.
  • CBh is a strong promoter, comprising CMV early enhancer fused to a modified chicken P-actin promoter (E. Rintz, et aL, Mol Ther Methods Clin Dev 24 (2022) 71-87).
  • CD1 lb is a myeloid cell-specific promoter from the alpha chain of Mac- 1 integrin (S. Dziennis, Blood 85 (1995) 319-329), while COL2A1 is a collagen type II specific promoter expressed in connective tissues and cartilage (H. Peng, et aL, J Cell Physiol 215 (2008) 562- 573).
  • TLRs toll-like receptors
  • CpG motifs dimerize TLR9 in plasmacytoid dendritic cells following binding, resulting in innate and adaptive immune responses. Moreover, high CpG contents in the expression cassette induce cytotoxic T cell (CTL) response eliminating transduced cells. Thus, hepato-immunotoxicity occurs after systemic administration of AAV vectors (Wicherska-Pawlowska and Wrobel, Diseases. IntJ Mol Sei. 2021 Dec 13;22(24):13397). CpG motifs were depleted in AAV vectors since TLR9 was known to be specific for DNA viruses and easily recognize unmethylated pathogen-related CpG motifs to activate transcription factors and related proteins.
  • LVs including integrase-deficient LVs and reverse transcriptase-deficient LVs, are known to be less immunogenic; however, the study showed that myeloid dendritic cells were activated immediately after lentiviral entry and reverse transcription in in vitro and in vivo although these cells are the absence of TLR9 molecules.
  • LC-MS/MS has a strong sensitivity, specificity, and accuracy in detecting each GAG, including KS, HS, DS, and CS in various specimens such as blood, urine, cerebrospinal fluid, dried blood spots etc. (Kubaski F, et al., Mol Genet Metab. 2017 Jan-Feb).
  • vector copies per cell might be considerable.
  • LV gene therapy is a promising, one-time, and permanent treatment for many diseases, including genetic disorders and cancers.
  • LV gene therapy in three cell lines was assessed under the effect of different promoters with increasing doses of viral load.
  • the inventors restored the GALNS enzyme activity within lysosomes and confirmed its secretion into the extracellular area.
  • the enzyme activity showed variations based on the cellular origin and promoter profile. It is crucial to investigate by in vivo studies on mouse models the gene expression profile under ubiquitous and -specific promoters at tissue levels. Improving the safety concerns of LV gene therapy will result in an attractive treatment for many diseases in the future
  • LV lentiviral
  • HSCs hematopoietic stem cells
  • ubiquitous-CBh collagen type II-COL2A1
  • hematopoietic stem cells-CDl lb hematopoietic stem cells
  • FIG. 14A-14C the inventors injected busulfan intraperitoneally into knockout (KO) newborn male mice to condition them for 24 hours. Then, HSCs were isolated from bone marrow of donor mice and transduced with LVs at a particular MOI.
  • HSCs were collected to treat intravenously busulfan-conditioned newborn male mice. Blood samples were collected biweekly following ex vivo infusion, and mice were autopsied at 16 weeks old to collect tissues. The therapeutic efficacy of ex vivo lentiviral gene therapy in MPS IVA mice was assessed. To do that, the inventors investigated vector copy number, enzyme activity levels, and the GAG concentrations in plasma, white blood cells, bone marrow cells, and tissue samples. Mouse studies demonstrated that HSCs treated with LVs under ubiquitous CBh promoter in KO newborn mice had the highest enzyme activity. Furthermore, following busulfan conditioning, the GALNS enzyme activity in plasma, WBCs, BM, and liver under each promoter was detected.
  • MPS IVA mice MPS IVA knock-out (KO) mice (GALNS ⁇ MKC2) were generated at Inotiv (West Lafayette, IN). This MPS IVA mouse model had a large deletion of ⁇ 6300bp (exons 2-5: 6359bp) at genomic coordinates.
  • sgRNAs for CRISPR-Cas9 mediated knock-out in C57BL/6J mouse zygotes. Briefly, we designed sgRNAs targeting upstream of exon 2 and upstream of exon 6 of mouse GALNS, which disrupted mGALNS and made the MPS IVA murine model.
  • the most potent sgRNA with minimal off-target potential was assembled into a ribonucleoprotein complex with Cas9 endonuclease and delivered into zygotes from C57BL/6J mice, followed by embryo transfer into pseudo-pregnant females.
  • Viable progeny was analyzed for the desired mutation by genomic PCR and DNA sequencing. Twenty-three transgenic founder mice with knock-out have been identified to be positive. The two founders of choice were backcrossed to wild-type mice to generate Fl heterozygous progeny for the GALNS” mice, as confirmed by PCR-mediated genotyping and DNA sequence analysis. Nine heterozygous mice (2 males) were identified (GALNS +/ ‘).
  • LVs were constructed as described in the in vitro example above.
  • LV-HST GT for patients with MPS IVA
  • the inventors designed 3 rd generation self-inactivating LVs with native human GALNS transgene driven by the ubiquitous CHh or collagen-specific COL2A1 promoters.
  • the final constructs for the transduction of HSCs were LV-CBh-hGALNS and LV-C0L2Al -hGALNS.
  • FIG. 14 B To confirm whether each LV has a therapeutic efficacy, we transduced fibroblasts (Gains’ ’) derived from MPS IVA patients at the MOI of 20 according to the company instructions (Fig. 2A-B) and HSCs derived from MPS IVA mice.
  • FIG. 15A-D Isolation and Transduction of Mouse HSCs: As shown in FIG. 14, Donor bone marrow was harvested from the femur and tibia of 8- to 12-week-old male GALNS’ 7 ’ mice for treatment groups (LV-HSC GT) and from wild-type donors for allogeneic HSCT (allo-HSCT) control group (FIG. 14 A).
  • GALNS GALNS’
  • wild-type donor HSCs were purified using EasySepTM mouse hematopoietic progenitor cell isolation kit (Stem Cell Technologies# 18000, Vancouver, Canada).
  • EasySepTM mouse hematopoietic progenitor cell isolation kit Stem Cell Technologies# 18000, Vancouver, Canada.
  • Dulbecco’s modified Eagle’s medium nutrient mixture F-12 (DMEM/F12, Gibco#l 1320033, Grand Island, NY) supplemented with 1% insulin/transferrin/selenium (ITS; Gibco#41400045), 1% penicillin/ streptomycin (Pen/Strep; Sigma- Al drich#P4333, Burlington, MA), 0.1% recombinant human albumin (RHA; SeraCare Life Sciences# 18600016, Milford, MA), 10 mM 2-[4-(2-hydroxyethy])piperazin-l-yl]ethane-l -sulfonic acid (HEPES;
  • SCF murine stem cell factor
  • TPO 100 ng/ml murine thrombopoietin
  • Fms-related tyrosine kinase-3 Fms-related tyrosine kinase-3
  • Ham’s F12 nutrient mix medium Gibco#l 1765-054, Waltham
  • 10 mM HEPES 1% penicillin-streptomycin-glutamine
  • PSG penicillin-streptomycin-glutamine
  • ITSX insulin- transferrin-selenium-ethanolamine
  • PVA polyvinyl alcohol
  • TPO TPO
  • 10 ng/ml SCF 10 ng/ml SCF as described in the published protocol. Wilkinson, A.C., et al., Nat Protoc 15 (2020) 628-648.
  • lin’ HSCs were seeded at a density of 0.9 x 10 6 cells per well into both complete medium 1 and 2 for further LV transduction.
  • APC anti-mouse c-Kit CD117; 0.2 mg/ml; #105811 , Biolegend
  • FITC anti-mouse Sca-1 Ly-6A/E; 0.5 mg/ml; #122506, Biolegend
  • Pacific Blue anti-mouse SLAM CD150; 0.5 mg/ml; #115924
  • APC Rat IgG2b K #400612, Biolegend
  • FITC Rat IgG2a K #400506, Biolegend
  • Busulfan Conditioning and HSCs Transplantation in MPS IVA Mice In vitro assays, e.g., cell culture assays, can be used to measure hGALNS expression from a cell transduced with the LV-hGALNS described herein, thus indicating, e.g., potency of the LV-hGALNS.
  • Cells utilized for the assay can include, but are not limited to, A549 , WEHI, 10T1/2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, HEK293, HEK293-T, HuH7, Saos, C2C12, L, HT1080, HepG2, primary fibroblast, hepatocyte, and myoblast cells.
  • the cells utilized in the cell culture assay comprise HuH7 cells.
  • cells transfected with the LV-hGALNS can be analyzed for hGALNS enzyme activity. (See FIG. 14)
  • WST-1 Toxicity Assay The WST -1 toxicity assay tests if LV transduction and further GALNS enzyme expression have a cytotoxic effect on MPS IVA patients’ fibroblasts at the MOI of 20. Cells were seeded at 1 x 10 4 cells/well in 100 pl culture medium and transduced for 48 h at 37°C and 5% CO2. Then, 10 pl of cell proliferation reagent WST-1
  • KS Keratan sulfate
  • LC-MS/MS liquid chromatography-tandem mass spectrometry
  • CFU Colony Forming Unit
  • MethoCultTM mixture containing cells were distributed evenly in 3 wells of 6-well plates (9.6 cm 2 /well; FalconTM353046; Fisherscientific#08-772-lB, Waltham) and incubated at 37°C, 5% CO with >95% humidity for 7-10 days according to the manufacturer’s instructions. Colonies were identified and counted by visual inspection using STEMgridTM-6 (StemCell
  • Pathology Mice were euthanized with CO2 gas 16 weeks post-transduction, and then the heart, liver, and knee joints were collected in 10% formalin. To evaluate lysosomal storage by light microscopy, these tissues were then fixed in 2% paraformaldehyde, 4% glutaraldehyde, and toluidine blue-stained 0.5-pm-thick sections were prepared. Bone pathology was quantified by using Image J (NIH). To evaluate storage quantitatively, the inventors measured cell number, cell size, organization of the growth plate, and epiphyseal and articular cartilage thickness (Sawamoto et al., 2020b). FIG. 18
  • MicroCT A micro-CT scan was performed on the femur using Sky Scan 1276 Micro- CT System (Bruker, Manning Park, MA). The femur samples were collected in 100% EtOH at 16 weeks post-transduction, and they were further wrapped in salinated (0.9% saline) gauze in preparation for the micro-CT imaging. The scanning was performed with high spatial resolution down to 2.8 pm pixel size, 528 projections, exposure time of 50 msec, photon energy of 80 keV, and current of 125 pA. Three-dimensional reconstruction of each bone was made (Azario et al., 2017; Pievani et al., 2015; Rowan et al., 2013).
  • Enzyme Assay with 4-Methylumbelliferone (4-MU) The GALNS enzyme activity in cells, plasma, and tissue extracts was meticulously assessed using a 4-methylumbelliferone (4- MU) assay (Melford Laboratories Ltd, Suffolk, UK). MPS IVA patients’ fibroblasts, wild-type mice HSCs, and Gains' " mice HSCs post-transduction were lysed in 60-100 pl of homogenization buffer (25 mM Tris-HCl, pH 7.2, 1 mM PMSF) via sonication for 30 sec and 10% amplitude. Then, cell lysates were centrifuged for 10 min at 4°C.
  • homogenization buffer 25 mM Tris-HCl, pH 7.2, 1 mM PMSF
  • the supernatant was transferred into a new tube and assayed for the GALNS enzyme activity.
  • tissue was dissected and immediately homogenized with Bead Mill Homogenizer (OMNI International, Kennesaw, GA) in homogenization buffer. Homogenates were centrifuged for 30 min at 4°C, and the supernatant was transferred into a new tube.
  • Both supernatants of cells/tissues and plasma samples underwent 4-MU enzyme assay and were incubated with 22 mM 4-methylumbelliferyl- P-galactopyranoside-6-sulfate (Research Products International, Mount Prospectm, IL) at 37°C for 16 h.
  • Immunohi stochemi stry The GALNS enzyme expression in the tibia and liver was confirmed by performing immunohistochemistry (IHC) of GALNS. Additionally, we analyzed KS and procollagen II levels via IHC. Collagen, KS, and GALNS were stained by antiprocollagen (Invitrogen#BTE0030202, Waltham, MA), anti-KS (Santa Cruz Biotechnology#sc- 73518, Dallas, TX), and custom-made monoclonal anti-GALNS antibodies (Creative Biolabs, NY). The liver and tibia were fixed in 10% formalin and sectioned with 5 pm-thickness for IHC.
  • IHC immunohistochemistry
  • KS, GALNS, procollagen II distribution, and intensity patterns were investigated immunohistochemically to determine any correlation with therapeutic effects.
  • Image J (NIH) software was used to evaluate the expression of GALNS and collagen and the reduction of KS.
  • NASH Image J
  • Anti-GALNS Antibodies Biweekly collected plasma samples were assayed to determine the antibody response against hGALNS (Milani et al., 2019; Schlimgen et al., 2016; Wang et al., 2011). To determine the anti-GALNS antibody response, ninety-six well polystyrene microplates were coated with 2 ug/ml of Vimizin® enzyme in the coating buffer (15 mM Na2CC>3, 35 mM NaHCOs, 0.021% NaN?, pH 9.6) and incubated overnight at 4°C.
  • the coated plates were blocked with 3% BSA in PBS for 1 h at room temperature and washed first with TTBS (10 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.5) and second with TBS (10 mM Tris, 150 mM NaCl, pH 7.5).
  • TTBS 10 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.5
  • TBS 10 mM Tris, 150 mM NaCl, pH 7.5
  • Biweekly collected plasma samples (1 : 100 dilution in TBST) and monoclonal anti-GALNS antibodies (Custom-made clone 2F5F2, Creative Biolabs, NY) were added into each well individually and incubated for 2.5 h at 37°C.
  • the enzyme reaction was stopped with 100 pl of 2% sodium dodecyl sulfate, and the absorbance was measured at 450 nm using a FLUOstar Omega plate reader stated above.
  • Plasma concentrations of anti-GALNS antibodies were derived by extrapolating the absorbance values from a calibration curve using monoclonal anti-GALNS antibodies mentioned above.
  • GAG Levels To confirm whether accumulated GAG levels decreased following the LV treatments, (KS) levels were measured in plasma and bone harvested at 16 weeks. (FIG.
  • Lineage-negative (Lin') HSCs were isolated from the bone marrow of GALNS " donor mice and wild-type donor mice via magnetic selection. The purity of lin' HSCs was measured immediately after isolation via flow cytometry by staining c-Kit (CD 117), Sca-1, and SLAM (CD150) surface markers, in which the final population of lin' cells was -21% of all isolated cells. Then, lin7c-Kit+/Sca-l+hematopoietic progenitor cells were gated among lin' (20.57%), 95.75% of which were positive for CD150 marker (Lin'Scal+c-kit+CD150+).
  • the isolated cells were transduced in media 1 and 2 and analyzed for the GALNS enzyme activity and VCN 24 h and 15 days post-transduction.
  • medium 1 transduced or untransduced HSCs were maintained up to 36-72 h; however, they were either differentiated, aggregated, or primarily dead after 72 h post-transduction. Only enzyme activity and VCN were analyzed in 24 h posttransduction in medium 1.
  • medium 2 the same transduction method was followed, and MOIs, in which the sternness of transduced HSCs was well-maintained and cultured for long-term to test the enzyme activity of GALNS, VCN, mono-sulfated KS levels, and other relevant experiments.
  • HSCs derived from MPS IVA mice were transduced with LVs to verify the GALNS enzyme activities compared to untreated and wild-type control HSCs.
  • transduced Gains' " HSCs the enzyme activity of GALNS at 24 h and 15 days post-transduction were compared. Following 24 h of transduction, the enzyme activity increased up to 0.9 ⁇ 0.04 nmol/h/mg under LV-CBh-hGALNS as compared to untreated Gains' " HSCs, and no statistical difference was found between LV-CBh-hGALNS -transduced and wild-type HSCs (0.7 ⁇ 0.1 nmol/h/mg).
  • LV-COL2Al-hGALNS elevated enzyme activity by 0.3 ⁇ 0.03 nmol/h/mg.
  • the data confirmed that LV-CBh-hGALNS had ⁇ 3-fold more enzyme activity than LV-COL2A1- hGALNS compared to untreated HSCs.
  • the enzyme activity reduced to 0.5 ⁇ 0.03 and 0.06 ⁇ 0.03 nmol/h/mg under LV-CBh-hGALNS and LV-COL2Al-hGALNS treatments. No statistical significance was found regarding this reduction between 24 h and 15 days.
  • the overexpression of the GALNS enzyme was also evaluated by utilizing HSCs isolated from wild-type mice. The results showed that the activity of the GALNS enzyme increased by 86.8% and 88.6% under LV-CBh-hGALNS (6.07 ⁇ 0.1 nmol/h/mg) and LV- COL2Al-hGALNS (7.02 ⁇ 0.3 nmol/h/mg), respectively compared to untransduced wild-type HSCs (0.8 ⁇ 0.06 nmol/h/mg).
  • the enzyme activity was elevated in the trachea under LV-CBh-hGALNS (0.3 ⁇ 0.09 nmol/h/mg) compared to that of untreated MPS IVA mice. However, it did not reach the enzyme activity of wild-type mice (4.7 ⁇ 0.6 nmol/h/mg) (Fig. 16B).
  • the enzyme activity via LV-COL2Al-hGALNS treatment was insufficient to detect in the trachea. Similar results were found in the lungs, bone (tibia), and heart under LV-COL2A1- hGALNS treatment.
  • LV-CBh-hGALNS treatment increased the GALNS enzyme activity in the lungs, bone (tibia), and heart by 0.4 ⁇ 0.2, 1.03 ⁇ 0.5, and 0.01 ⁇ 0.006 nmol/h/mg, respectively, compared to wild-type levels (1.6 ⁇ 0.4, 2.6 ⁇ 0.6 and 0.1 ⁇ 0.02 nmol/h/mg, respectively).
  • wild-type levels 1.6 ⁇ 0.4, 2.6 ⁇ 0.6 and 0.1 ⁇ 0.02 nmol/h/mg, respectively.
  • the GALNS enzyme activity increased under both vectors.
  • Treatment with LV-CBh-hGALNS showed an increase by 2.3 ⁇ 0.4 nmol/h/mg, while LV-COL2A1- hGALNS treated group showed a slight elevation by 0.07 ⁇ 0.05 nmol/h/mg as compared to that of untreated group (Fig. 16E).
  • the GALNS enzyme activity was normalized to the wild-type level (4.2 ⁇ 0.9 nmol/h/mg) in the liver and bone (tibia). No statistical significance was found between LV-CBh-hGALNS and wild-type groups. In the spleen, both LVs drove the GALNS expression, increasing enzyme activity.
  • Treatment with LV-CBh-hGALNS showed a significant increase in the GALNS enzyme activity by 5.1 ⁇ 1.2, which was -2.7 times higher than the wildtype level (1.9 ⁇ 0.2 nmol/h/mg). Additionally, the enzyme activity under treatment with LV- COL2Al-hGALNS (0.08 ⁇ 0.02 nmol/h/mg) was significant compared to the wild-type level.
  • the GALNS enzyme was highly expressed under LV-CBh-hGALNS over 16 weeks (the highest, 74.6 ⁇ 24.1 nmol/h/mg, and the lowest, 8.4 ⁇ 1.5 nmol/h/mg), which was found statistically significant compared to wild-type levels (-0.12 ⁇ 0.05 nmol/h/mg).
  • HSC-transplanted mice with LV-COL2Al-hGALNS elevated the GALNS enzyme activity in WBCs by -0.7 ⁇ 0.5 nmol/h/mg.
  • the GALNS enzyme activity was normalized to the wild-type level (Fig. 161). This expression of the GALNS enzyme under treatment with LV- C0L2A1 -hGALNS was further detected in BMCs.
  • Tissue-specific COL2A1 LVs reduced the accumulation of GAGs under lower expression of hGALNS.
  • allo-HSCT group had no statistical difference compared to the untreated, which results in no change in mono-sulfated KS levels in plasma.
  • LV-COL2 Al -hGALNS (6.09 ⁇ 0.8 ng/ml) reduced KS level by 56.2% compared to untreated control (13.93 ⁇ 1.02 ng/ml) and no statistical significance was found between the group treated with this vector and wild-type (7.40 ⁇ 1.07 ng/ml).
  • mono-sulfated KS concentrations between the LV-CBh-hGALNS group (14.90 ⁇ 3.50 ng/ml) and the untreated group.
  • KS level In the bone, the differences in KS level by 40.7% between untreated Gains 7- (0.05 ⁇ 0.003 ng/mg) and wild-type group (0.03 ⁇ 0.003 ng/mg).
  • the group treated with LV-CBh-hGALNS showed a 14.1% reduction in mono-sulfated KS levels (0.04 ⁇ 0.004 ng/mg), but it was insignificant compared to the untreated group.
  • KS level decreased by 40.2% under treatment with LV-COL2 Al -hGALNS (0.03 ⁇ 0.002 ng/mg) compared to untreated MPS IVA, which was statistically significant.
  • LV-COL2A1 -hGALNS treatment normalized mono-sulfated KS level to that of wild-type. Additionally, the monosulfated KS level of WBCs and bone were found to be similar under each treatment compared to untreated and wild-type controls at 16 weeks. Concerning mono-sulfated KS levels under allo- HSCT, neither WBCs nor bone showed a significant reduction. As a result, LV-HSC-GT under each promoter significantly reduced mono-sulfated KS levels in plasma and bone. Importantly, LV-COL2Al-hGALNS significantly reduced mono-sulfated KS levels in bone compared to those in LV-CBh-hGALNS.
  • LVs under ubiquitous or tissue-specific promoters were similarly inserted into the genome of HSCs.
  • LV VCNs were found by 0.75 ⁇ 0.02 and 0.74 ⁇ 0.02 relative to 2-copy control of Tfrc under LV-CBh-hGALNS and LV-COL2Al-hGALNS, respectively while VCN on day 15 was 0.24 ⁇ 0.003 and 0.74 ⁇ 0.003 per relative to 2-copy control of Tfrc, respectively. This reduction in the LV-CBh-hGALNS group on day 15 was significant compared to 24 h post-transduction.
  • VCN was 0.36 ⁇ 0.001 and 0.7 ⁇ 0.005 relative to 2-copy control of Tfrc in LV-CBh and LV-COL2A1 -modified Gains’ 7 ’ HSCs, while it was 0.37 ⁇ 0.005 and 0.7 ⁇ 0.003 relative to 2-copy control of Tfrc in LV-CBh and LV-COL2A1 -modified wild-type HSCs (Fig. 15H). Overall, VCN was similar in Gains' 7 ’ HSCs and wild-type HSCs and did not show significant alterations before and after the CFU assay.
  • VCN was further analyzed in liver samples at 16 weeks, which was 0.03 ⁇ 0.007 and 0.03 ⁇ 0.01 relative to 2-copy controls of Tfrc under LV-CBh-hGALNS and LV-COL2Al-hGALNS, respectively.
  • LV-GT did not affect the colony formation of transduced HSCs while increasing the GALNS enzyme activity in the pooled CFU colonies.
  • the treatment with LV-COL2 Al -hGALNS showed complete correction of disease progression except for one mouse, with partial correction among all groups, compared to untreated MPS IVA mice. No statistical differences were found between the group treated with LV-COL2A1 -hGALNS and the wild-type control group. Moreover, the group treated with LV-CBh-hGALNS showed a partial correction in the vacuolization of heart structures, which did not reach that of the wild-type level.
  • the LV-C OL2A1 -hGALNS - treated group had an elevation in the protein concentration compared to the untreated group, and this slight elevation was found to be significant compared to the wild-type.
  • the expression of the GALNS enzyme significantly increased under treatment with LV-CBh- hGALNS (-204%), which was followed by LV-COL2 Al -hGALNS by -90% compared to the wild-type level. This increase was found to correlate with the enzyme activity in liver, but not with VCN since both vectors had similar copy numbers per genome.
  • ELISA was performed using plasma samples of wild-type, treated, and untreated MPS IVA mice.
  • Treated groups with LV-COL2A1 -hGALNS showed significant elevation in the circulating anti-GALNS antibodies overtime starting from 6 weeks of age by 3.06 ⁇ 0.7 compared to control groups (untreated MPS IVA mice: -0.01 ⁇ 0.02 and wild-type mice: -0.11 ⁇ 0.12).
  • mice treated with LV-CBh-hGALNS showed significant up-and-down variations during treatment.
  • anti-GALNS antibodies were undetectable in the allo- HSCT group. It was anticipated that the undetectable/weak production of the GALNS enzyme might not trigger a strong immune reaction in allo-HSCT compared to LV-HSC GT groups.
  • mice from each group were measured weekly.
  • the data showed that the LV-COL2 Al -hGALNS or LV-CBh-hGALNS modified HSCs treated group significantly increased the body weight from the day of injection to the 3rd week. This initial increase in body weight could indicate an initial positive response to the treatment.
  • the subsequent decrease in body weight compared to untreated and wild-type controls from the 4th to the 16th week suggests that the treatment may not sustain body weight.
  • no significant difference was found between the LV-C0L2Al-hGALNS modified HSCs treated or allo-HSCT group and an untreated group from the 4th to the 16th week.
  • LV-CBh-hGALNS modified HSCs treated group remained under the body weight level of the untreated group. It is thought that busulfan administration might have a detrimental effect on low body weight over 16 weeks.
  • LVs carrying the native GALNS gene were produced under three promoters (ubiquitous - CBh, collagen type II - COL2A1 , hematopoietic stem cells - CD1 lb). Furthermore, LVs carrying the native GALNS gene under CD1 lb promoter tagged with D8 octapeptide and codon-optimized GALNS gene under CBh promoter, respectively, were created. Then, the inventors treated GALNS knockout (KO) mice intravenously at newborns and 4 weeks old with different doses of LVs (5xl0 9 and 5x10" TU/kg) under three different promoters.
  • KO GALNS knockout mice
  • KS levels were normalized to KS levels in wild-type mice. Bone pathology and biochemical analyses, including p24, cytokine, and anti-GALNS antibody assays, are underway to investigate therapeutic efficacy. Furthermore, KS levels were reduced to wildtype levels in the livers of MPS IVA mice compared to untreated mice.
  • MPS IVA mice MPS IVA knockout mice (GALNS"" generated from C57BL/6 background were used in this study. The colony was housed in a pathogen-free facility on a 12- hour light/dark cycle. Newborn (1-2 days old) and 4-week-old mice were injected with lentiviral vectors through the superficial temporal vein. All mouse care and handling procedures were in compliance with the rules of the Institutional Animal Care and Use Committee (IACUC) of Nemours Hospital Delaware Valley under the protocol number RSP20-12482-005.
  • IACUC Institutional Animal Care and Use Committee
  • LVs were constructed as described in the in vitro example above. The inventors will also design a lentiviral vector expressing EGFP fluorescence protein under CBh promoter.
  • MKC2 which is a knockout mouse model generated by Dr. Shunji Tomatsu’ lab, was treated with 5 x 10 9 TU/kg or 1 x 10 11 TU/kg GALNS LV vector starting at newborns (day 1-2) or 4 weeks old. Mice were treated via a superficial temporal vein (newborn) or tail vein (4-week-old). As controls, untreated MKC2, and wild-type mice at the same ages were provided with saline.
  • mice per group were euthanized, and tissues were collected, including brain, heart, lung, liver, kidney, spleen, muscle, femur-tibia, arm, eye, and trachea (FIG. 19).
  • Blood samples were collected at baseline and biweekly points, and enzyme activity, KS concentration, and anti-GALNS antibody were determined.
  • enzyme activity, KS concentration, and anti-GALNS antibody were determined.
  • LC-MS/MS was used.
  • the enzyme was analyzed with a 4MU assay, and the antibody was measured with ELISA.
  • Therapeutic effects The inventors evaluated the therapeutic efficacy by measuring GALNS activity and GAG levels in blood and tissues, examining pathology, and conducting micro-CT analysis. The inventors determined that MED could provide enough therapeutic effects for MPS IVA mice.
  • GALNS Enzyme Assay GALNS activity in plasma and tissue extracts was performed using a 4-methylumbelliferone (4-MU) assay (Melford Laboratories Ltd, Suffolk, UK). Briefly, tissues were dissected and immediately homogenized for 30 sec on ice (Omni homogenizer; Kennesaw GA) in 5 vols of homogenization buffer (25 mM Tris-HCl, pH 7.2, 1 mM PMSF). Activity was expressed as nanomoles of 4-methylumbelliferone released per milligram of protein per hr. Protein concentrations were determined using a BCA kit (Thermo Fisher Scientific). Enzyme activity in samples treated with a dose of 1x10 11 TU/kg. As shown in FIG.
  • the enzyme activity fluctuated in the plasma of all groups treated with lentiviruses under CBh and C0L2A1 promoters.
  • LV-CBh-hGALNS and LV-CBh- hGALNSco LV-CBh-hGALNSco vector had a stable expression over time starting at 6 weeks when compared to other groups.
  • LV-COL2 Al -hGALNS treated group had an increased trend over time while starting to drop after 12 weeks.
  • the secreted enzyme activity in plasma was elevated over time (FIG.
  • LV-CBh- hGALNSco and LV-COL2Al-hGALNS groups were 16 weeks old in liver (FIG 20B); however, it was reduced in LV-CBh- hGALNSco and LV-COL2Al-hGALNS groups at 16 weeks old in liver (FIG 20B), heart (FIG. 20C), and bone (FIG. 20D).
  • hGALNS enzyme activity was detected in all visceral organs including brain, heart, lung, liver, kidney, spleen, and muscle at variable levels. The trachea, arm and bone had undetectable enzyme levels in all lentivirus-treated groups. The highest enzyme activity was detected in liver of mice treated with LV-CBh-hGALNS, which was followed by LV-CBh-hGALNSco and LV-COL2A1 -hGALNS.
  • the enzyme activity was 5-fold higher in the wild-type level.
  • the inventors analyzed lymph nodes and thymus to identify the enzyme expression levels, which would guide us in analyzing immune reactions due to the enzyme levels.
  • GAG and Other Biomarkers KS in plasma (FIG. 21A-C) and tissues (FIG. 22A-C) from untreated and treated mice were measured by LC-MS/MS to evaluate the reduction of accumulated GAG (Hintze et al., 2011; Kubaski et al., 2016; Kubaski et aL, 2017; Oguma et al., 2007; Rowan et al., 2013; Shimada et al., 2014; Shimada et al., 2015; Tomatsu et al., 2010a). Tissue GAGs were extracted by the acetone precipitation method (Long et al., 2016).
  • KS keratan sulfate
  • KS levels were normalized to wild-type mice KS levels, except the group treated with LV-CBh-hGALNS. Furthermore, KS levels were found to be reduced to wild-type levels in the livers of MPS IVA- affected mice when compared to untreated (Fig 22B). Muscle KS levels had similar data with liver KS levels (Fig 22C).
  • Vector Copy Number Viral genome and mRNA were extracted at autopsy from brain, heart, lung, liver, kidney, muscle, and spleen to identify tissues transduced by the vectors expressing human GALNS. Quantification of vectors was done by digital PCR using the primers specific to the LV vector, 5'-CGACTGGTGAGTACGCCAAA-3' and 5'- CCCGCTTAATACTGACGCTCTC-3' and produced an 82-bp PCR-product (Probe;
  • ddPCR Digital droplet PCR
  • Pathology Mice were euthanized with CO2 gas after treatment, and then the following tissues were collected: brain, heart, lung, liver, kidney, muscle, femur, tibia, and eye to evaluate the reduction of stored KS. To evaluate lysosomal storage by light microscopy, tissues collected was fixed in 2% paraformaldehyde, 4% glutaraldehyde, and toluidine blue-stained 0.5- Lim-thick sections prepared. Bone pathology was quantified by using Image J (NIH). To evaluate storage quantitatively, the inventors measured cell number, cell size, organization of the growth plate, and epiphyseal and articular cartilage thickness (Sawamoto et al., 2020b).
  • Immunohistochemistry The inventors confirmed enzyme expression in bone and cartilage by performing immunohistochemistry (IHC) of GALNS. Additionally, the inventors analyzed anti-KS and anti-collagen levels following IHC. Tissues, including cartilage, were fixed in 10% formalin and sectioned with 5 pm-thickness for immunohistochemistry. The distribution and intensity patterns of KS and GALNS were investigated immunohistochemically to determine any correlation with therapeutic effects. Collagen, KS, and GALNS were stained by anti-KS (1 :50, sc-73518, Santa Cruz Biotechnology) anti-GALNS antibodies (Ghezzi et al., 2017).
  • MicroCT A micro-CT scan was performed on the femur using SkyScan 1276 Micro- CT System (Bruker, Manning Park, MA). The bone was wrapped in salinated (0.9% saline) gauze in preparation for the micro-CT imaging, performed with high spatial resolution down to 2.8 .m pixel size, 528 projections, exposure time of 50 msec, photon energy of 80 keV, and current of 125 pA. Three-dimensional reconstruction of each bone was made (Azario et al., 2017; Pievani et al., 2015; Rowan et al., 2013).
  • liver Toxicity Levels To determine the potential side effect of the lentiviral gene therapy, levels of alanine transaminase (ALT, EALT- 100, BioAssay Systems, Hayward, CA, USA) and aspartate transaminase (ALT, EASTR-100, BioAssay Systems, Hayward, CA, USA) in liver cells were evaluated. All the reactions were conducted on 96-well plates and read in a FLUOstar Omega microplate reader (BMG LabTech, Weston Parkway, NC, USA) at the wavelength specified by the supplier. No significant differences between groups (T-test) suggest that treatment failed to induce chronic hepatic toxicity. T statical analysis was done according to normality distribution determined using the Shapiro test (Fig. 24A-B). Additionally, the inventors measured the liver toxicity in newborn-treated low-dose groups, in which there were no differences between groups.

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Abstract

Provided herein are gene therapy methods for the treatment of mucopolysaccharidosis type IVA (MPS IVA) involving the use of recombinant lentivirus (LV) to deliver human N- acetylgalactosamine-6-sulfate sulfatase (hGALNS) to the bone of a human subject diagnosed with MPS IVA. Also provided herein are LV-hGALNS that can be used in Hematopoietic Stem Cell Gene Therapy (HSC-GT) methods.

Description

LENTIVIRAL GENE THERAPY OF MUCOPOLYSACCHARIDOSIS IVA
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/601,001, filed November 20, 2023, which is incorporated by reference herein in their entireties.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] This application incorporates by reference a st.26 Sequence Listing submitted with this application entitled “045009-0061-WO-OO st26.xml” created on November 19, 2024 and having a size of 70KB.
1. FIELD
[0003] The field relates to the treatment of mucopolysaccharidosis type IVA (MPS IVA). Provided herein are methods and compositions for treatment of MPS IVA involving novel lentiviral vectors (LVs) gene therapy to produce and secrete active N-acetylgalactosamine-6- sulfate sulfatase (GALNS).
2. BACKGROUND
[0004] Mucopolysaccharidosis type IVA (MPS IVA; Morquio A Syndrome) is an autosomal recessive lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-6- sulfate sulfatase (GALNS) (Khan, et al., Mol Genet Metab., 2017; 120(l-2):78-95). Deficiency of the enzyme results in a progressive accumulation of the glycosaminoglycans (GAGs), chondroitin 6-sulfate (C6S), and keratan sulfate (KS) leading to a systemic and unique skeletal dysplasia with incomplete ossification and successive imbalance of growth resulting in a short neck and trunk, cervical spinal cord compression, tracheal obstruction, pectus carinatum, laxity of joints, kyphoscoliosis, coxa valga, and genu valgum. Other clinical manifestations of the disease can include hearing loss, heart valve involvement, and corneal opacity. Over 200 different mutations have been identified in patients, and the prevalence in the United States is approximately 1 in 250,000.
[0005] Patients with a severe type MPS IVA die of airway compromise, cervical spinal cord complications or heart valve disease in their 20s or 30s if untreated (Khan, et al., Mol Genet Metab., 2017; 120(1 -2):78-95); Tomatsu, S., et al. Mol. Genet. Metab. 2016; 1 17, 150-156; Montano, A.M., et al. J. Inherit. Metab. Dis. 2007; 30, 165-174; Tomatsu, S., et al. Res. Rep. Endocr. Disord. 2012; 2012, 65-77; Pizarro, C., et al. Ann. Thorac. Surg. 2016; 102, e329-331). Enzyme replacement therapy (ERT), hematopoietic stem cell transplantation (HSCT), and various surgical interventions are currently available as supportive therapy for patients with MPS IVA in clinical practice. In February of 2014, the FDA approved the use of an ERT (elosulfase- alpha) (Hendriksz, et al., J Inherit Metab Dis., 2014; 37(6): 979-990). ERT, the current standard of care, results in partial improvement in soft tissue pathology and activity of daily living (ADL) of patients with MPS IVA; however, these therapies provide very limited impact in bone and cartilage due to the avascular character of these lesions. Current limitations of ERT include: i) weekly injections for 5-6 hours are required, ii) drug is rapidly cleared from the circulation, iii) the treatment cost is very expensive ($500,000 per year per patient), and v) the drug shows limited penetration to bone (Algahim and Almassi, Ther Clin Risk Manag., 2013;9:45-53;
Tomatsu et al., Curr Pharm Biotechnol., 2011; 12:931-945). For MPS IVA, weekly administration of recombinant human N-acetylgalactosamine-6-sulfate sulfatase (rhGALNS: Vimizim™, elosulfase alfa) currently provides no impact on bone and cartilage lesions of patients with MPS IVA. While HSCT may provide a better impact than ERT on bone, this cellbased therapy may not be applicable to all patients because of limited matched donors, the agelimit for effective treatment, a lack of well-trained facilities, the mortality risk of the procedure such as graft-versus-host disease (GVHD), infection, and other complications (Tomatsu et al., Drug Des Devel Then, 2015; 9: 1937-1953). In this sense, a novel drug for MPS IVA, particularly a novel drug for treating skeletal dysplasia in patients with MPS IVA, is urgently required.
[0006] Current treatments for MPS IVA include enzyme replacement therapy (ERT) with the elosulfase alfa (Vimizin®; recombinant human GALNS enzyme) and hematopoietic stem cell transplantation (HSCT) (Akyol, et al., Orphanet J Rar Dis 14 (2019) 137). ERT improves endurance, exercise capacity, and oxygen utilization, resulting in improved ADL to some extent; however, its impact on bone remains limited due to the avascularity of cartilage and the growth plate of bone. Other limitations include immune reactions of infused enzymes and the short halflife of infused enzymes in the circulation (Sawamoto, et al , Int J Mol Sci 21 (2020); Akyol, et al., Orphanet J Rar Dis 14 (2019) 137; Hughes et al , Orphanet J Rare Dis 12 (2017) 98). Allogeneic HSCT has been considered a promising treatment for MPS IVA. It improves pulmonary function, cardiovascular involvement, ADL, bone mineral density, and laxity of joints, in addition to reducing surgical interventions (Sawamoto, et al., Int J Mol Sci 21 (2020); Wang, et al., Biol of Blood and Marrow Transplantation 22 (2016) 2104-2108; Yabe, et al., Mol Genet Metab 117 (2016) 84-94). However, it is not standard of care for patients with MPS IVA due to the lack of evidence in bone and cartilage, risk of transplantation rejection, graft-versus- host-disease (GVHD), limited age of transplantation, and the issues of finding human l eukocyte antigen (HLA)-matched donors (Akyol, el al., Orphanet J Rar Dis 14 (2019) 137; Dede, et al., J of Bone and Joint Surgery 95 (2013) 1228-1234). Overall, ERT and HSCT have limited or no impact in ameliorating skeletal complications, although they improve ADL (Sawamoto, et al., Int J Mol Sci 21 (2020)). Surgical interventions may still be required to improve clinical outcomes even after ERT or HSCT Preclinical studies have extensively evaluated alternatives to ERT and HSCT: pharmacological chaperones, substrate reduction therapy, gene therapy (GT) with adeno- associated viral vectors (AAV), clustered regularly interspaced palindromic repeats (CRISPR/Cas9), or nanoparticles (Sawamoto and Tomatsu, Int J Mol Sci 20 (2019) 4139; Almeciga-Diaz, et al., J Med Chem 62 (2019) 6175-6189; Leal, et al., Int J Mol Sci 24 (2023) 16148; Sawamoto, et al., Mol Ther Methods Clin Dev 18 (2020) 50-61; Leal, et al.. Scientific Rpts 2022 12: 1 1-15). AAV gene therapy is an option for many genetic disorders; however, the episomal characteristic of AAV vectors has been affected by dilution factors over time.
[0007] Lentiviral (LV) mediated hematopoietic stem cell gene therapy (HSC-GT) has been attempted in other lysosomal storage diseases (LSDs) and blood-related disorders. However, off-target effects and potential immune reactions against infused LVs have been observed when delivered via in vivo direct infusion.
[0008] Despite the efforts described above, there is currently still no effective treatment for this skeletal disease, and there continues to be a need for more effective treatments and/or cure. While LV-mediated gene therapy shows potential, there remains a need for improvement in the LV-mediated HSC-GT approach to achieve LV-modification of HSCs in treating skeletal involvement in MPS IVA patients. The invention described herein addresses this continued need.
[0009] Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure. 3. SUMMARY
[0010] The present invention involves the use of novel lentiviral vectors (LVs) HSC-GT to produce and secrete the active GALNS enzyme supraphysiologic levels in cells of different organs of a subject diagnosed with MPS IVA. Provided herein are gene therapy methods for the treatment of mucopolysaccharidosis type IVA (MPS IVA) involving the use of recombinant lentiviral vectors (rLVs) to deliver human N-acetylgalactosamine-6-sulfate sulfatase (hGALNS) to the bone of a human subject diagnosed with MPS IVA. Also provided herein are rLVs that can be used in the gene therapy methods, methods of making such rLVs, as well as polynucleotides, plasmids, and cells that can be used for making such rLVs.
[0011] In one aspect, provided herein is a recombinant lentivirus (LV) has a human N- acetylgalactosamine-6-sulfate sulfatase (hGALNS) gene stably integrated in the LV genome (LV-hGALNS).
[0012] In another aspect, provided herein is a hematopoietic stem cell transduced with LV- hGALNS.
[0013] In another aspect, provided herein is a pharmaceutical composition comprising an LV-hGALNS provided herein and a pharmaceutically acceptable carrier.
[0014] In another aspect, provided herein, A recombinant lentivirus (LV) comprising a recombinant LV genome comprising a human N-acetylgalactosamine-6-sulfate sulfatase (hGALNS) expression cassette wherein said hGALNS expression cassette comprises a promoter operably linked to a nucleotide sequence encoding the hGALNS protein.
[0015] In another aspect, the LV -hGALNS, wherein the promoter of the hGALNS expression cassette is a tissue-specific promoter.
[0016] In another aspect, the LV -hGALNS, wherein the tissue-specific promoteris a collagen-specific promoter; or comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90% identical, at least 95%, at least 98%, or at least 100% identical to SEQ ID NO: 6. [0017] In another aspect, the LV -hGALNS, wherein the expression cassette comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90% identical, at least 95%, at least 98%, or at least 100% identical to SEQ ID NO: 10.
[0018] In another aspect, the LV -hGALNS, wherein the promoter of the hGALNS expression cassette is a ubiquitous promoter. [0019] In another aspect, the LV -hGALNS where the ubiquitous promoter comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90% identical, at least 95%, at least 98%, or at least 100% identical to SEQ ID NO: 5.
[0020] In another aspect, the LV -hGALNS, wherein the expression cassette comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90% identical, at least 95%, at least 98%, or at least 100% identical to SEQ ID NO: 8.
[0021] In another aspect, the LV -hGALNS, wherein the nucleotide sequence encoding hGALNS or the nucleotide sequence encoding the fusion protein is codon-optimized.
[0022] A hematopoietic stem cell transduced with a LV -hGALNS.
[0023] A pharmaceutical composition comprising the hematopoietic stem cell transduced with a LV -hGALNS.
[0024] A method for treating a human subject diagnosed with mucopolysaccharidosis type IVA (MPS IVA), comprising administering to the human subject the LV-hGALNS or the hematopoietic stem cell transduced with LV-hGALNS or pharmaceutical composition comprising the hematopoietic stem cell transduced with LV-hGALNS.
[0025] A method for treating a human subject diagnosed with MPS IVA, comprising delivering to the bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle, and/or heart valve of said human subject a therapeutically effective amount of a fusion protein that is hGALNS by administering to the human subject the LV- hGALNS or the hematopoietic stem cell transduced with LV-hGALNS or pharmaceutical composition comprising the hematopoietic stem cell transduced with LV-hGALNS.
ABBREVIATIONS
MPS IVA mucopolysaccharidosis type IVA
GALNS N-acetylgalactosamine-6-sulfate sulfatase hGALNS human N-acetylgalactosamine-6-sulfate sulfatase
GAG glycosaminoglycan
C6S chondroitin 6-sulfate
KS keratan sulfate
ERT enzyme replacement therapy
HSC-CT hematopoietic stem cell gene therapy
HSCT hematopoietic stem cell transplantation
D8 aspartic acid octapeptide
ELISA enzyme-linked immunosorbent assay
HS heparan sulfate
IS internal standard
LC-MS/MS liquid chromatography/tandem mass spectrometry
MOI multiplication of infections
OD optical density
PBS phosphate buffered saline
KO knockout
BMC bone marrow cells
BRIEF DESCRIPTION OF THE FIGURES
[0026] The foregoing and other objects, features, and advantages will be apparent from the following description of particular embodiments of the invention, as illustrated in the accompanying drawings. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the invention.
[0027] FIG. 1. Schematic diagram of in vitro experiments. AB+/-: With or without Geneticin antibiotic.
[0028] FIG. 2. Schematic diagram of the experiment to confirm the uptake of secreted GALNS enzyme by MPS IVA patient’s fibroblasts. [0029] FIGS. 3A and 3B. Bar graphs depicting enzyme activity in fibroblasts derived from the MPS IVA patient. A. Media enzyme activity. The GALNS enzyme activities of each vector were compared to the wild-type level through two-way ANOVA analysis. The inventors have shown the statistical significance of LV-COL2Al-hGALNS over time, but other vectors showed statistical significance as well at variations (p < 0.05), especially on day 8 (data not shown). B. Intracellular enzyme activity. The enzyme activities of each vector on day 8 and 30 were compared through Sidak’s multiple comparison test and two-way ANOVA analysis (*: < 0.05, **: < 0.005, ***: < 0.001) (n=4).
[0030] FIGS. 4A-4C. Graphs depicting enzyme activity in media of HepG2 cultures treated under different MOIs of LVs. A: LV-CBh-hGALNS, B: LV-COL2Al-hGALNS, C: LV- CD1 Ib-hGALNS. Two-way ANOVA and Tukey multiple comparison tests were used to compare each MOI on the collection days (*: < 0.05, ***: < 0.001) (n=3).
[0031] FIGS. 5A-5C. Bar graphs depicting intracellular enzyme activity of HepG2 cells treated under different MOIs of LVs. A: LV-CBh-hGALNS, B: LV-COL2Al-hGALNS, C: LV- CD1 Ib-hGALNS. Two-way ANOVA and Sidak multiple comparison tests were used to compare each MOI on the collection days (*: < 0.05) (n=3).
[0032] FIGS. 6A-6E. Bar graphs depicting enzyme activity in media of HEK293T cells treated under different MOIs with LVs. A: LV-CBh-hGALNS, B: LV-COL2Al-hGALNS, C: LV-CD1 Ib-hGALNS, D: LV-CD1 lb*D8-hGALNS, E: LV-CBh-hGALNSco. Two-way ANOVA and Tukey multiple comparison tests were applied, but no statistical difference was found among MOIs over time (n=3 for CBh, COL2A1, CD1 lb, CD1 lb*D8, and CBh- hGALNSco).
[0033] FIGS. 7A-7E. Bar graphs depicting intracellular enzyme activity of HEK293T cells treated under different MOIs with LVs. A: LV-CBh-hGALNS, B: LV-COL2Al-hGALNS, C: LV-CD1 Ib-hGALNS, D: LV-CD1 lb*D8-hGALNS, E: LV-CBh-hGALNSco. Two-way ANOVA and Sidak multiple comparison test were used to compare each MOI on the collection days (*: < 0.05, **: < 0.005, ***: < 0.001, ****: < 0.0001) (n=3 for CBh, COL2A1, CDl lb, CDl lb*D8, and CBh-hGALNSco).
[0034] FIG. 8. Bar graph depicting KS levels of MPS IVA fibroblasts after administering LVs. Comparison between untreated and treated groups was made according to Tukey multiple comparison test and two-way ANOVA (*: < 0.05, ***: < 0.001). Furthermore, each treatment was compared with wild-type, in which no statistical difference was found, while the inventors compared the cell collection days (8 and 30) via Tukey multiple comparison test and two-way ANOVA. Each treatment significantly reduced KS levels on days 8 and 30 (p < 0.0001).
[0035] FIGS. 9A-9C. Bar graphs depicting vector copy numbers in MPS IVA fibroblasts. A: MPS IVA fibroblasts (MOI 20). Copy numbers were compared to untreated levels and each other using one-way ANOVA and Tukey multiple comparison test. Since the VCN is the only factor affected by the treatments, the inventors used one-way ANOVA. B: HEK293T (MOI 5,10,15, respectively) C: HepG2 cells (MOI 5,10,15, respectively). Statistical methods used in Fig. 9B and C were Tukey multiple comparison and two-way ANOVA tests since the VCNs were affected by both LV treatments and MOIs. Compared to untreated, copy numbers in all treated groups significantly increased in HEK293T and HepG2 cells (p < 0.0001). In addition, the vector copies in B and C under each viral vector showed a significant increase compared to increasing MOIs in treated groups (*: < 0.05, **: < 0.005, ***: < 0.001, ****: < 0.0001) (n=2). [0036] FIGS. 10A-10B. Lysosomal mass in MPS IVA fibroblasts treated with LVs; LV- CDl lb-hGALNS, LV-CBh-hGALNS, LV-CBh-hGALNSco, and LV-COL2Al-hGALNS at MOI 15. A. A representative histogram from wild-type, untreated, and treated MPS IVA patient’s skin fibroblasts. B. Two-way ANOVA for the means of two independent experiments. (**: < 0.005, ****■ < 0.0001).
[0037] FIGS. 11A-11B. Graphical depiction of Enzyme activity levels following uptake experiments. A: Media enzyme activities before and after adding into culture media in MPS IVA fibroblasts. B: Intracellular enzyme activity of MPS IVA fibroblasts after adding media, including the secreted GALNS from previous experiments; wild type (9.24 ± 0.4 nmol/h/mg). The comparison was made using Tukey multiple comparison and one-way ANOVA tests (**: < 0.005, ****: < 0.0001) to determine the effect of different LV gene therapies on the GALNS enzyme activity (the one variable factor) (n=3).
[0038] FIGS. 12A-12B. Immunohistochemistry analysis of GALNS enzyme expression. A: IHC pictures from each experimental group. B: Statistical analysis of GALNS enzyme-positive areas after eliminating the background. Tukey multiple test and one-way ANOVA were used (****: < 0.0001). (n=4). [0039] FIG. 13. Bar graph depicting statistical analysis of KS GAG-positive areas in immunohistochemistry. Tukey multiple test and one-way ANOVA were used (**: < 0.005, ****:
< 0.0001) (n=4).
[0040] FIG. 14A-14C. Schematic of general workflow of Ex vivo gene therapy procedures. Isolation and modifications of HSCs removed from MPS IVA mice (A). Lentiviral vector constructs contained either the CBh or COL2A1 promoter driving native human GALNS (B). Isolation and transplantation of wild-type HSCs from healthy donors (C). WT: Wild-type, VSVG: Vesicular stomatitis virus G protein.
[0041] FIG. 15A-15H. Graphs depicting GALNS enzyme activity in MPS IVA patients’ fibroblasts and media (A-B) and the percentage of cell survival 8 days LV-post-transduction (C), the GALNS enzyme activity in MPS IVA mouse HSCs 24h and 15 days and LVs-transduced wild-type HSCs (overexpression experiments) 15 days post-transduction and (D), number of CFU colonies 7 days post-transduced HSCs (E), VCNs; Gains'" HSCs 24h vs 15 days posttransduction, wild-type HSCs 15 days post-transduction, and MPS IVA fibroblasts 8 days posttransduction (F), and the GALNS enzyme activity of pooled CFU colonies (G), VCN of pooled CFU colonies originated from Gains'" HSCs and wild-type HSCs (H). One-way ANOVA with Tukey’s post-hoc test; *: < 0.05, **: < 0.005, ***: < 0.001, ****: < 0.0001.
[0042] FIGS. 16A-16I. Graphs depictingeEnzyme activities in tissues after LV-modified HSCs transplantation and wild-type allo-HSCT (A-G); brain (A), trachea (B), lung (C), heart (D), liver (E), spleen (F) and bone (G) at 16 weeks old, and in plasma and WBCs over 16 weeks (H-I) One-way ANOVA with Tukey’s post-hoc test for plasma and WBCs week-by-week; *: LV-CBh-hGALNS vs. wild-type ; *: < 0.05, **: < 0.005, ***: < 0.001, < 0.0001 and #:
LV-CBh-hGALNS vs Untreated; #: < 0.05, ##: < 0.005, ###: < 0.001, ####: < 0.0001). No statistical differences were detected in LV-COL2Al-hGALNS vs. wild-type and wild-type allo- HSCT vs. wild-type.
[0043] FIGS. 17A-17D. Bar graphs depicting mono-sulfated keratan sulfate (KS) concentrations in plasma (A), WBCs (B), and bone (humerus) (C) at 16 weeks. VCN in the liver 16 weeks post-transplantation (D). One-way ANOVA with Tukey’s post-hoc test; *: < 0.05, **:
< 0.005, ***: < 0.001.
[0044] FIGS. 18B-18C. Bar graphs depicting Bone pathology of lentiviral vector (LV-CBh- hGALNS or LV-COL2Al-hGALNS)-treated and control groups. Chondrocyte vacuolization in the tibia growth plate (B) and chondrocyte column structure in the tibia growth plate (C).
Kruskal-Wallis with Dunn’s test; *: < 0.05, **: < 0.005, ***: < 0.001, ****: < 0.0001..
[0045] FIGS. 19A-19X. Bar graphs depicting bone morphometric analysis. Trabecular bone morphology (A-L) and cortical bone morphology (M-X). One-way ANOVA with Tukey’s post- hoc test; *: < 0.05, **: < 0.005, ***: < 0.001.
[0046] FIG. 20. Schematic of workflow of in vivo experiments
[0047] FIGS. 21A-21D. Bar graphs depicting GALNS enzyme activity in (A) plasma, (B) liver, (C) heart, and (D) bone of treated and untreated mice at 16 weeks old.
[0048] FIGS. 22A-22C. Bar graphs depicting plasma KS levels in plasma of (A) low-dose treated 4-week-old, (B) low-dose treated newborn, and (C) high-dose treated newborn mice.
[0049] FIGS. 23A-23C. GAG level (KS) in liver (A), muscle (B) and bone (humerus) (C) samples treated with a dose of IxlO11 TU/kg.
[0050] FIG. 24. Bar graph depicting VCN in liver samples.
[0051] FIGS. 25A-25B. Bar graph depicting liver toxicity levels (A) AST and (B) ALT levels of mice treated with a dose of IxlO11 TU/kg
[0052] FIG 26. Construct map for
[0053] FIG 27 Construct map for
[0054] FIG. 28. Construct map fo
[0055] FIG. 29. Construct map fo
[0056] FIG. 30 Construct map fo
Figure imgf000012_0001
4. DETAILED DESCRIPTION
[0057] MPS IVA is one of the lysosomal storage disorders with no effective cure to date, which affects multiple tissues, including bone and cartilage (Khan,S., et al. Mol Genet Metab 2017, 120, 78-95; Sawamoto, K., et al., IntJMol Set 2020, 21; Melbouci, M. et al., Mol Genet Metab 2018, 124, 1-10). Diagnosis and treatment of MPS IVA with skeletal dysplasia at an early stage of life had a crucial impact on disease progression. Although ERT and HSCT have little impact on bone, these treatments cannot fully recover hard-to-reach tissues. AAV gene therapy is a highlighted option for many genetic disorders; however, the episomal characteristic of AAV vectors has been affected by dilution factors over time. In contrast, LV gene therapy provides stable and permanent transgene expression, a one-time treatment. Disclosed herein are recombinant LVs for the treatment of MPS IVA. The LV-hGALNS disclosed herein show effectiveness in increasing GALNS enzyme activity in a variety of cell types.
[0058] Lentiviral (LV) gene therapy provides stable and permanent transgene expression in a one-time treatment. Described herein is LV-mediated human stem cell gene therapy (HSC-GT) in the treatment of MPS IVA.
[0059] Viral vectors have been widely explored in vivo and ex vivo. The first ex vivo gene therapy with a recombinant retroviral vector (LGSN) was tested in the human MPS IVA fibroblasts, increasing GALNS enzyme activity for up to 6 days (Toietta, G., et al., Hum Gene Ther. 12(16):2007-2016, 2001). The effect of CMV and EFla promoters driving the GALNS gene was tested with the co-expression of SUMF1 in HEK293 cells. The results showed that HEK293 cells transfected with EFla-pIRES- GALNS had a normal GALNS enzyme activity by 8 days, while co-transfection with SUMF1 plasmid increased the GALNS enzyme activity nearly 2.6-fold. Furthermore, the study showed that the EFla promoter stably drove the GALNS gene (C.J. Almeciga-Diaz, M.A, et al., Mol Biol Rep 36 (2009) 1863-1870). Nevertheless, 6- or 8- days post-transduction is too short of a period to precisely state whether viral vectors stably and permanently expressed the proteins. The LV-hGALNS viral vectors disclosed herein showed long-term, stable expression of hGALNS in transformed cells. Exemplary LV-hGALNS viral vectors of the present disclosure are represented in the construct maps of FIG. 26, 27, 28, 29 and 30
[0060] Described herein are LV-hGALNSs for use in the treatment of MPS IVA in a human subject in need of treatment. These LV-hGALNS comprise a recombinant LV genome encoding for hGALNS. The LV-hGALNS can be administered to an MPS IVA patient, resulting in the synthesis of hGALNS and the delivery of hGALNS to the affected tissues, such as bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle, and/or heart valve, thereby improving pathology, and preventing the progression of the disease. [0061] Also provided herein are polynucleotides comprising an hGALNS expression cassette as described herein. Further provided are plasmids and cells (e.g., ex vivo host cells) comprising a polynucleotide provided herein for making the LV-hGALNS for use with the methods and compositions provided herein. [0062] Also provided herein are methods for treating a human subject diagnosed with mucopolysaccharidosis type IVA (MPS IVA). In one aspect, the method comprises administering an LV-hGALNS described herein to the human subject. In another aspect, the method comprises delivering hGALNS to the affected tissue(s) in the human subject. In another aspect, the method comprises delivering hGALNS through the delivery of transduced cells to the human subject.
[0063] Further provided herein are pharmaceutical compositions and kits comprising LV- hGALNS and/or transduced cells described herein.
[0064] Without being bound by theory, the manufacture, composition, and method of use of the LV-hGALNS can be modified such that it still results in the delivery of the hGALNS enzyme to the bone, cartilage, ligament, meniscus, and/or heart valve of a human subject as a treatment for MPS IVA.
LENTIVIRUS
[0065] Provided herein are LVs useful for the treatment of MPS IVA in a human subject in need thereof, which LVs comprise an hGALNS expression cassette (LV-hGALNS). Preferably, the LVs of the present invention may be used in HST-GT for patients with MPS-IVA.
[0066] In one aspect, provided herein are LV-hGALNS resulting in stable long-term GALNS enzyme activity in transfected hematopoietic stem cells.
[0067] In contrast, the first ex vivo gene therapy with a recombinant retroviral vector (LGSN) was tested in the human MPS IVA fibroblasts, increasing GALNS enzyme activity for up to 6 days (Toietta G, et al., Hum Gene Ther. 12(16):2007-2016, 2001). The effect of CMV and EFla promoters driving the GALNS gene was tested with the co-expression of SUMF1 in HEK293 cells. The results showed that HEK293 cells transfected with EFl a-pIRES- GALNS had a normal GALNS enzyme activity by 8 days, while co-transfection with SUMF1 plasmid increased the GALNS enzyme activity nearly 2.6-fold. Furthermore, the study showed that the EFla promoter stably drove the GALNS gene (C.J. Almeciga-Diaz, et al., Mol Biol Rep 36 (2009) 1863-1870). Nevertheless, 6- or 8-days post-transduction is a short period to precisely state whether viral vectors stably and permanently expressed the proteins. The present LVs exhibit GALNS enzyme activity past the prior 8-day threshold previously achieved. [0068] In one aspect, provided herein is an LV-hGALNS comprising an hGALNS expression cassette, said hGALNS expression cassette comprising a nucleotide sequence encoding a hGALNS transgene. In one aspect, the transgene encodes a fusion protein that is hGALNS fused to an acidic oligopeptide. The hGALNS expression cassette may further comprise a nucleotide sequence encoding a tissue-specific promoter, wherein the nucleotide sequence encoding the tissue-specific promoter is operably linked to the nucleotide sequence encoding the hGALNS protein.
[0069] Preferably, the hGALNS expression cassette comprises a nucleotide sequence encoding a tissue-specific promoter operably-linked to the nucleotide sequence encoding hGALNS.
[0070] The different components of LV-hGALNS provided herein are described in detail below. hGALNS
[0071] In certain embodiments, the nucleotide sequence encoding hGALNS or the hGALNS portion of the fusion protein comprises the sequence of SEQ ID NO: 1 or 2. In certain embodiments, the nucleotide sequence encoding hGALNS or the hGALNS portion of the fusion protein is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence set forth in SEQ ID NO: 1 or 2.
[0072] In certain embodiments, the nucleotide sequence encoding the fusion protein comprises the sequence of SEQ ID NO: 1 or 2. In certain embodiments, the nucleotide sequence encoding the fusion protein is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence set forth in SEQ ID NO: 1 or 2. [0073] In certain embodiments, the nucleotide sequence encoding hGALNS or the hGALNS portion of the fusion protein comprises the cDNA sequence of hGALNS. In certain embodiments, the nucleotide sequence encoding hGALNS or the hGALNS portion of the fusion protein is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the cDNA sequence of hGALNS. [0074] In certain embodiments, the nucleotide sequence encoding the fusion protein comprises the cDNA sequence of the fusion protein. In certain embodiments, the nucleotide sequence encoding the fusion protein is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the cDNA sequence of the fusion protein.
[0075] In certain embodiments, the nucleotide sequence encoding hGALNS or the nucleotide sequence encoding the fusion protein is codon-optimized, for example, via any codonoptimization technique known to one of skill in the art (see, e.g., review by Quax et al., 2015, Mol Cell 59: 149-161).
[0076] In certain embodiments, CpG sites are depleted in the nucleotide sequence encoding hGALNS or the nucleotide sequence encoding the fusion protein.
Acidic Oligopeptide
[0077] Acidic oligopeptides have high binding affinities for hydroxyapatite, a major component of bones and cartilage. The term “acid oligopeptide,” as used herein, refers to an oligopeptide with a repeating amino acid sequence of glutamic acid (E) and/or aspartic acid (D) residues. The number of amino acid residues in an acidic oligopeptide may be, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In specific embodiments, the number of amino acid residues in an acidic oligopeptide is 4-8. In specific embodiments, the number of amino acid residues in an acidic oligopeptide is 6-8. In a specific embodiment, the number of amino acid residues in an acidic oligopeptide is 6. In another specific embodiment, the number of amino acid residues in an acidic oligopeptide is 8.
[0078] In a preferred embodiment, the acidic oligopeptide is D8 (i.e., an oligopeptide with an amino acid sequence of eight aspartic acid residues. In another embodiment, the acidic oligopeptide is E6 i.e., an oligopeptide with an amino acid sequence of six glutamic acid residues. The E6 sequence is described in Tomatsu et al., 2010, Molecular Therapy, 18(6): 11094-1102, which is incorporated by reference herein in its entirety.
[0079] In a preferred embodiment, the acidic oligopeptide is fused to the N-terminus of hGALNS. In another embodiment, the acidic oligopeptide is fused to the C-terminus of hGALNS. [0080] In a specific embodiment, the acidic oligopeptide is fused directly to hGALNS, with no intervening amino acid sequence. In another specific embodiment, the acidic oligopeptide is fused to hGALNS via a linker amino acid sequence e.g., an amino acid sequence that is 1-10, 2- 8, or 4-6 amino acid residues in length).
[0081] In certain embodiments, the hGALNS enzyme can be delivered to the lysosomes in the bone and cartilage area to improve bone and cartilage pathology.
Promoters and Modifiers of Gene Expression:
[0082] In certain embodiments, the hGALNS expression cassette described herein comprises components that modulate gene delivery or gene expression (e.g., “expression control elements”). In certain embodiments, the hGALNS expression cassette described herein comprises components that modulate gene expression. In certain embodiments, the hGALNS expression cassette described herein comprises components that influence binding or targeting cells. In certain embodiments, the hGALNS expression cassette described herein comprises components that influence the localization of the hGALNS within the cell after uptake. In certain embodiments, the hGALNS expression cassette described herein comprises components that can be used as detectable or selectable markers, e.g., to detect or select cells that have taken up the hGALNS expression cassette. In certain embodiments, the hGALNS expression cassette described herein comprises nucleotide sequence(s) encoding one or more promoters, at least one of which is operably linked to the nucleotide sequence encoding hGALNS or the fusion protein that is hGALNS fused to an acidic oligopeptide. In certain embodiments, the promoter can be a constitutive promoter. In alternate embodiments, the promoter can be an inducible promoter. [0083] In certain embodiments, the promoters are selected from known housekeeping promoters, including PGK, CMV, EFla, MND, MCU3, SFFV, and CBh. In certain embodiments, the promoters are selected from tissue-specific promoters, including CDl lb, ALB, TBG, MHC, MLC2v, and cTnT. In one preferred embodiment, the promoter is the COL2A1 promotion, a collagen type II specific promoter expressed in connective tissues and cartilage. In one preferred embodiment, the promoter is the CBh promoter, which comprises CMV early enhancer fused to a modified chicken [Lactin promoter. In one preferred embodiment, the promoter is the CD1 lb promotion, a myeloid cell-specific promoter from the alpha chain of Mac-1 integrin. [0084] In certain embodiments, the promoter is a ubiquitous promoter, such as CBh. In certain embodiments, the promoter is a collagen type II promoter, such as COL2A1, to provide for expression in cartilage and other connective tissues. In certain embodiments, the promoter is a hematopoietic promoter, such as CD1 lb, which can provide expression in hematopoietic stem cells.
[0085] In certain embodiments, the promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5. In certain embodiments, the promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6. In certain embodiments, the promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:7. In certain embodiments, the liver-specific promoter is SEQ ID NO:5. In certain embodiments, the liver-specific promoter is SEQ ID NO:6. In certain embodiments, the liver-specific promoter is SEQ ID NO:7.
[0086] In certain embodiments, the promoter comprises one or more elements that enhance the expression of hGALNS or the fusion protein. In certain embodiments, the promoter comprises a TATA box.
[0087] In certain embodiments, one or more promoter elements can be inverted or moved relative to one another. In certain embodiments, the elements of the promoter can be positioned to function cooperatively. In certain embodiments, the elements of the promoter can be positioned to function independently. In certain embodiments, the hGALNS expression cassette described herein comprises one or more ubiquitous promoters. In certain embodiments, the hGALNS expression cassette provided herein comprises one or more tissue-specific promoters. [0088] In a specific embodiment, the hGALNS expression cassette comprises a fusion protein that is hGALNS fused to an acidic oligopeptide (preferably D8).
Untranslated Regions
[0089] In certain embodiments, the hGALNS expression cassette described herein comprises one or more untranslated regions (UTRs), e.g., 3’ and/or 5’ UTRs. In certain embodiments, the UTRs are optimized for the desired level of protein expression. In certain embodiments, the UTRs are optimized for the mRNA half-life of the hGALNS. In certain embodiments, the UTRs are optimized for the stability of the mRNA of the hGALNS. In certain embodiments, the UTRs are optimized for the secondary structure of the mRNA of the hGALNS.
Pharmaceutical Compositions and Kits
[0090] In certain embodiments, provided herein are pharmaceutical compositions comprising an LV-hGALNS provided herein and a pharmaceutically acceptable carrier. The pharmaceutical composition may be prepared as individual, single unit dosage forms. The pharmaceutical compositions provided herein can be formulated for, for example, parenteral, subcutaneous, intramuscular, intravenous, intraperitoneal, intranasal, intrathecal, or transdermal administration. In a specific embodiment, the pharmaceutical composition is formulated for intravenous administration. A suitable pharmaceutically acceptable carrier (e.g., for intravenous administration and transduction in liver cells) would be readily selected by one of skill in the art.
[0091] In certain embodiments, provided herein are pharmaceutical compositions comprising cells transduced with LV-hGALNS.
[0092] Provided herein are kits comprising a pharmaceutical composition described herein, contained in one or more containers. The containers that the pharmaceutical composition can be packaged in can include but are not limited to, bottles, packets, ampoules, tubes, inhalers, bags, vials, and containers. In certain embodiments, the kit comprises instructions for administering the pharmaceutical administration. In certain embodiments, the kit comprises devices that can be used to administer the pharmaceutical composition, including, but not limited to, syringes, needle-less injectors, drip bags, patches, and inhalers.
[0093] Also provided are devices and blood circulation systems that can be utilized when treating MPS IVA using an LV-hGALNS described herein by gene therapy. Such devices and systems would be readily selected by one of skill in the art.
MANUFACTURE OF LV-hGALNS TRANSDUCED HSC
[0094] Also provided herein are hematopoietic stem cells transduced with an LV containing a hGALNS expression cassette as described herein.
[0095] Provided herein are hematopoietic stem cells comprising an hGALNS expression cassette. [0096] In one aspect, said hGALNS expression cassette comprises a nucleotide sequence encoding a transgene, such as the transgene encoding a fusion protein that is hGALNS. The hGALNS expression cassette may further comprise a nucleotide sequence encoding a collagenspecific promoter (for example, a COL2A1 promoter), wherein the nucleotide sequence encoding the collagen-specific promoter is operably linked to the nucleotide sequence encoding the fusion protein. In certain embodiments, the collagen-specific promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6. In certain embodiments, the expression cassette comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 10.
[0097] In one aspect, said hGALNS expression cassette comprises a nucleotide sequence encoding a transgene, such as the transgene encoding a fusion protein that is hGALNS. The hGALNS expression cassette may further comprise a nucleotide sequence encoding a ubiquitous promoter (for example, a COL2A1 promoter), wherein the nucleotide sequence encoding the ubiquitous promoter is operably linked to the nucleotide sequence encoding the fusion protein. In certain embodiments, the ubiquitous promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:5. In certain embodiments, the expression cassette comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 8.
Methods of Making LV-hGALNSs
[0098] Methods for preparation of recombinant lentivirus are known to those of skill in the art. Additionally, lentivirus packaging of one or more genes of interest may be performed through vector packaging vendors. The LV-hGALNS provided herein were prepared through the lentivirus packaging service, Vector Builder.
Assessment of Efficacy
[0099] In vitro assays, e.g., cell culture assays can be used to measure hGALNS expression from a cell transduced with the LV-hGALNS described herein, thus indicating, e.g., the potency of the LV-hGALNS. Cells utilized for the assay can include, but are not limited to, A549, WEHI, 10T1/2, BHK, MDCK, C0S1, COS7, BSC 1 , BSC 40, BMT 10, VERO, W138, HeLa, HEK293, HEK293-T, HuH7, Saos, C2C12, L, HT1080, HepG2, primary fibroblast, hepatocyte, and myoblast cells. In a specific embodiment, the cells utilized in the cell culture assay comprise HuH7 cells. In certain embodiments, cells transfected with the LV-hGALNS can be analyzed for hGALNS enzyme activity.
[00100] Animal models may also be used to assess the expression of hGALNS from an LV- hGALNS described herein and its efficacy. Mouse models for MPS IVA have been described (see, e.g., Tomatsu etal., 2003, Hum Mol Genet 12(24):3349-3358). The mouse model for MPS IVA has a targeted disruption of Exon 2 of mouse GALNS. These mice have no detectable GALNS enzyme activity, and increased levels of GAGs are detected in the urine. At 2 months old, increased storage of GAGs is seen in the reticuloendothelial cells, Kupffer cells, and the sinusoidal cells which line the spleen. At 12 months old, vacuolar change is observed in the visceral epithelial cells of glomeruli and cells at the base of heart valves, but it is not present in parenchymal cells such as hepatocytes and renal tubular epithelial cells. Lysosomal storage of GAGs is seen in hippocampal and neocortical neurons and meningeal cells. Keratan sulfate (KS) and chondroitin-6-sulfate (C6S) is increased in the corneal epithelial cells of this mouse model compared to wild type; however, no skeletal indications become evident in the mouse model. Additionaly, a mouse model for MPS IVA tolerant to human GALNS has also been described (see, e.g., Tomatsu et al., 2005, Hum Mol Genet 14(22):3321-3335). See Examples below for exemplary assays to assess the hGALNS expression from an LV-hGALNS described herein and its efficacy.
[00101] According to some embodiments, the methods include gene therapy vectors, e.g., the combination of regulatory elements and transgenes that provide increased expression of a functional hGALNS protein. Such expression may be measured 1) by several proteins (hGALNS) determination assays known to the skilled person, not limited to sandwich ELISA, Western Blot, histological staining, and liquid chromatography tandem mass spectrometry (LC- MS/MS); 2) by several protein activity assays, such as enzymatic assays or functional assays; and/or 3) by several substrate detection assays, not limited to keratan sulfate (KS), glycosaminoglycans (CAG), and/or chondroitin-6-sulfate (C6S) detection, and be determined as efficacious and suitable for human treatment (Hintze, J.P. et al, Biomarker Insights 2011 :6 69- 78). Assessment of the quantitative and functional properties of hGALNS using such in vitro and in vivo cellular, blood, and tissue studies have been shown to correlate to the efficacy of certain therapies (Hintze, J.P. et al., 2011, supra) and were utilized to evaluate response to gene therapy treatment of MPS IVA with the vectors described herein.
[00102] The invention thus provides methods and gene therapy vectors that increase intracellular hGALNS enzyme activity in tissue cells, e.g. including hepatic, muscle, white blood cells, kidney, lung, spleen cardiac, bone, or cartilage cells of the subject to levels compared to wild-type levels, or that increase intracellular hGALNS enzyme activity to about 2-fold wildtype hGALNS activity levels, or about 5-fold wild-type hGALNS activity levels, about 10-fold wild-type hGALNS activity levels, about 25-fold wild-type hGALNS activity levels, about 40- fold wild-type hGALNS activity levels, about 50-fold wild-type hGALNS activity levels, about 60-fold wild-type hGALNS activity levels, about 70-fold wild-type hGALNS activity levels, about 75-fold wild-type hGALNS activity levels, about 80-fold wild-type hGALNS activity levels, about 85-fold wild-type hGALNS activity levels, about 90-fold wild-type hGALNS activity levels, about 95-fold wild-type hGALNS activity levels, or about 100-fold wild-type hGALNS activity levels, as measured by a hGALNS enzymatic activity assay, e.g. using an assay format as described in the Examples herein, or a substantially similar assay. In some embodiments, gene therapy provides a method of increasing hGALNS activity levels in the subject two weeks after administration of the gene therapy as compared to the levels prior administration or the average levels in the untreated subjects. In some embodiments, gene therapy provides a method of increasing hGALNS activity levels in the subject two weeks after administration of the gene therapy. In some embodiments, gene therapy provides a method of increasing hGALNS activity levels in blood or tissues, for example, liver, muscle, kidney, lung, spleen, heart, bone, or cartilage of the subject two weeks after administration of the gene therapy. In some embodiments, the increase in hGALNS activity levels in the subject is measured ten weeks after administration of the gene therapy.
[00103] The invention also provides methods and gene therapy vectors that reduce blood (e.g., plasma or serum) levels or tissue levels of KS in the subject to levels compared to the levels of KS in untreated wild-type subjects or that reduce KS levels to about 1.1 -fold wild-type KS levels, or about 1.2-fold wild-type KS levels, about 1.3 -fold wild-type KS levels, about 1.4-fold wild-type KS levels, about 1.5-fold wild-type KS levels, about 1.6-fold wild-type KS levels, about 1.7-fold wild-type KS levels, about 1.8-fold wild-type KS levels, about 1.9-fold wild-type KS levels, about 2-fold wild-type KS levels, about 2.5-fold wild-type KS levels, about 3-fold wild-type KS levels, about 3.5-fold wild-type KS levels, or about 4-fold wild-type KS levels, as measured by a KS assay, e.g., using an assay format as described in the Examples herein, or a substantially similar assay. In some embodiments, gene therapy provides a method of reducing KS levels in the subject two weeks after administration of the gene therapy. In some embodiments, the gene therapy provides a method of reducing tissue levels of KS in the subject two weeks after administration of the gene therapy. In some embodiments, the KS assay comprises measurement of mono-sulfated KS in blood or tissue, and the gene therapy provides a method of reducing mono-sulfated KS levels in the subject two weeks after administration of the gene therapy.
METHODS FOR TREATMENT
[00104] Provided herein are methods for treating a human subject diagnosed with MPS IVA. [00105] In one aspect, the method comprises administering LV-hGALNS in conjunction with Hematopoietic Stem Cell Transplant (HSCT). HSCs are collected from a subject’s donor cells (autologous) and are modified with LV-hGALNS in vitro. Patients are pre-conditioned with an applicable method (such as administration of busulfan or fludarabine) to eliminate existing blood cells before transplanting modified HSCs to engraft in the bone marrow. Following transplantation, engineered HSCs expressing hGALNS are delivered into circulation and, thus, into many tissues at different rates.
[00106] In another aspect, the method comprises administering to the human subject an LV- hGALNS described herein, or cells transduced with LV-hGALNS or a pharmaceutical composition described herein.
[00107] In another aspect, the method comprises delivering to the bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle, and/or heart valve (e.g., delivering to the bone and/or cartilage) of said human subject a therapeutically effective amount of a hGALNS, by administering to the human subject an HSC capable of expressing the hGALNS gene through incorporation of the LV-hGALNS expression casette provided herein. In a preferred embodiment, the LV-hGALNS expression cassette comprises a nucleotide sequence encoding a tissue-specific promoter, wherein the nucleotide sequence encoding the tissuespecific promoter is operably linked to a nucleotide sequence encoding the fusion protein. In a preferred embodiment, the tissue-specific promoter is a COL2A1 promoter. In certain embodiments, the LV-hGALNS comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 10. In a preferred embodiment, the tissue-specific promoter is a CD1 Ib-hGALNS promoter. In certain embodiments, the tissue-specific promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 11. In a preferred embodiment, the promoter is a ubiquitous promoter. In a preferred embodiment, the ubiquitous promoter is a CBh promoter. In certain embodiments, the ubiquitous promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 8 or to SEQ ID NO:9.
[00108] In another aspect, the method comprises delivering to the bone, cartilage, ligament, growth plate, meniscus, liver, spleen, lung, kidney, trachea, heart muscle, and/or heart valve (e.g, delivering to the bone and/or cartilage) of said human subject a therapeutically effective amount of a fusion protein that is hGALNS fused to an acidic oligopeptide (such as an acidic oligopeptide, for example, D8), wherein the fusion protein is produced from a genome in which the hGALNS gene cassette has been stably integrated from the LV-hGALNS. The genome may comprise a portion of the hGALNS expression cassette.
[00109] In another aspect, the method comprises delivering to the bone, cartilage, ligament, growth plate, meniscus, liver, spleen, lung, kidney, trachea, heart muscle, and/or heart valve (e.g., delivering to the bone and/or cartilage) of said human subject a therapeutically effective amount of hGALNS protein through administration of LV-hGALNS. In another aspect, the method comprises delivering to the bone, cartilage, ligament, growth plate, meniscus, liver, spleen, lung, kidney, trachea, heart muscle, and/or heart valve (e.g., delivering to the bone and/or cartilage) of said human subject a therapeutically effective amount of a fusion protein that is hGALNS fused to an acidic oligopeptide (such as an acidic oligopeptide, for example, D8), through administration of LV-hGALNS. The LV-hGALNS may comprise an hGALNS expression cassette. In a preferred embodiment, the LV-hGALNS expression cassette comprises a nucleotide sequence encoding a tissue-specific promoter, wherein the nucleotide sequence encoding the tissue-specific promoter is operably linked to a nucleotide sequence encoding the fusion protein. In a preferred embodiment, the tissue-specific promoter is a COL2A1 promoter. In certain embodiments, the LV-hGALNS comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 10. In a preferred embodiment, the tissue-specific promoter is a CD1 lb promoter. In certain embodiments, the tissue-specific promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 11. In a preferred embodiment, the promoter is a ubiquitous promoter. In a preferred embodiment, the ubiquitous promoter is a CBh promoter. In certain embodiments, the ubiquitous promoter comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 8 or to SEQ ID NOV.
Target Patient Populations
[00110] According to the invention, the human subject or patient is an individual who has been diagnosed with MPS IVA (Morquio A syndrome). In specific embodiments, the patient has one or more of the following symptoms of MPS IVA: abnormal heart valve morphology, carious teeth, cervical myelopathy, cervical subluxation, chondroitin sulfate excretion in urine, coarse facial features, constricted iliac wings, coxa valga, disproportionate short-trunk, short stature, epiphyseal deformities of tubular bones, flaring of rib cage, genu valgum, grayish enamel, hearing impairment, hepatomegaly, hyperlordosis, hypoplasia of the odontoid process, inguinal hernia, joint laxity, juvenile onset, keratin sulfate excretion in urine, kyphosis, large elbow, mandibular prognathism, metaphyseal widening, opacification of the corneal stroma, osteoporosis, ovoid vertebral bodies, platyspondyly, pointed proximal second through fifth metacarpals, prominent sternum, recurrent upper respiratory tract infection, restrictive ventilator defect, scoliosis, ulnar deviation of the wrist, wide mouth, and widely spaced teeth.
[00111] In certain embodiments, the patient has been identified as responsive to treatment with hGALNS.
[00112] In a specific embodiment, the patient has a severe and rapidly progressive, early-onset form of MPS IVA. In another specific embodiment, the patient has a slowly progressive, later- onset form of MPS IVA.
[00113] In a specific embodiment, the patient is an adult (at least age 16). In another specific embodiment, the patient is an adolescent (age 12-15). In another specific embodiment, the patient is a child (under age 12).
[00114] In a specific embodiment, the patient is under age 6.
Administration and Dosage
[00115] The route of administration of an LV described herein and the amount of LV to be administered to the human patient can be determined based on the severity of the disease, condition of the human patient, and the knowledge of the treating physician.
(a) Therapeutic Dosage
[00116] In preferred embodiments, the amount of LV-hGALNS administered to a human subject is sufficient to supply a therapeutically effective amount of hGALNS to the affected tissue (bone, cartilage, ligament, meniscus, and/or heart valve).
[00117] In certain embodiments, dosages are measured by the number of genome copies administered to the human subject via LV-hGALNS provided herein. In a specific embodiment, 1 x 1010 to 1 x 1016 genome copies are administered. In another specific embodiment, 1 x 1010 to 1 x 1011 genome copies are administered. In another specific embodiment, 1 x 1011 to 1 x 1012 genome copies are administered. In another specific embodiment, 1 x 1012 to 1 x 1013 genome copies are administered. In another specific embodiment, 1 x 1013 to 1 x 1014 genome copies are administered. In another specific embodiment, 1 x 1014 to 1 x IO15 genome copies are administered. In another specific embodiment, 1 x IO13 to 1 x 1016 genome copies are administered.
(b) Routes of administration
[00118] In a specific embodiment, the LV-hGALNS can be present in a pharmaceutical composition in order to be administered to the human subject.
[00119] The LV-hGALNS can be administered, for example, by parenteral, subcutaneous, intramuscular, intravenous, intraperitoneal, intranasal, intrathecal, or transdermal administration. In a specific embodiment, the LV-hGALNS is administered by intravenous administration.
COMBINATION THERAPIES
Co-therapy with Immune Suppression
[00120] While the delivery of LV-hGALNS should minimize immune reactions, the clearest potential source of toxicity related to gene therapy is generating immunity against the expressed hGALNS protein in human subjects who are genetically deficient for hGALNS and, therefore, potentially not tolerant of the enzyme or the LV-hGALNS. Thus, in a certain embodiment, it is advisable to co-treat the patient with immune suppression therapy — especially when treating patients with severe disease who have close to zero levels of hGALNS. Immune suppression therapies involving a regimen of tacrolimus or rapamycin (sirolimus) in combination with mycophenolic acid or other immune suppression regimens used in tissue transplantation procedures can be employed. Such immune suppression treatment may be administered during the course of gene therapy, and in certain embodiments, pre-treatment with immune suppression therapy may be preferred. Immune suppression therapy can be continued subsequent to the gene therapy treatment, based on the judgment of the treating physician, and may thereafter be withdrawn when immune tolerance is induced; e.g., after 180 days.
[00121] In certain embodiments, the methods of treatment provided herein further comprise administering to the human patient an immune suppression regimen comprising prednisolone, mycophenolic acid, and tacrolimus. In certain embodiments, the methods of treatment provided herein further comprise administering to the human patient an immune suppression regimen comprising prednisolone, mycophenolic acid, and rapamycin (sirolimus). In certain embodiments, the methods of treatment provided herein further comprise administering to the human patient an immune suppression regimen that does not comprise tacrolimus. In certain embodiments, the methods of treatment provided herein further comprise administering to the human patient an immune suppression regimen comprising one or more corticosteroids such as methylprednisolone and/or prednisolone, as well as tacrolimus and/or sirolimus. In certain embodiments, the immune suppression therapy comprises administering a combination of (a) tacrolimus and mycophenolic acid, or (b) rapamycin and mycophenolic acid to said subject before or concurrently with the hGALNS treatment and continuing thereafter. In certain embodiments, the immune suppression therapy is withdrawn after 180 days. In certain embodiments, the immune suppression therapy is withdrawn after 30, 60, 90, 120, 150, or 180 days.
Co-therapy with Other Treatments
[00122] Combination therapy involving the administration of the LV-hGALNS as described herein to the human subject accompanied by the administration of other available treatments are encompassed by the methods of the described embodiment. The additional treatments may be administered before, concurrently, or after the gene therapy treatment. Available treatments for MPS IVA that could be combined with the gene therapy of the invention include but are not limited to enzyme replacement therapy (ERT) and/or HSCT therapy.
DISEASE MARKERS AND TREATMENT ASSESSMENT
[00123] In certain embodiments, efficacy of a treatment method as described herein may be monitored by measuring reductions in biomarkers of disease (such as GAG, KS, and C6S storage) and/or increase in hGALNS enzyme activity in bone, cartilage, ligament, meniscus, heart valve, urine, and/or serum. Signs of inflammation and other safety events may also be monitored.
[00124] In certain embodiments, the efficacy of a treatment method as described herein is monitored by measuring the level of a disease biomarker in the patient. In certain embodiments, the level of the disease biomarker is measured in the serum of the patient. In certain embodiments, the level of the disease biomarker is measured in the urine of the patient. In certain embodiments, the disease biomarker is GAG. In certain embodiments, the disease biomarker is KS. In certain embodiments, the disease biomarker is C6S. In certain embodiments, the disease biomarker is hGALNS enzyme activity.
[00125] In certain embodiments, the efficacy of a treatment method as described herein can be monitored by measuring physical characteristics associated with lysosomal storage deficiency in the patient. In certain embodiments, the physical characteristics can be storage lesions. In certain embodiments, the physical characteristic can be short neck and trunk. In certain embodiments, the physical characteristic can be pectus carinatum. In certain embodiments, the physical characteristic can be the laxity of joints. In certain embodiments, the physical characteristic can be kyphoscoliosis. In certain embodiments, the physical characteristic can be tracheal obstruction. In certain embodiments, the physical characteristic can be spinal cord compression. In certain embodiments, the physical characteristic can be hearing impairment. In certain embodiments, the physical characteristic can be corneal opacity. In certain embodiments, the physical characteristics can be bone and joint deformities. In certain embodiments, the physical characteristic can be cardiac valve disease. In certain embodiments, the physical characteri sties can be restrictive/obstructive airway. Such physical characteristics may be measured by any method known to one of skill in the art.
EXAMPLES
[00126] Certain embodiments provided herein are illustrated by the following non-limiting examples.
Example 1. Design of LVs and in vitro transfection assays
[00127] LVs carrying the native GALNS encoding sequence (cDNA) were produced under three different promoters: CBh, COL2A1, and CDl lb. Moreover, the inventors designed LVs carrying the native GALNS cDNA under CD1 lb promoter tagged with D8 octapeptide and codon-optimized GALNS cDNA under CBh promoter, respectively. These LVs were transduced in MPS IVA patient fibroblasts, HEK293T, and HepG2 cells at an increasing multiplicity of infections. Transduced cells were cultured for 8 and 30 days, respectively, and media was collected every three days. The enzyme activity and GAG levels in these cells and media were analyzed using fluorometric and LC-MS/MS methods. Vector copy numbers (VCN) were detected by ddPCR in each cell, and lysosomal mass was measured in MPS IVA patient fibroblasts. Data showed that the highest enzyme activity was generated in MPS IVA patient fibroblasts, HEK293T, and HepG2 cells transduced with LVs under COL2A1 promoter, followed by LVs under CBh promoter. VCNs were higher in MPS IVA patient fibroblasts under LV-CBh-hGALNS and HepG2 cells under LV-CD1 Ib-hGALNS than HEK293T cells. GAG concentrations and lysosomal mass were normalized to wild-type levels. These in vitro findings have demonstrated that LV gene therapy is a promising strategy for MPS IVA.
Materials and Methods
[00128] Construction of LVs: LVs were provided by VectorBuilder (Chicago, IL). The LVs were designed under different promoters: ubiquitous CBh, collagen targeting COL2A1, myeloid cell targeting CD1 lb, and D8 octapeptide tagged CD1 lb. Furthermore, LVs expressing the native hGALNS gene and codon-optimized hGALNS (hGALNSco) cDNA under the CBh promoter were also compared. The final constructs for the treatment of MPS IVA were:
Figure imgf000029_0001
Figure imgf000030_0001
[00129] Cell Culture: MPS IVA fibroblasts, HEK293T, and HepG2 cells were analyzed. Fibroblasts derived from MPS IVA patients were cultured in complete Dulbecco’s modified Eagle’s medium nutrient mixture F-12 (DMEM/F12, Gibco#l 1320033, Grand Island, NY, USA) supplemented with 20% fetal bovine serum (FBS; Gibco#10082147), 1% streptomycin/penicillin. HEK293T cells were cultured in chemically defined medium Pro293s™CDM (Lonza#12-765Q, Rockland, ME) supplemented with 15% FBS and 1% streptomycin/penicillin. HepG2 cells were cultured in Eagle’s minimum essential medium (EMEM; ATCC# 30-2003, Manassas, VA) supplemented with 15% FBS and 1% streptomycin/penicillin. Cultured cells were incubated at 37°C and 5% CO2.
[00130] Characterizations of mutations in the GALNS gene from MPS IVA patients’ fibroblasts were performed through exon sequencing using Sanger sequencing chemistry. Sequencing was aligned to NCBI reference sequence NG_008667.1 using Sequencher software (Gene Codes Corporation), and variant status was determined using dbSNP and ClinVar.
[00131] Transduction of LVs: The effect of LV gene therapy was first evaluated on MPS IVA fibroblasts, HEK293T, and HepG2 following transduction at different multiplication of infections (MOIs). HEK293T and HepG2 cells were transduced at MOI 5, 10, and 15, and MPS IVA fibroblasts at MOI 20. The transduction process is shown in FIG. 1. Briefly, previously cultured 90% confluent cells were harvested, and 3 x 105 cells per well of each cell line were seeded into 6-well culture plates (Day 0). After 24 h incubation, 6-well culture plates were transduced with LVs (Day 1). Following 48 h incubation, media was replaced, and Geneticin antibiotics were added to select transduced colonies (Day 3). The next day, antibiotics-added media were replaced with fresh media (Day 4). Starting day 4, media were collected into separate Eppendorf tubes every three days and stored at -80°C. On day 8, half of the cells were collected and stored at -80°C, and the other half were transferred into 10 cm diameter culture plates for 30 days, on which the inventors continued to collect media every three days. On day 30, all cells and media were collected and stored, as explained above, for further processing. [001321 Enzyme Assay with 4-Methylumbelliferone (4-MU): GALNS activity in cell homogenates was performed using a 4-methylumbelliferone (4-MU) assay (Melford Laboratories Ltd, Suffolk, UK). Briefly, cells were sonicated in 30-60 ul of homogenization buffer (25 mM Tris-HCl, pH 7.2, 1 mM PMSF) for 30 seconds on ice. The activity was expressed as nanomoles of 4-methylumbelliferone released per milligram of protein per hr. Protein concentrations were determined using Pierce™ BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL).
[00133] Glycosaminoglycans (GAGs) assay with Liquid Chromatography Mass Spectrometry (LC-MS/MS): KS levels in untreated and treated cells were measured by LC-MS/MS (J.P. Hintze, et al., Biomark Insights 6 (2011) 69-78; S. Tomatsu, et al., J Inherit Metab Dis 33 Suppl 3 (2010). GAGs were extracted from the cell lysates (S.A. Khan, et al., Mol Genet Metab 125 (2018) 44-52; S.A. Khan, et al., Methods Mol Biol 2619 (2023) 3-24). Crude GAGs were digested by heparitinase, chondroitinase B, and keratanase II (each enzyme 1 mU per sample) and incubated at 37°C for 15 h overnight. Then, the sample solution was injected into LC- MS/MS. The inventors evaluated the therapeutic efficacy by measuring GALNS enzyme activity and GAG levels in transduced cells.
[00134] Vector Copy Numbers: Viral genome and mRNA were extracted on day 30 from each cell line transduced by the LVs expressing hGALNS at different MOIs. Quantification of vectors was done by digital droplet PCR (ddPCR) using the primers specific to p24 capsid protein of LV, 5'-CGACTGGTGAGTACGCCAAA-3' and 5'-CCCGCTTAATACTGACGCTCTC-3' and produce an 82-bp PCR-product (Probe; AGCGGAGGCTAGAAGGAGAGAGATGGGT).
[00135] Lysosomal Mass: Lysosomal mass was determined using the pH-responsive broad LysoTracker™ Deep Red (Thermo Fisher Scientific #L 12492, Carlsbad, CA) by flow cytometry on MPS IVA fibroblasts treated with LV-CBh-hGALNS, LV-CBh-hGALNSco, LV-CD11b- hGALNS, and LV-COL2A1 -hGALNS over 30 days. Briefly, MPS IVA fibroblasts in monolayer were stained with 50 pM LysoTrackerTM Deep Red in supplemented DMEM. After one hour of incubation, cells were washed three times with IX PBS and harvested by trypsinization. Pelleted cells were washed twice and resuspended on 500 pL IX warmed Hank’s buffer salt solution (HBSS) for analysis. A Novocyte 3000 Flow Cytometer (Exc/Em: 647/668 nm) was used to acquire at least 50,000 events following the protocol described previously [47], Propidium iodide (PI; Img/mL, Sigma-Aldrich#P4864, Saint Louis, MO) was used to identify viable cells. The mean fluorescence intensity (MFI) from singlets was included for the analysis on FlowJo® software.
[00136] Uptake of GALNS: To confirm the uptake of secreted GALNS enzyme by MPS IVA patient’s fibroblasts, LV vector transduced culture’s media was collected on day 30 and transferred into new untreated MPS IVA fibroblast cultures as follows in FIG. 2. Before and after media transferring, the GALNS enzyme activity was measured in both media and cells.
[00137] Immunohi stochemi stry : To confirm enzyme expression in each cell, the inventors performed immunohistochemistry (IHC) for GALNS (anti-GALNS). Additionally, the inventors analyzed KS levels (anti-KS) following IHC. Harvested cells on day 30 were fixed in 10% formalin and sectioned with 5 pm-thickness for immunohistochemistry. The distribution and intensity patterns of GALNS and KS were investigated to determine any correlation with therapeutic effects. KS and GALNS were stained by anti-KS (1 :50, Santa Cruz Biotechnology#sc-73518, Santa Cruz, CA) and anti-GALNS antibodies (Leal, A. F. , et al., Mol Ther Methods Clin Dev 2023 , 31, 101153).
[00138] Statistical Analysis: The mean ± the standard deviation of the mean (SD) is presented by a two-way ANOVA test for variance differences. Post-hoc analysis using the Sidak or Tukey’s test was implemented for multiple comparisons between treatments when appropriate, p < 0.05 was considered significant.
Results
[00139] Mutation Analysis: To confirm the mutational profile of MPS IVA patients’ skin fibroblasts, 14 exons in the GALNS gene were sequenced. The data analysis showed that exon 2 had a homozygous c,122T>A (p.Met41Lys) transversion, known as a pathogenic variant in MPS IVA patients (National Center for Biotechnology Information. ClinVar; [VCV000520822.10], (2023); S. Tomatsu, J Med Genet 41 (2004) e98-e98 ). No mutations were detected in the rest of the exons.
[00140] Enzyme Activity in MPS IVA Fibroblasts: Normal skin fibroblasts (as wild-type) and MPS IVA patient’s skin fibroblasts (as treated or untreated) were used to confirm hGALNS enzyme activity and the effectiveness of LVs under CBh, CD1 lb, COL2A1, and CD1 lb*D8 at MOI 20. Following LV transduction, treated and control groups were cultured in two sets over 8 and 30 days. The secreted enzyme activity in the media of each group was analyzed 30 days post-transduction (FIG. 3A). The results showed that the intracellular enzyme activity was elevated in all treated groups compared to untreated MPS IVA and wild-type fibroblasts; ~3.5-, 5-, 17- and 7.5-fold on day 30 for LV-CBh-hGALNS, LV-CBh-hGALNSco, LV-COL2A1- hGALNS, and LV-CD1 Ib-hGALNS, respectively. Regarding the LV-CD1 lb*D8-hGALNS vector, the enzyme activity was below the wild-type level. The inventors found that the GALNS enzyme activity gradually increased over time, and the media enzyme activity treated with LV- COL2Al-hGALNS was at the highest level, nearly 12 nmol/h/ml in MPS IVA patient fibroblasts on day 29, which was significant compared to the wild-type control (1.271 ± 0.1 nmol/h/ml) (p = 0.0366). Each LV, except the CD1 lb*D8-related LV, was expressed at similar levels above wild-type; however, no significant difference was detected among these treatments on day 29. After 8 and 30 days of transduction, the intracellular hGALNS enzyme activity in transduced cells was measured in individual experiments (FIG. 3B). LV-COL2A1 -hGALNS vector had the highest expression level (138.9 nmol/h/mg on day 8 and 170.0 nmol/h/mg on day 30), although the LV-CD1 lb*D8-hGALNS vector was expressed at the lowest level (0.54 nmol/h/mg on day 8 and 0.06 nmol/h/mg on day 29). There were no statistical differences regarding intracellular enzyme activities between days 8 and 30 under COL2A1, CD1 lb, and CBh vectors. Compared to the intracellular GALNS enzyme activities on day 8, the enzyme activity under LV-CBh- hGALNS decreased by 38.06% on day 30 (p = 0.0027) while increasing by 71.43% under LV- CBh-hGALNSco (p = 0.0313). The GALNS enzyme was stably expressed at the highest level in MPS IVA fibroblasts treated with LV-COL2A1 -hGALNS (FIG. 3A-3B).
[00141] Enzyme Activity in HepG2: To confirm if LV could be transduced and expressed in the cells originating from different organs, the inventors also used HEK293T and HepG2 cells originating from the kidney and liver, respectively. In these experiments, the inventors tested three different MOIs (5, 10, 15) and analyzed the cells at 8- and 30-days post-transduction. The secreted enzyme levels in the media of HepG2 cells treated with LVs illustrated a similar tendency among groups and MOIs (FIG. 4A-C). All three vectors were expressed in HepG2 cells, but LV-CD1 Ib-hGALNS lost the expression over time (FIG. 4C). Although the inventors found the highest enzyme activity at MOI 15 under different promoters, there were no statistical differences among MOIs (FIG. 4A-C). When closely looked into each treatment compared to untreated HepG2 media where the enzyme activity was 0.17 ± 0.08 nmol/h/ml, the media enzyme activity under LV-CBh-hGALNS was -5.80 and 16.71 nmol/h/ml at MOI 5; -11.50 and 31.64 nmol/h/ml at MOI 10, and -20.78 and 36.50 nmol/h/ml at MOI 15 on day 8 and day 26, respectively. In LV-COL2Al-hGALNS, the hGALNS enzyme activity was found -60.98 and 22.95 nmol/h/ml at MOI 5, -78.97 and 39.17 nmol/h/ml at MOI 10, and -75.74 and 49.22 nmol/h/ml at MOI 15 on day 8 and day 30, respectively. The media enzyme activity in the treatment with LV-CD1 Ib-hGALNS was -3.00 and 0.69 nmol/h/ml at MOI 5, -3.75 and 2.49 nmol/h/ml at MOI 10, and -11.99 and 3.9 nmol/h/ml at MOI 15 on day 8 and day 30, respectively. In HepG2 cells under each LV treatment, although the GALNS enzyme activity peaked on days 11 to 14, it was stabilized by the end of treatments.
[00142] In HepG2 cells, LV-COL2A1 -hGALNS expression was the highest in all groups. At day 30 with MOI 15, this vector reached approximately 500 nmol/h/mg hGALNS enzyme expression (FIG. 5B). Increased MOIs also showed higher GALNS enzyme activity in HepG2 cells. Nevertheless, the inventors could not detect this phenomenon in all other vectors or MOIs. Depending on the promoters, the enzyme activity had variations among treatments. HepG2 cells had the highest intracellular GALNS enzyme activity at MOI 15 under the COL2A1 promoter on day 30, while it was -164.0 nmol/h/mg under the CBh promoter and -39.53 nmol/h/mg under the CD1 lb promoter, respectively, compared to the enzyme activity in untreated HepG2 cells (9.25 ± 11.7 nmol/h/mg). The inventors did not find statistical differences among MOIs regarding intracellular enzyme activity in HepG2 cells. The GALNS enzyme activity was found to be stable over time in these cells (FIG. 5A-C). The GALNS enzyme activity at MOI 15 increased 47.4-fold by LV-COL2 Al -hGALNS, 17.7-fold by LV-CBh-hGALNS, and 4.27-fold by LV-CD1 Ib-hGALNS in HepG2 cells on day 30.
[00143] Enzyme Activity in HEK293 Cells: In HEK293T cells, the secreted enzyme levels in media showed several variations among treatment groups and MOIs (FIG. 6A-C). All five vectors were expressed in HEK293T cells, but LV-CD1 Ib-hGALNS and LV-CD1 lb*D8- hGALNS had less expression over time (FIG. 6D-E). Compared to untreated HEK293T media (0.38 ± 0.32 nmol/h/ml), the media enzyme activity in HEK293T cells treated with LV-CBh- hGALNS was -0.71 and 3.61 nmol/h/ml at MOI 5 and -2.92 and 2.89 nmol/h/ml at MOI 15 on day 8 and day 29, respectively. Even though there were statistical differences in media enzyme activity among media collection days (p < 0.05), no statistical differences were found among different MOIs over time (FIG. 6A). In LV-COL2A1 -hGALNS, the hGALNS enzyme activity was found -0.15 and 2.23 nmol/h/ml at MOI 5, -0.33 and 0.75 nmol/h/ml at MOI 10, and -3.28 and 6.80 nmol/h/ml at MOI 15 on day 8 and day 29, respectively (FIG. 6B). The media enzyme activity in the treatment with LV-CD1 Ib-hGALNS was -0.31 and 0.54 nmol/h/ml at MOI 5, -0.56 and 0.13 nmol/h/ml at MOI 10, and -1.80 and 0.79 nmol/h/ml at MOI 15 on day 8 and day
29, respectively (FIG. 6C). The media enzyme activity under the LV-CD1 lb*D8-hGALNS vector was -0.75 and 1.04 nmol/h/ml at MOI 5, -0.61 and 0.80 nmol/h/ml at MOI 10, and -0.58 and 0.93 nmol/h/ml at MOI 15 on day 8 and day 29, respectively while the LV-CBh-hGALNSco LV provided the enzyme activity in media by 0.53 and -5.63 nmol/h/ml at MOI 5, 0.51 and 7.94 nmol/h/ml at MOI 10, and -0.20 and 6.24 nmol/h/ml at MOI 15 on day 8 and day 29, respectively (FIG. 6D-E). In conclusion, LVs under the CBh promoter with either native or codon-optimized hGALNS had relatively higher expression in HEK293T culture media than wild-type controls and other treatment groups. However, the lowest GALNS enzyme activity was detected in HEK293T media under each treatment compared to MPS IVA fibroblasts and HepG2.
[00144] The intracellular GALNS enzyme activity of HEK293T cells was confirmed under each treatment with LVs. The inventors detected untreated intracellular GALNS enzyme activity by 1.26 ± 0.33 nmol/h/mg. LV-CBh-hGALNS, LV-COL2A1 -hGALNS, and LV-CBh- hGALNSco LVs were expressed higher than CD 11 -related LVs (FIG. 7A-E). The enzyme activity via LV-CBh-hGALNS was approximately the same under MOI 5 (-23.6 and -23.5 nmol/h/mg on day 8 and 30, respectively) and MOI 10 (-27.8 and 32.2 nmol/h/mg on day 8 and
30, respectively). LV-COL2A1 -hGALNS vector showed the highest expression (-77.90 nmol/h/mg) at MOI 5 among treated groups on day 30, while it was -32.17 nmol/h/mg on day 8. At MOI 10, the enzyme activity was -41.73 and 74.26 nmol/h/mg on days 8 and 30, respectively. Although no statistical difference was found between MOI 5 and 10 under COL2A1 -related LV treatment, the elevated intracellular enzyme activity under each LV was significant compared to that of untreated cells (p = 0.04 and p = 0.0018 for days 8 and 30 at MOI 5; p = 0.0187 and p = 0.0022 for days 8 and 30 at MOI 10) (Fig. 7B). Additionally, increased enzyme activity levels under LV-CBh-hGALNS or LV-CD1 lb*D8-hGALNS were not significant compared to untreated HEK293T cells. Regarding the treatment with LV-CD11b- hGALNS, the elevated enzyme activity on day 8 was significant compared to untreated cells (p = 0.0023 for MOI 5 and p = 0.0005 for MOI 10). Since the cells treated with LV-CBh-hGALNS, LV-CD1 Ib-hGALNS, and LV-COL2A1 -hGALNS were lost at MOI 15 under the same conditions, no data in these cells was presented. In HEK293T cells treated with LV-CBh- hGALNSco, the inventors detected the intracellular enzyme activity by -6.06 and 10.6 nmol/h/mg at MOI 5, -9.1, and 20.0 nmol/h/mg at MOI 10, and -14.8 and 23.06 nmol/h/mg at MOI 15 on days 8 and 30, respectively. Compared to untreated HEK293T cells, the enzyme activity by LV-CBh-hGALNS co was significant under different MOIs, especially MOI 10 (p < 0.0001) and 15 (p < 0.0001) on days 8 and 30. Among CBh-related LVs carrying native hGALNS or codon-optimized hGALNS genes expressed in HEK293T cells, no statistical differences were found (FIG. 7A-E). Taken all together, the GALNS enzyme activity at MOI 5 increased by 61.8-, 18.6-, 8.4-, 2.47-, and 2.14-fold, and at MOI 10, increased by 58.9-, 25.5-, 15.9-, 2.33-, and 2.85-fold with LV-COL2A1 -hGALNS, LV-CBh-hGALNS, LV-CBh- hGALNSco, LV-CD1 Ib-hGALNS, and LV-CD1 lb*D8-hGALNS vectors in HEK293T cells on day 30, respectively.
[00145] GAG Levels of MPS IVA Fibroblasts Following Lentiviral Gene Therapy: To confirm whether accumulated GAG levels decreased following the LV treatments, MPS IVA fibroblasts were harvested separately on days 8 and 30. After isolating GAGs from the cells according to the methods described above, LC-MS/MS was performed. KS is one of the clinical biomarkers of MPS IVA (J.P. Hintze, et al., Biomark Insights 6 (2011) 69-78; S. Tomatsu, et aL, J Inherit Metah Dis 33 Suppl 3 (2010). Thus, KS levels were analyzed and reported (FIG. 8). The inventors also tested other GAGs, including heparan sulfate and dermatan sulfate (data not shown). The results indicated that LV gene therapy under CBh, COL2A1, and CD1 lb promoters reduced KS levels in MPS IVA fibroblasts compared to untreated (25.9 ± 1.6 ng/mg) and wildtype control ones (8.25 ± 1.35 ng/mg). Decreased KS levels by LV-CBh-hGALNSco and LV- CD1 Ib-hGALNS were found significant (p = 0.042 and 0.044, respectively), while no statistical differences were found in LV-CBh-hGALNS and LV-COL2A1 -hGALNS compared to the level in untreated MPS IVA fibroblasts (data not shown). Regarding the changes in KS levels over time, the inventors found that CBh-related LVs reduced KS on day 30 compared to day 8 (p < 0.0001). However, KS levels were found to increase by 1.4-fold in CD1 lb (p < 0.0001) and 1.3- fold in COL2A1 -related promoters (p < 0.0001) on day 30 compared to day 8 (FIG. 8). [00146] Vector Copy Numbers: To determine LV copy numbers, LV-specific p24 protein was tested via digital droplet PCR (ddPCR) in the LV-treated MPS IVA fibroblasts (MOI 20), HEK293T (MOI 5,10,15, respectively), and HepG2 cells (MOI 5,10,15, respectively) at day 30 (FIG. 9A-C). Each lentiviral treatment had similar copy numbers in MPS IVA fibroblasts; 4.6 ± 0.007, 3.8 ± 0.007, 3.6 ± 0.02, 3.6 ± 0.02, and 3.5 ± 0.01 copies per diploid cells by LV-CBh- hGALNS, LV-CBh-hGALNSco, LV-COL2Al-hGALNS, LV-CD1 Ib-hGALNS, and LV- CD1 lb*D8-hGALNS, respectively. VCN was found significant in all treated groups compared to untreated (p < 0.0001) (FIG. 9A). Moreover, as compared to all treated groups, LV-CBh- GALNS and LV-CBh-hGALNSco vectors had the highest copies (p < 0.0001). The lowest copies were detected in HEK293T cells, varying from 0.2 to 1.8 copies per diploid cell (FIG. 9B), and the highest vector copies were detected in HepG2 cells ranging from 0.5 to 12.5 copies per diploid cell (FIG. 9C). The inventors tested three different MOIs in HEK293T and HepG2 cells treated with LVs under CBh, CD1 lb, and COL2A1 promoters, which resulted in the increased copy numbers as MOI increased. In HEK293T cells, VCNs were found at 0.7 ± 0.13, 0.2 ± 0.04, 1.15 ± 0.03, 0.8 ± 0.02, and 0.9 ± 0.08 copies per diploid cell at MOI 5, while 0.9 ± 0.12, 0.4 ± 0.007, 1.2 ± 0.007, 1.1 ± 0.007, and 1.8 ± 0.02 copies per diploid cell at MOI 15 for LV-CBh-hGALNS, LV-CBh-hGALNSco, LV-COL2Al-hGALNS, LV-CD1 Ib-hGALNS, and LV-CD1 lb*D8-hGALNS, respectively (p < 0.05). In HepG2 cells, the inventors detected VCN only for LV-CBh-hGALNS, LV-COL2Al-hGALNS, and LV-CD1 Ib-hGALNS, which was 0.3 ± 0.0009, 3.05 ± 0.006, and 3.3 ± 0.005 copies per diploid cell at MOI 5 and 0.8 ± 0.0005, 6.7 ± 0.008, 12.3 ± 0.03 copies per diploid cell at MOI 15, respectively (p < 0.05). -CD1 Ib-hGALNS provided the highest level, approximately 12.5 copies per diploid cell, although nearly 1.1 copies per diploid cell in HEK293T cells (FIG. 9B-C). These results revealed that both HEK293T and HepG2 cells had the highest copy numbers of LVs under different promoters at increasing MOIs. [00147] Lysosomal Mass: To confirm GAG reduction in lysosomes, flow cytometry analysis was performed on days 8 and 30. Following a 3.3-fold difference between wild-type and untreated MPS IVA fibroblasts, the inventors found that all LV treatments significantly reduced the lysosomal mass on day 30. Moreover, LV-CD1 Ib-hGALNS (p = 0.1909) and LV-COL2A1- hGALNS (p = 0.0925) were indistinguishable from WT levels, suggesting that those vectors led to the normalization of the lysosomal mass on day 30 (FIG. 10A-B). No significant differences between treatment and untreated control on day 8 post-transduction were observed. [00148] Uptake of GALNS: To test the uptake of GALNS enzyme following secretion in the extracellular area, culture media from LV-transduced MPS IVA fibroblasts were transferred into cultured plates in untreated MPS IVA fibroblasts. The inventors measured the enzyme activity in media before and after transferring into the untreated MPS IVA fibroblasts. Following 13-hour incubation in secreted hGALNS enzyme, the cells were harvested, and enzyme activity was measured. The media enzyme activities under each promoter were found at -0.75, 0.41, 1.92, and 1.34 nmol/h/ml before treatment, while at -0.17, 0.27, 1.38, and 0.85 nmol/h/ml after treatment with LV-CBh-hGALNS, LV-CBh-hGALNSco, LV-COL2A1 -hGALNS, and LV- CD1 Ib-hGALNS at MOI 20, respectively. The uptake of GALNS enzymes into the cells accounted for 76.9, 34.07, 28.1, and 36.3% of the secreted GALNS under treatment with LV- CBh-hGALNS, LV-CBh-hGALNSco, LV-COL2A1 -hGALNS, and LV-CD1 Ib-hGALNS, respectively (FIG. 11 A). Statistical difference was not found in the media enzyme activity before and after uptake experiment. The intracellular enzyme activity was followed as 1.31 ± 0.8, 0.94 ± 0.5, 2.27 ± 0.3, and 2.24 ± 0.7 nmol/h/mg for LV-CBh-hGALNS, LV-CBh-hGALNSco, LV-COL2A1 -hGALNS, and LV-CD1 Ib-hGALNS, respectively. As compared to untreated MPS IVA fibroblasts, the inventors found that the enzyme activity was elevated in MPS IVA fibroblasts incubated with culture media, including the secreted GALNS enzyme (p < 0.05) (FIG. 11B).
[00149] Immunohistochemistry: The inventors performed IHC to show the localization of the GALNS enzyme. Compared to untreated and wild-type MPS IVA fibroblasts, CBh-related LV- treated groups were intensively stained with monoclonal anti-GALNS antibodies (p < 0.0001 for both vectors). No statistical difference was found between neither LV-CD1 Ib-hGALNS vs. untreated nor LV-COL2A1 -hGALNS vs. untreated (FIG. 12A-B). Furthermore, the inventors analyzed KS localization through IHC and found a significant reduction in KS levels following LV gene therapies compared to untreated controls (p < 0.05) (FIG. 13). Overall, anti-KS IHC results were parallel to KS level obtained from LC-MS/MS.
[00150] The results illustrate that the extracellular GALNS enzyme activity under CBh, COL2A1, and CD1 Ib-related LVs had variations up to 14-days but then stabilized and gradually increased until 30-days in MPS IVA fibroblasts cultures and the intracellular GALNS enzyme activity was found parallel each other from day 8 to day 30, even though lowering under LV- CBh-hGALNS vector on day 30. However, the inventors did not find statistical differences in HEK293T cells regarding the enzyme activity on days 8 and 30. Although the enzyme activity in HepG2 cells increased under CBh (at MOI 5; p = 0.0237) and COL2A1 (MOI 15; p = 0.0428)- related LVs from day 8 to day 30, no statistical difference was found in the rest of the treatment with LVs. Hence, LV transduced-MPS IVA fibroblasts and HepG2 cells stably expressed the hGALNS transgene in the cells and secreted into the extracellular area better than HEK293T cells over time.
[00151] Puentes-Tellez et al. tested MOI 1, 5, and 10 for Lenti-GALNS and Lenti-SUMFl in MPS IVA patient fibroblasts. The data showed that the intracellular GALNS enzyme activity decreased in both treatments if MOI was higher than one (Puentes-Tellez et aL, Gene 780 (2021) 145527). Therefore, the inventors decided to test MOI 20 for MPS IVA fibroblasts since lower MOIs were already tested in MPS IVA fibroblasts. Increasing MOIs (5, 10, 15) were also assessed in HEK293T and HepG2 cells. As a result, the GALNS enzyme activity in MPS IVA fibroblasts was elevated ~40-fold with LV-CBh-hGALNS, ~54-fold with LV-CBh-hGALNSco, ~170-fold with LV-COL2A1 -hGALNS, ~75-fold with LV-CD1 Ib-hGALNS and 0.7-fold with LV-CD1 lb*D8-hGALNS as compared to untreated fibroblasts. When closely investigating the results from their group and other group’s study, promoters have been found to impact transgene expression in MPS IVA fibroblasts. Although the inventors used a higher MOI than Puentes- Tellez et al., the enzyme activity was not affected. In HEK293T and HepG2 cells, it was found that the enzyme activity increased at increasing MOIs; however, this was not significant.
Furthermore, the inventors expected that increased MOIs might have toxic effects on cellular GALNS expression. For example, in HepG2 cells on day 30, the intracellular enzyme activity increased 15.8-fold at MOI 5 and 17.7-fold at MOI 15 under LV-CBh-hGALNS. The difference is too small, while MOI is three times higher in the same cell line. LV-COL2A1 -hGALNS treated HepG2 cells showed a 30.6-fold and 47.4-fold increase at MOI 5 and MOI 15, respectively. LV-CD1 Ib-hGALNS vector increased the GALNS enzyme activity 2.9-fold at MOI 5 and 4.3-fold at MOI 15 in HepG2 cells. In conclusion, the cellular expression and fold changes are not only affected by toxicity due to viral load but also promoter considerations. To increase the effectiveness of LVs, choosing an efficient promoter to integrate multiple regions in the genome and strongly express transgene in either multiple tissues or only tissue-specific regions is crucial. [00152] In another prior study, AAV vectors under CMV, EFl , and al -antitrypsin (AAT) promoters driving the GALNS gene were transduced into HEK293, human MPS IVA fibroblasts and murine MPS IVA chondrocytes. The GALNS enzyme activity showed a 13-fold to 30-fold increase in HEK293 cells with AAV vectors under CMV, EFla, and AAT promoters. However, the efficiency of the CMV promoter remained poor since HEK293 cells did not show any GALNS activity between 2 days to 10 days post-transduction. Following co-transfection with the AAV-CMV-SUMF1 vector, the GALNS enzyme activity increased 1.8-fold, 3.5-fold, and 4.0- fold under CMV, AAT, and EFla promoters, respectively. MPS IVA human fibroblasts transduced with AAV under CMV, AAT, and EFla showed 36.5%, 54.6%, and 15.3% increase in the GALNS enzyme activity, respectively, and the co-transduction with SUMF1 elevated the enzyme activity by 1.5-fold (C.J. Almeciga-Diaz, et al., Mol Biol Rep 36 (2009) 1863-1870). In the inventors’ study, the enzyme activity in HEK293T cells was lower than in HepG2 cells under each LV treatment; 18.6-fold in LV-CBh-hGALNS, 61.8-fold in LV-COL2Al-hGALNS, 2.5- fold in LV-CD1 Ib-hGALNS, 2.1-fold in LV-CD1 lb*D8-hGALNS and 8.4-fold in LV-CBh- hGALNSco treatment of HEK293T cells at MOI 5 on day 30. These results indicate that an appropriate promoter selection significantly influences transgene expression and cell target specificity. The binding of relevant proteins, RNA polymerase, and transcription factors to the promoter upstream of the interest gene initiates transgene transcription. In addition, enhancers and other DNA regions working with promoters increase gene transcription (C.J. Almeciga-Diaz, et al., FEBS J 277 (2010) 3608-3619). Promoters themselves or in combination with enhancers strengthen the expression of genes. PGK, CMV, EFla, MND, MCU3, SFFV, and CBh have been used as housekeeping promoters in a variety of LSDs, whereas CD1 lb, ALB, TBG, MHC, MLC2v, cTnT as tissue-specific promoters (E. Rintz, et al., Mol Ther Methods Clin Dev 24 (2022) 71-87). Among them, CBh is a strong promoter, comprising CMV early enhancer fused to a modified chicken P-actin promoter (E. Rintz, et aL, Mol Ther Methods Clin Dev 24 (2022) 71-87). CD1 lb is a myeloid cell-specific promoter from the alpha chain of Mac- 1 integrin (S. Dziennis, Blood 85 (1995) 319-329), while COL2A1 is a collagen type II specific promoter expressed in connective tissues and cartilage (H. Peng, et aL, J Cell Physiol 215 (2008) 562- 573).
[00153] To improve the protein expression and reduce the sequence complexity of transgene, the inventors designed a codon-optimized hGALNS gene and tested it compared to the native hGALNS gene in each cell line. No statistical significance was found regarding enzyme activities of codon-optimized and native hGALNS in vitro (Fig. 3). This proved the safety of their gene construct regarding transgene susceptibility to CpG transitional mutations. CpG motifs are immunostimulatory elements called pathogen-associated molecular patterns (PAMPs) in microorganisms (J.F. Wright, Molecular Therapy 28 (2020) 1756-1758). The presence or absence of CpG dinucleotide led to variations in the DNA sequence and DNA methylation at the cytosine residue, producing 5 -methylcytosine, resulting in a cytosine to thymine transitional point mutation. Furthermore, 10-60% of point mutations in genetic disorders have resulted from CpG-related point mutations. In terms of MPS IVA, this ratio accounted for over 20% (S. Tomatsu, J Med Genet 41 (2004) e98-e98). Another study showed that more than 35 % of IDS point mutations were found at CpG hot spots (Tomatsu, S., et al., Hum Mutat. 2004 Jun; 23(6): 590-8). The inventors’ study determined that the GALNS gene from MPS IVA patients’ skin fibroblasts had C.122T>A (p.Met41Lys) mutation on exon 2 in which CpG sites were abundant. The study reported that the methylation of CpG cytosines was abundant between exon 2 and 14, which caused the GALNS gene to be susceptible to mutations. Only exon 1 had unmethylated CpG cytosines, which correlated with the absence of transitional mutations (Tomatsu, S., et al., Hum Mutat. 2004 Jun; 23 (6): 590-8). All in all, methylated CpG sites in native hGALNS are exposed to the high risks of C-to-G mutations. Therefore, it may optimize the transgene sequence to eliminate CpG sites for the safety of gene therapy. Besides the adverse effects on transgene expression, CpG motifs lead to a robust immune response when interacting with toll-like receptors (TLRs). TLRs are abundant in macrophages, mast cells, dendritic cells, B and T cells, endothelial cells, epithelial cells, and fibroblasts (Wicherska-Pawlowska and Wrobel, Diseases. IntJ Mol Sei. 2021 Dec 13;22(24):13397 Shirley, JL, et al., Mol Ther. 2020 Mar 4;28(3):709-722). In the prior art AAV vector, CpG motifs dimerize TLR9 in plasmacytoid dendritic cells following binding, resulting in innate and adaptive immune responses. Moreover, high CpG contents in the expression cassette induce cytotoxic T cell (CTL) response eliminating transduced cells. Thus, hepato-immunotoxicity occurs after systemic administration of AAV vectors (Wicherska-Pawlowska and Wrobel, Diseases. IntJ Mol Sei. 2021 Dec 13;22(24):13397). CpG motifs were depleted in AAV vectors since TLR9 was known to be specific for DNA viruses and easily recognize unmethylated pathogen-related CpG motifs to activate transcription factors and related proteins. The results indicated that the depletion of CpG motifs improved transgene expression, avoided immunity, and minimized infiltration of effector cells upon skeletal gene transfer in mice (Shirley, JL, et al., Mol Ther. 2020 Mar 4;28(3):709- 722; Faust SM, et al. J Clin Invest. 2013 Jul;123(7):2994-3001). Furthermore, CpG depletion of the AAV vector genome expressing human coagulation factor IX reduced CD8+ T cell infiltration after intramuscular gene transfer in hemophilia B mice (Bertolini TB, et al., Front Immunol. 2021 May 31;12:672449). Another study confirmed that the depletion of CpG motifs in a cationic lipid-plasmid DNA eliminated the interaction with TLR9 and thus reduced acute toxic response (de Wolf, HK, et al., Pharm Res. 2008 Jul;25(7); 1654-62). LVs, including integrase-deficient LVs and reverse transcriptase-deficient LVs, are known to be less immunogenic; however, the study showed that myeloid dendritic cells were activated immediately after lentiviral entry and reverse transcription in in vitro and in vivo although these cells are the absence of TLR9 molecules. Further experiments revealed that LVs interacted with TLR3, which may be activated by viral double-stranded RNA, and TLR7, which has doublestranded life cycle intermediates, may activate reverse transcription (Dela Justina V, et al., Clin Sei (Lond). 2020 Jan 31 ; 134(2):303-313). In conclusion, the inventors tested a codon-optimized expression cassette to confirm GALNS transgene expression in vitro, and it will further be used in mouse models.
[00154] The absence of the GALNS enzyme led to the accumulation of GAGs within lysosomes, resulting in MPS IVA disorder (Khan S, et al., Mol Genet Metab. 2017 Jan-Feb;120 (1-2). The elevated GALNS enzyme in MPS IVA patients’ skin fibroblasts following LV gene therapy reduced KS GAG levels through LC-MS/MS, supported by IHC results (FIG. 8 and FIG. 13). Additionally, the inventors found a negative correlation between the GALNS enzyme activity and KS levels under each LV treatment. However, it was not significant (data not shown). LC-MS/MS has a strong sensitivity, specificity, and accuracy in detecting each GAG, including KS, HS, DS, and CS in various specimens such as blood, urine, cerebrospinal fluid, dried blood spots etc. (Kubaski F, et al., Mol Genet Metab. 2017 Jan-Feb). The inventors’ study indicated that LC-MS/MS was a precise method for identifying GAGs in cellular concepts. [00155] To increase the transgene expression along with promoters and enhancers, vector copies per cell might be considerable. Some studies indicated that high vector copies resulted in elevated transgene expression. Thus, the inventors assessed each LV-transduced cell under different MOIs in terms of VCN. Although insignificant, the data showed a positive correlation between VCNs and enzyme activity levels in fibroblasts. However, increasing MOI increased VCN in HepG2 and HEK293T cells, which was significant (p < 0.05). Nevertheless, the nature of LVs should be taken into consideration. Since the viral genome is randomly integrated into the genome, there is a chance to bind to a hot spot contributing to oncogenic effects in treated groups. A cancer incident occurred following LV administration in a clinical trial for sickle cell disease. Researchers found that viral DNA was integrated into the MDS-linked site, resulting in abnormally proliferated blood stem cells (Espinoza DA, et al., Mol Ther. 2019 Jun 5;27(6): 1074- 1086). The dose justifications and viral integration features might be considered for in vivo studies and clinical trials to develop a safe and efficient LV gene therapy.
[00156] LV gene therapy is a promising, one-time, and permanent treatment for many diseases, including genetic disorders and cancers. LV gene therapy in three cell lines was assessed under the effect of different promoters with increasing doses of viral load. The inventors restored the GALNS enzyme activity within lysosomes and confirmed its secretion into the extracellular area. The enzyme activity showed variations based on the cellular origin and promoter profile. It is crucial to investigate by in vivo studies on mouse models the gene expression profile under ubiquitous and -specific promoters at tissue levels. Improving the safety concerns of LV gene therapy will result in an attractive treatment for many diseases in the future
Example 2. Ex vivo Gene Therapy
[00157] The inventors anticipated that ex vivo lentiviral (LV) gene therapy could produce the active GALNS enzyme continuously by the infused hematopoietic stem cells (HSCs) treated with LVs carrying the native GALNS gene under three different promoters (ubiquitous-CBh, collagen type II-COL2A1, hematopoietic stem cells-CDl lb) into the circulation and it would significantly impact bone and cartilage abnormalities in MPS IVA. As shown experimental design illustrated in FIG. 14A-14C the inventors injected busulfan intraperitoneally into knockout (KO) newborn male mice to condition them for 24 hours. Then, HSCs were isolated from bone marrow of donor mice and transduced with LVs at a particular MOI. Following incubation for 20 h, HSCs were collected to treat intravenously busulfan-conditioned newborn male mice. Blood samples were collected biweekly following ex vivo infusion, and mice were autopsied at 16 weeks old to collect tissues. The therapeutic efficacy of ex vivo lentiviral gene therapy in MPS IVA mice was assessed. To do that, the inventors investigated vector copy number, enzyme activity levels, and the GAG concentrations in plasma, white blood cells, bone marrow cells, and tissue samples. Mouse studies demonstrated that HSCs treated with LVs under ubiquitous CBh promoter in KO newborn mice had the highest enzyme activity. Furthermore, following busulfan conditioning, the GALNS enzyme activity in plasma, WBCs, BM, and liver under each promoter was detected.
Methods and Results
[00158] MPS IVA mice: MPS IVA knock-out (KO) mice (GALNS \ MKC2) were generated at Inotiv (West Lafayette, IN). This MPS IVA mouse model had a large deletion of ~6300bp (exons 2-5: 6359bp) at genomic coordinates. We designed, generated, and tested cellular assay sgRNAs for CRISPR-Cas9 mediated knock-out in C57BL/6J mouse zygotes. Briefly, we designed sgRNAs targeting upstream of exon 2 and upstream of exon 6 of mouse GALNS, which disrupted mGALNS and made the MPS IVA murine model. The most potent sgRNA with minimal off-target potential was assembled into a ribonucleoprotein complex with Cas9 endonuclease and delivered into zygotes from C57BL/6J mice, followed by embryo transfer into pseudo-pregnant females. Viable progeny was analyzed for the desired mutation by genomic PCR and DNA sequencing. Twenty-three transgenic founder mice with knock-out have been identified to be positive. The two founders of choice were backcrossed to wild-type mice to generate Fl heterozygous progeny for the GALNS” mice, as confirmed by PCR-mediated genotyping and DNA sequence analysis. Nine heterozygous mice (2 males) were identified (GALNS+/‘). Cohort breeding, cryopreservation, in vivo assays, and molecular analyses were made for these mice. After mating heterozygous male and female mice, we obtained homozygous mice (GALNS’7’, MKC2). The age-matched male (wild-type and untreated) mice were used as controls. The colony was housed in a pathogen-free facility on a 12-hour light/dark cycle. All mouse care and handling procedures were in accordance with the rules of the Institutional Animal Care and Use Committee (IACUC) of Nemours Hospital Delaware Valley. [00159] Construction of LVs: LVs were constructed as described in the in vitro example above. To develop LV-HST GT for patients with MPS IVA, the inventors designed 3rd generation self-inactivating LVs with native human GALNS transgene driven by the ubiquitous CHh or collagen-specific COL2A1 promoters. The final constructs for the transduction of HSCs were LV-CBh-hGALNS and LV-C0L2Al -hGALNS. (FIG. 14 B) To confirm whether each LV has a therapeutic efficacy, we transduced fibroblasts (Gains’ ’) derived from MPS IVA patients at the MOI of 20 according to the company instructions (Fig. 2A-B) and HSCs derived from MPS IVA mice. We evaluated enzyme activities before moving to ex vivo experiments compared to healthy controls (healthy human skin fibroblasts and wild-type mice HSCs) (FIG. 15A-D) [00160] Isolation and Transduction of Mouse HSCs: As shown in FIG. 14, Donor bone marrow was harvested from the femur and tibia of 8- to 12-week-old male GALNS’7’ mice for treatment groups (LV-HSC GT) and from wild-type donors for allogeneic HSCT (allo-HSCT) control group (FIG. 14 A). GALNS’ ’ and wild-type donor HSCs were purified using EasySep™ mouse hematopoietic progenitor cell isolation kit (Stem Cell Technologies# 18000, Vancouver, Canada). We utilized two different medium recipes to culture isolated HSCs: Medium 1. Dulbecco’s modified Eagle’s medium nutrient mixture F-12 (DMEM/F12, Gibco#l 1320033, Grand Island, NY) supplemented with 1% insulin/transferrin/selenium (ITS; Gibco#41400045), 1% penicillin/ streptomycin (Pen/Strep; Sigma- Al drich#P4333, Burlington, MA), 0.1% recombinant human albumin (RHA; SeraCare Life Sciences# 18600016, Milford, MA), 10 mM 2-[4-(2-hydroxyethy])piperazin-l-yl]ethane-l -sulfonic acid (HEPES;
ThermoSci entific#Jl 6924. AE, Waltham), and the growth factors (PeproTech, Cranbury, NJ) - 10 ng/ml murine stem cell factor (SCF; #25003, PeproTech), 100 ng/ml murine thrombopoietin (TPO; #31514), and 100 ng/ml Fms-related tyrosine kinase-3 (Flt-3; #25031L). Medium 2. Ham’s F12 nutrient mix medium (Gibco#l 1765-054, Waltham) supplemented with 10 mM HEPES, 1% penicillin-streptomycin-glutamine (PSG; Gibco#10378-016, Waltham), 1% insulin- transferrin-selenium-ethanolamine (ITSX; Gibco#51500-056, Waltham), 1 mg/ml polyvinyl alcohol (PVA; Sigma-Aldrich#P8136), 100 ng/ml TPO, and 10 ng/ml SCF as described in the published protocol. Wilkinson, A.C., et al., Nat Protoc 15 (2020) 628-648. Then, lin’ HSCs were seeded at a density of 0.9 x 106 cells per well into both complete medium 1 and 2 for further LV transduction.
[00161] To modify GALNS ” HSCs by LVs, we followed the MOI of 60 based on the literature. Sergijenko, A., et al., Molecular Therapy 21 (2013) 1938-1949; Gleitz, H.F., et al., EMBO Mol Med 10 (2018). GALNS’ ’HSCs were transduced with LVs expressing the GALNS transgene at the MOI of 60 and incubated for 20 h at 37°C 5% CO2. The isolated HSCs were stained with HSC markers c-Kit, Sca-1, and SLAM. Following selected antibodies and isotypes were used (Biolegend, San Diego, CA): APC anti-mouse c-Kit (CD117; 0.2 mg/ml; #105811 , Biolegend), FITC anti-mouse Sca-1 (Ly-6A/E; 0.5 mg/ml; #122506, Biolegend), Pacific Blue anti-mouse SLAM (CD150; 0.5 mg/ml; #115924), APC Rat IgG2b K (#400612, Biolegend), FITC Rat IgG2a K (#400506, Biolegend) and analyzed through flow cytometry to confirm the purity.
[00162] Busulfan Conditioning and HSCs Transplantation in MPS IVA Mice: In vitro assays, e.g., cell culture assays, can be used to measure hGALNS expression from a cell transduced with the LV-hGALNS described herein, thus indicating, e.g., potency of the LV-hGALNS. Cells utilized for the assay can include, but are not limited to, A549 , WEHI, 10T1/2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, HEK293, HEK293-T, HuH7, Saos, C2C12, L, HT1080, HepG2, primary fibroblast, hepatocyte, and myoblast cells. In a specific embodiment, the cells utilized in the cell culture assay comprise HuH7 cells. In certain embodiments, cells transfected with the LV-hGALNS can be analyzed for hGALNS enzyme activity. (See FIG. 14)
[00163] WST-1 Toxicity Assay: The WST -1 toxicity assay tests if LV transduction and further GALNS enzyme expression have a cytotoxic effect on MPS IVA patients’ fibroblasts at the MOI of 20. Cells were seeded at 1 x 104 cells/well in 100 pl culture medium and transduced for 48 h at 37°C and 5% CO2. Then, 10 pl of cell proliferation reagent WST-1
(Abcam#ab 155902, Cambridge, UK) was added to each well, mixed by pipetting gently, and incubated for 4 h at 37°C and 5% CO2. A blank control: 10 pl of WST-1 reagent was added to 100 pl of culture medium prepared parallel to experimental groups. The absorbance of each group was measured in a plate reader (FLUOstar Omega plate reader; BMG LABTECH Inc, NC) at an optical density of 450 nm, and the reference wavelength was set to 650 nm. The results were evaluated by averaging the duplicate reading for each sample and then subtracting the culture medium-related background from each sample to obtain the final absorbance. The equation to calculate the percentage of cytotoxicity using final absorbance is as follows: %Cytotoxicity=(100x(Control-Samples))/Control.
[00164] Keratan sulfate (KS) assay by liquid chromatography-tandem mass spectrometry (LC-MS/MS): LC-MS/MS analysis allowed us to evaluate the reduction of accumulated mono- sulfated-KS. The plasma and tissues of treated, untreated, and wild-type mice. FIG. 17A-17D. [00165] Vector Copy Number (VCN): To confirm the biodistribution of LVs in cultured Gains' " HSCs 24 h and 15 days post-transduction and in the liver 16 weeks post-transplantation, DNA was extracted from the cultured mouse HSCs and liver samples with the Qiagen Gentra Puregene Tissue Kit, and DNA was digested with Proteinase K at 55°C. Following Rnase treatment, DNA was resuspended in Tris-EDTA buffer, and DNA concentration was measured. Quantification of VCN was performed by digital PCR (dPCR; ThermoFisher QuantStudio Absolute Q) as single plex (LV and Tfrc on 2 separate chips) using the primers (Integrated DNA Technologies) specific to the LV vector psi gene, resulting in an 82-bp PCR fragment (Table SI). QuantStudio™ Absolute Q™ MAP16 Plate Kit and Master Mix (ThermoFisher#A53301) were used to prepare the reaction. The quantification was done with the following formulation: (LV cps/pl*DNA dilution)/(Tfrc cps/pl/2). Results are shown in FIG. 15F
[00166] Colony Forming Unit (CFU) Assay: To set up the CFU assay for triplicate cultures, we thawed a vial of previously aliquoted 4 ml MethoCult™ GF M3434 complete media (StemCell Technologies#03434, Vancouver, Canada) overnight at 4°C. A total of 1 x 105 cells/ml from each group (untreated, LV-CBh and LV-COL2A1 -treated groups) was diluted in the Iscove’s Modified Dulbecco’s medium (IMDM; Gibco#12440053) with 2% FBS. Afterward, 400 pl of diluted cell suspensions were added to a 4 ml MethoCult™ tube previously thawed and mixed thoroughly via vortexing. Following removal of bubble formation, MethoCult™ mixture containing cells were distributed evenly in 3 wells of 6-well plates (9.6 cm2/well; Falcon™353046; Fisherscientific#08-772-lB, Waltham) and incubated at 37°C, 5% CO with >95% humidity for 7-10 days according to the manufacturer’s instructions. Colonies were identified and counted by visual inspection using STEMgrid™-6 (StemCell
Technologies#27000) via an inverted light microscope at 4X and 10X magnification. After completing colony counting, HSCs in each well were collected in separate vials to confirm the GALNS enzyme activity and VCN of these pooled CFU colonies. Results are shown at FIG. 15E, 15G, and 15H
[00167] Pathology: Mice were euthanized with CO2 gas 16 weeks post-transduction, and then the heart, liver, and knee joints were collected in 10% formalin. To evaluate lysosomal storage by light microscopy, these tissues were then fixed in 2% paraformaldehyde, 4% glutaraldehyde, and toluidine blue-stained 0.5-pm-thick sections were prepared. Bone pathology was quantified by using Image J (NIH). To evaluate storage quantitatively, the inventors measured cell number, cell size, organization of the growth plate, and epiphyseal and articular cartilage thickness (Sawamoto et al., 2020b). FIG. 18
[00168] MicroCT: A micro-CT scan was performed on the femur using Sky Scan 1276 Micro- CT System (Bruker, Manning Park, MA). The femur samples were collected in 100% EtOH at 16 weeks post-transduction, and they were further wrapped in salinated (0.9% saline) gauze in preparation for the micro-CT imaging. The scanning was performed with high spatial resolution down to 2.8 pm pixel size, 528 projections, exposure time of 50 msec, photon energy of 80 keV, and current of 125 pA. Three-dimensional reconstruction of each bone was made (Azario et al., 2017; Pievani et al., 2015; Rowan et al., 2013). The inventors evaluated bone structure (trabecular and cortical bones) by measuring the following parameters: bone mineral density, total volume (TV), bone volume, thickness, BV/TV, etc. (Azario et al., 2017; Pievani et al., 2015). Results are shown in FIG. 19A-19H.
[00169] Enzyme Assay with 4-Methylumbelliferone (4-MU): The GALNS enzyme activity in cells, plasma, and tissue extracts was meticulously assessed using a 4-methylumbelliferone (4- MU) assay (Melford Laboratories Ltd, Suffolk, UK). MPS IVA patients’ fibroblasts, wild-type mice HSCs, and Gains' " mice HSCs post-transduction were lysed in 60-100 pl of homogenization buffer (25 mM Tris-HCl, pH 7.2, 1 mM PMSF) via sonication for 30 sec and 10% amplitude. Then, cell lysates were centrifuged for 10 min at 4°C. The supernatant was transferred into a new tube and assayed for the GALNS enzyme activity. As stated above, tissue was dissected and immediately homogenized with Bead Mill Homogenizer (OMNI International, Kennesaw, GA) in homogenization buffer. Homogenates were centrifuged for 30 min at 4°C, and the supernatant was transferred into a new tube. Both supernatants of cells/tissues and plasma samples underwent 4-MU enzyme assay and were incubated with 22 mM 4-methylumbelliferyl- P-galactopyranoside-6-sulfate (Research Products International, Mount Prospectm, IL) at 37°C for 16 h. The next day, lo mg/ml of -galactosidase from Aspergillus oryzae (Sigma-Aldrich, Saint Louis, MO) was added into the reaction and incubated for 1 h at 37°C. The reaction was stopped with 1 M of glycine buffer (pH 10.5 adjusted by NaOH). The FLUOstar Omega plate reader (BMG LABTECH Inc. NC) was used to measure the enzyme activity at an excitation wavelength of 336 nm and an emission wavelength of 450 nm. The activity was expressed as nanomoles of 4-methylumbelliferone released per hr per milligram of protein (nmol/h/mg). Protein concentrations were determined using a Pierce™ BCA protein assay kit (Thermo Fisher Scientific #23225, Waltham, MA) Results are shown at FIG. 16A-I.
[00170] Immunohi stochemi stry : The GALNS enzyme expression in the tibia and liver was confirmed by performing immunohistochemistry (IHC) of GALNS. Additionally, we analyzed KS and procollagen II levels via IHC. Collagen, KS, and GALNS were stained by antiprocollagen (Invitrogen#BTE0030202, Waltham, MA), anti-KS (Santa Cruz Biotechnology#sc- 73518, Dallas, TX), and custom-made monoclonal anti-GALNS antibodies (Creative Biolabs, NY). The liver and tibia were fixed in 10% formalin and sectioned with 5 pm-thickness for IHC. KS, GALNS, procollagen II distribution, and intensity patterns were investigated immunohistochemically to determine any correlation with therapeutic effects. To evaluate the expression of GALNS and collagen and the reduction of KS, we used Image J (NIH) software and analyzed each slide, selecting only the proliferative region of the growth plate using software tools. After selection, we utilized the software color intensity tools to quantify each section and compare it with untreated and wild-type controls.
[00171] Anti-GALNS Antibodies: Biweekly collected plasma samples were assayed to determine the antibody response against hGALNS (Milani et al., 2019; Schlimgen et al., 2016; Wang et al., 2011). To determine the anti-GALNS antibody response, ninety-six well polystyrene microplates were coated with 2 ug/ml of Vimizin® enzyme in the coating buffer (15 mM Na2CC>3, 35 mM NaHCOs, 0.021% NaN?, pH 9.6) and incubated overnight at 4°C. Then, the coated plates were blocked with 3% BSA in PBS for 1 h at room temperature and washed first with TTBS (10 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.5) and second with TBS (10 mM Tris, 150 mM NaCl, pH 7.5). Biweekly collected plasma samples (1 : 100 dilution in TBST) and monoclonal anti-GALNS antibodies (Custom-made clone 2F5F2, Creative Biolabs, NY) were added into each well individually and incubated for 2.5 h at 37°C. After 5 times washing the plates with TTBS (x3) and TBS (x2) successively, the plates were incubated with peroxidase- conjugated goat anti-mouse secondary antibodies (Invitrogen#656120, Waltham) in TBST at room temperature for 1 h according to manufacturer’s instructions. Following 4 washes, first with TBST and the rest with TBS, 100 pl of peroxidase substrate 2,2’-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid) (ABTS; Invitrogen#002024) was added into the reaction and incubate at room temperature for 30 min to develop the signal. The enzyme reaction was stopped with 100 pl of 2% sodium dodecyl sulfate, and the absorbance was measured at 450 nm using a FLUOstar Omega plate reader stated above. Plasma concentrations of anti-GALNS antibodies were derived by extrapolating the absorbance values from a calibration curve using monoclonal anti-GALNS antibodies mentioned above.
[00172] GAG Levels: To confirm whether accumulated GAG levels decreased following the LV treatments, (KS) levels were measured in plasma and bone harvested at 16 weeks. (FIG.
17A-B)
[00173] Evaluation of Gains " (Lin ) HSCs Transduced with LVs
[00174] Lineage-negative (Lin') HSCs were isolated from the bone marrow of GALNS " donor mice and wild-type donor mice via magnetic selection. The purity of lin' HSCs was measured immediately after isolation via flow cytometry by staining c-Kit (CD 117), Sca-1, and SLAM (CD150) surface markers, in which the final population of lin' cells was -21% of all isolated cells. Then, lin7c-Kit+/Sca-l+hematopoietic progenitor cells were gated among lin' (20.57%), 95.75% of which were positive for CD150 marker (Lin'Scal+c-kit+CD150+). The isolated cells were transduced in media 1 and 2 and analyzed for the GALNS enzyme activity and VCN 24 h and 15 days post-transduction. In medium 1, transduced or untransduced HSCs were maintained up to 36-72 h; however, they were either differentiated, aggregated, or primarily dead after 72 h post-transduction. Only enzyme activity and VCN were analyzed in 24 h posttransduction in medium 1. In medium 2, the same transduction method was followed, and MOIs, in which the sternness of transduced HSCs was well-maintained and cultured for long-term to test the enzyme activity of GALNS, VCN, mono-sulfated KS levels, and other relevant experiments. The data showed no statistical differences in GALNS enzyme activity, VCN, or mono-sulfated KS levels in both media after 24 h; however, the maintenance of lin' HSCs was well-established without any differentiation in medium 2 during procedures. Therefore, the rest of the experiments were continued in medium 2 to culture and analyzed these cells for the long term. We then evaluated the purity and proliferation of cultured lin' HSCs on 20 days post-LV- transduction as described above. The data showed that the percentage of untreated Gains' ', LV- CBh-hGALNS, and LV-COL2Al-hGALNS treated lin' HSCs reached 52.39%, 46.62%, and 59.74%, respectively, among which Scal+c-kit+cells accounted for 97.57%, 96.39%, and 97.93%, respectively. In all groups, lin'Scal+c-kit+CD150+cells were found at 97.33%, 96.44%, and 96.28% for untreated, LV-CBh-hGALNS, and LV-COL2Al-hGALNS, respectively. These lin' cells were then evaluated for the production and secretion of the GALNS enzymes, which might result from a possible engraftment potential. However, an engraftment experiment was not performed in the present study. In medium 2 culturing conditions, LV-transduced HSCs were cultured for up to 30-to-45 days with no differentiation; however, such long-term cultures may have poor engraftment efficiency. Thus, no further analysis regarding this process was performed. In murine experiments, all conditioned Gains' " newborn recipients received LV- transduced HSCs 20-24 h post-transduction. Overall, even though the culturing conditions and maintenance of HSCs were improved, transduction with the LVs was not efficient in cultures as expected at the MOI of 60.
[00175] Ubiquitous CBh promoter elevated the GALNS enzyme activity rather than tissue-specific COL2A1 .
[00176] HSCs derived from MPS IVA mice were transduced with LVs to verify the GALNS enzyme activities compared to untreated and wild-type control HSCs. In transduced Gains' " HSCs, the enzyme activity of GALNS at 24 h and 15 days post-transduction were compared. Following 24 h of transduction, the enzyme activity increased up to 0.9 ± 0.04 nmol/h/mg under LV-CBh-hGALNS as compared to untreated Gains' " HSCs, and no statistical difference was found between LV-CBh-hGALNS -transduced and wild-type HSCs (0.7 ± 0.1 nmol/h/mg). At the same time, LV-COL2Al-hGALNS elevated enzyme activity by 0.3 ± 0.03 nmol/h/mg. The data confirmed that LV-CBh-hGALNS had ~3-fold more enzyme activity than LV-COL2A1- hGALNS compared to untreated HSCs. However, with 15 days of culturing LV-transduced cells, the enzyme activity reduced to 0.5 ± 0.03 and 0.06 ± 0.03 nmol/h/mg under LV-CBh-hGALNS and LV-COL2Al-hGALNS treatments. No statistical significance was found regarding this reduction between 24 h and 15 days.
[00177] The overexpression of the GALNS enzyme was also evaluated by utilizing HSCs isolated from wild-type mice. The results showed that the activity of the GALNS enzyme increased by 86.8% and 88.6% under LV-CBh-hGALNS (6.07 ± 0.1 nmol/h/mg) and LV- COL2Al-hGALNS (7.02 ± 0.3 nmol/h/mg), respectively compared to untransduced wild-type HSCs (0.8 ± 0.06 nmol/h/mg).
[00178] Confirming the GALNS enzyme activity in Gains' " donor HSCs under LVs, the LV- HSCs or allo-healthy HSCs were intravenously transplanted into busulfan myeloablated Gains" newborn recipients. [00179] After transplantation and autopsies at 16 weeks, plasma, WBCs, and tissue enzyme activities was analyzed, including brain, trachea, lung, heart, liver, spleen, and bone (tibia) (Fig. 16A-I). In the brain, the enzyme activity under each treatment was undetectable compared to that of wild-type mice (2.7± 0.09 nmol/h/mg). The enzyme activity was elevated in the trachea under LV-CBh-hGALNS (0.3 ± 0.09 nmol/h/mg) compared to that of untreated MPS IVA mice. However, it did not reach the enzyme activity of wild-type mice (4.7 ± 0.6 nmol/h/mg) (Fig. 16B). The enzyme activity via LV-COL2Al-hGALNS treatment was insufficient to detect in the trachea. Similar results were found in the lungs, bone (tibia), and heart under LV-COL2A1- hGALNS treatment. However, LV-CBh-hGALNS treatment increased the GALNS enzyme activity in the lungs, bone (tibia), and heart by 0.4 ± 0.2, 1.03 ± 0.5, and 0.01 ± 0.006 nmol/h/mg, respectively, compared to wild-type levels (1.6 ± 0.4, 2.6 ± 0.6 and 0.1 ± 0.02 nmol/h/mg, respectively). In the liver, the GALNS enzyme activity increased under both vectors. Treatment with LV-CBh-hGALNS showed an increase by 2.3 ± 0.4 nmol/h/mg, while LV-COL2A1- hGALNS treated group showed a slight elevation by 0.07 ± 0.05 nmol/h/mg as compared to that of untreated group (Fig. 16E). The GALNS enzyme activity was normalized to the wild-type level (4.2 ± 0.9 nmol/h/mg) in the liver and bone (tibia). No statistical significance was found between LV-CBh-hGALNS and wild-type groups. In the spleen, both LVs drove the GALNS expression, increasing enzyme activity. Treatment with LV-CBh-hGALNS showed a significant increase in the GALNS enzyme activity by 5.1 ± 1.2, which was -2.7 times higher than the wildtype level (1.9 ± 0.2 nmol/h/mg). Additionally, the enzyme activity under treatment with LV- COL2Al-hGALNS (0.08 ± 0.02 nmol/h/mg) was significant compared to the wild-type level.
[00180] In plasma, treatment with LV-CBh-hGALNS normalized the enzyme activity to wildtype level starting from 6 to 16 weeks, and no statistical difference was found between LV-CBh- hGALNS and wild-type mice, except for 4 and 6 weeks. The enzyme activity under LV- COL2Al-hGALNS was not detectable in plasma. Furthermore, the allo-HSCT group was found to be -0.003 in plasma all over the experiment compared to LV-HSC GT.
[00181] In WBCs, the GALNS enzyme was highly expressed under LV-CBh-hGALNS over 16 weeks (the highest, 74.6 ± 24.1 nmol/h/mg, and the lowest, 8.4 ± 1.5 nmol/h/mg), which was found statistically significant compared to wild-type levels (-0.12 ± 0.05 nmol/h/mg). In addition, HSC-transplanted mice with LV-COL2Al-hGALNS elevated the GALNS enzyme activity in WBCs by -0.7 ± 0.5 nmol/h/mg. The GALNS enzyme activity was normalized to the wild-type level (Fig. 161). This expression of the GALNS enzyme under treatment with LV- C0L2A1 -hGALNS was further detected in BMCs.
[00182] In the LV-HSC GT group, the enzyme activity of BMCs was analyzed under only LV-COL2Al-hGALNS and allo-HSCT groups because the collection of BMCs in the group treated with LV-CBh-hGALNS was missed. Relatively low enzyme activity levels were detected in BMCs and slightly higher levels in WBCs of the same individual mice.
[00183] In conclusion, the findings suggest that most enzymes might have been captured in the spleen due to its role in the local and systemic regulation of immunity. This may negatively affect the delivery of the GALNS enzymes to the targeted tissues, specifically the bone.
[00184] Tissue-specific COL2A1 LVs reduced the accumulation of GAGs under lower expression of hGALNS.
[00185] To confirm the effectiveness of LV-GT in KS reduction, LC-MS/MS was performed to analyze the bone (humerus) and plasma mono-sulfated KS levels. In the plasma, statistical significance was found with a 50.1% reduction in mono-sulfated KS level in mice treated with LV-CBh-hGALNS (38.82 ± 1.9 ng/ml) and 44.4% reduction with LV-COL2A1 -hGALNS (43.86 ± 4.8 ng/ml) treatment compared to untreated MPS IVA mice (78.9 ± 6.1 ng/ml). There were no statistical differences between the LV-treated and wild-type groups (23.79 ± 3.02 ng/ml). Additionally, allo-HSCT group had no statistical difference compared to the untreated, which results in no change in mono-sulfated KS levels in plasma. In WBCs, LV-COL2 Al -hGALNS (6.09 ± 0.8 ng/ml) reduced KS level by 56.2% compared to untreated control (13.93 ± 1.02 ng/ml) and no statistical significance was found between the group treated with this vector and wild-type (7.40 ± 1.07 ng/ml). In contrast, there was no statistical significance in mono-sulfated KS concentrations between the LV-CBh-hGALNS group (14.90 ± 3.50 ng/ml) and the untreated group. In the bone, the differences in KS level by 40.7% between untreated Gains7- (0.05 ± 0.003 ng/mg) and wild-type group (0.03 ± 0.003 ng/mg). The group treated with LV-CBh-hGALNS showed a 14.1% reduction in mono-sulfated KS levels (0.04 ± 0.004 ng/mg), but it was insignificant compared to the untreated group. On the other hand, KS level decreased by 40.2% under treatment with LV-COL2 Al -hGALNS (0.03 ± 0.002 ng/mg) compared to untreated MPS IVA, which was statistically significant. These data showed that LV-COL2A1 -hGALNS treatment normalized mono-sulfated KS level to that of wild-type. Additionally, the monosulfated KS level of WBCs and bone were found to be similar under each treatment compared to untreated and wild-type controls at 16 weeks. Concerning mono-sulfated KS levels under allo- HSCT, neither WBCs nor bone showed a significant reduction. As a result, LV-HSC-GT under each promoter significantly reduced mono-sulfated KS levels in plasma and bone. Importantly, LV-COL2Al-hGALNS significantly reduced mono-sulfated KS levels in bone compared to those in LV-CBh-hGALNS.
[00186] LVs under ubiquitous or tissue-specific promoters were similarly inserted into the genome of HSCs.
[00187] To determine the VCN, dPCR was performed targeting the psi gene LV packaging signal sequence in HSCs 24 h and 15 days post-transduction following each treatment. In LV- modified Gains'7’ HSCs after 24 h, LV VCNs were found by 0.75 ± 0.02 and 0.74 ± 0.02 relative to 2-copy control of Tfrc under LV-CBh-hGALNS and LV-COL2Al-hGALNS, respectively while VCN on day 15 was 0.24 ± 0.003 and 0.74 ± 0.003 per relative to 2-copy control of Tfrc, respectively. This reduction in the LV-CBh-hGALNS group on day 15 was significant compared to 24 h post-transduction. No change was detected in LV-COL2A1-HGALNS-HSC group. Then, VCN was performed in LV-transduced wild-type HSCs on day 15, which was found that LV- CBh had 0.25 ± 0.005 while LV-COL2A1 was 0.73 ± 0.003 relative to 2-copy control of Tfrc. Furthermore, we analyzed VCN in pooled CFU colonies originating from both LV-modified Gains’7’ and LV-modified wild-type HSCs. The results confirmed that the VCN was 0.36 ± 0.001 and 0.7 ± 0.005 relative to 2-copy control of Tfrc in LV-CBh and LV-COL2A1 -modified Gains’7’ HSCs, while it was 0.37 ± 0.005 and 0.7 ± 0.003 relative to 2-copy control of Tfrc in LV-CBh and LV-COL2A1 -modified wild-type HSCs (Fig. 15H). Overall, VCN was similar in Gains'7’ HSCs and wild-type HSCs and did not show significant alterations before and after the CFU assay. VCN was further analyzed in liver samples at 16 weeks, which was 0.03 ± 0.007 and 0.03 ± 0.01 relative to 2-copy controls of Tfrc under LV-CBh-hGALNS and LV-COL2Al-hGALNS, respectively.
[00188] LV-GT did not affect the colony formation of transduced HSCs while increasing the GALNS enzyme activity in the pooled CFU colonies.
[00189] To determine whether transduction of LVs negatively affects the colony formation of Gains’7’ and wild-type HSCs, 7 days post-transduced HSCs were seeded at the recommended number and incubated for 7-10 days. Following colony counting, CFU-GM, CFU-GEMM, and BFU-E were found under each condition. Further evaluations showed no significance in colony formation between untreated and LV-HSC GT groups. Furthermore, we pooled the CFU colonies and analyzed the GALNS enzyme activity. The results confirmed that the enzyme activity of pooled colonies originated from LV-modified Gains7- HSCs was determined by 11.4 ± 2.8, 4.4 ± 0.7, and 8.4 ± 2.5 nmol/h/mg for LV-CBh-hGALNS, LV-COL2A1 -hGALNS, and wild-type group, respectively. In contrast, it was found in LV-modified wild-type HSCs by 19.7 ± 6.3 and 12.8 ± 1.8 nmol/h/mg under LV-CBh-hGALNS and LV-COL2Al-hGALNS, respectively. Overall, LV-HSCs continuously produced the GALNS enzyme before and after differentiation. [00190] COL2A1 LVs completely corrected heart pathology and partially improved bone pathology.
[00191] To evaluate the effect of expressed GALNS enzyme, the heart and knee joint were examined by toluidine blue staining. In the bone, both treatments under CBh and COL2A1 promoters driving hGALNS expression showed partial correction in vacuolization and column structure of chondrocytes compared to untreated groups. However, they did not reach normalization. Furthermore, the size of chondrocytes was measured in the growth plates of the tibia and femur to confirm whether LV treatment reduces the storage materials. The data showed that the size of chondrocytes was nomialized to the wild-type level with both LV-CBh-hGALNS and LV-COL2A1 -hGALNS vectors.
[00192] In the heart, the treatment with LV-COL2 Al -hGALNS showed complete correction of disease progression except for one mouse, with partial correction among all groups, compared to untreated MPS IVA mice. No statistical differences were found between the group treated with LV-COL2A1 -hGALNS and the wild-type control group. Moreover, the group treated with LV-CBh-hGALNS showed a partial correction in the vacuolization of heart structures, which did not reach that of the wild-type level.
[00193] Trabecular and cortical bone morphology
[00194] To analyze the trabecular and cortical bone structure following treatments with LVs, micro-CT was performed in the femur of treated and untreated Gains7- and wild-type control groups at 16 weeks. The representative group of data demonstrated that the LV-treated and control groups did not show apparent statistical differences in trabecular bone volume (BV), trabecular number (Tb.N), trabecular separation (Tb.Sp), trabecular thickness (Tb.Th), bone mineral density (BMD) and degree of anisotropy (DA). Similarly, no statistical difference was determined between LV-treated and untreated Gains7 groups regarding the cortical bone parameters, except for an increase in the BMD of cortical bone, which was found statistically significant. Overall, the treatment with LV-C0L2A1 -hGALNS showed a slight improvement in trabecular and cortical bone formation compared to untreated and wild-type groups; however, no statistical significance was found.
[00195] The expression of the hGALNS enzyme reduced the accumulation of KS, increasing the expression of procollagen in bone and liver.
[00196] To explore the expression of GALNS enzymes and alterations of KS and collagen levels under each LV, we stained the liver and bone (tibia) samples with anti-GALNS (Fig. 8A- C), anti-KS, and anti-collagen antibodies. In the tibia growth plate, we found that the GALNS positive area was approximately 50% under both LV-CBh-hGALNS and LV-COL2A1 -hGALNS compared to the wild- type level (100%). However, this expression was not sufficient to fully correct bone pathology. According to protein concentration in bone, the LV-C OL2A1 -hGALNS - treated group had an elevation in the protein concentration compared to the untreated group, and this slight elevation was found to be significant compared to the wild-type. In the liver, the expression of the GALNS enzyme significantly increased under treatment with LV-CBh- hGALNS (-204%), which was followed by LV-COL2 Al -hGALNS by -90% compared to the wild-type level. This increase was found to correlate with the enzyme activity in liver, but not with VCN since both vectors had similar copy numbers per genome.
[00197] A total anti-GALNS antibody elevation was observed under LV-HSC GT.
[00198] To investigate the immune reaction following the GALNS transgene expression, ELISA was performed using plasma samples of wild-type, treated, and untreated MPS IVA mice. Treated groups with LV-COL2A1 -hGALNS showed significant elevation in the circulating anti-GALNS antibodies overtime starting from 6 weeks of age by 3.06 ± 0.7 compared to control groups (untreated MPS IVA mice: -0.01 ± 0.02 and wild-type mice: -0.11 ± 0.12). However, mice treated with LV-CBh-hGALNS showed significant up-and-down variations during treatment. Moreover, anti-GALNS antibodies were undetectable in the allo- HSCT group. It was anticipated that the undetectable/weak production of the GALNS enzyme might not trigger a strong immune reaction in allo-HSCT compared to LV-HSC GT groups.
[00199] Body weight did not represent the effectiveness of the treatment.
[00200] After treatment, the body weight of mice from each group was measured weekly. The data showed that the LV-COL2 Al -hGALNS or LV-CBh-hGALNS modified HSCs treated group significantly increased the body weight from the day of injection to the 3rd week. This initial increase in body weight could indicate an initial positive response to the treatment. However, the subsequent decrease in body weight compared to untreated and wild-type controls from the 4th to the 16th week suggests that the treatment may not sustain body weight. In addition, no significant difference was found between the LV-C0L2Al-hGALNS modified HSCs treated or allo-HSCT group and an untreated group from the 4th to the 16th week. LV-CBh-hGALNS modified HSCs treated group remained under the body weight level of the untreated group. It is thought that busulfan administration might have a detrimental effect on low body weight over 16 weeks.
Example 3. Direct in vivo gene therapy
[00201] LVs carrying the native GALNS gene were produced under three promoters (ubiquitous - CBh, collagen type II - COL2A1 , hematopoietic stem cells - CD1 lb). Furthermore, LVs carrying the native GALNS gene under CD1 lb promoter tagged with D8 octapeptide and codon-optimized GALNS gene under CBh promoter, respectively, were created. Then, the inventors treated GALNS knockout (KO) mice intravenously at newborns and 4 weeks old with different doses of LVs (5xl09 and 5x10" TU/kg) under three different promoters. Blood samples were collected biweekly following direct in vivo infusion, and mice were autopsied at 16 weeks old to collect tissues. The inventors investigated vector copy number, enzyme activity levels, GAG concentrations in blood and tissue samples, pathology, and bone morphology. In vivo experiment data demonstrated that LVs under ubiquitous CBh promoter with a IxlO11 TU/kg dose in KO newborn mice had the highest enzyme activity. The hGALNS enzyme activity was detected in all visceral organs. KS levels were normalized to KS levels in wild-type mice. Bone pathology and biochemical analyses, including p24, cytokine, and anti-GALNS antibody assays, are underway to investigate therapeutic efficacy. Furthermore, KS levels were reduced to wildtype levels in the livers of MPS IVA mice compared to untreated mice.
Methods and Results
[00202] MPS IVA mice: MPS IVA knockout mice (GALNS"") generated from C57BL/6 background were used in this study. The colony was housed in a pathogen-free facility on a 12- hour light/dark cycle. Newborn (1-2 days old) and 4-week-old mice were injected with lentiviral vectors through the superficial temporal vein. All mouse care and handling procedures were in compliance with the rules of the Institutional Animal Care and Use Committee (IACUC) of Nemours Hospital Delaware Valley under the protocol number RSP20-12482-005.
[00203] Construction of LVs: LVs were constructed as described in the in vitro example above. The inventors will also design a lentiviral vector expressing EGFP fluorescence protein under CBh promoter.
[00204] Direct Infusion of Lentiviral Vectors under Different Promoters and Doses: The effects of lentiviral gene therapy in MPS IVA mice were evaluated on 1) therapeutic efficacy (enzyme activity, GAG levels, pathology), 2) MED, 3) age dependency, 4) immune responses. MKC2, which is a knockout mouse model generated by Dr. Shunji Tomatsu’ lab, was treated with 5 x 109 TU/kg or 1 x 1011 TU/kg GALNS LV vector starting at newborns (day 1-2) or 4 weeks old. Mice were treated via a superficial temporal vein (newborn) or tail vein (4-week-old). As controls, untreated MKC2, and wild-type mice at the same ages were provided with saline. At 16 weeks old, 5 mice per group were euthanized, and tissues were collected, including brain, heart, lung, liver, kidney, spleen, muscle, femur-tibia, arm, eye, and trachea (FIG. 19). Blood samples were collected at baseline and biweekly points, and enzyme activity, KS concentration, and anti-GALNS antibody were determined. For the biological determination of KS, LC-MS/MS was used. The enzyme was analyzed with a 4MU assay, and the antibody was measured with ELISA.
[00205] Therapeutic effects: The inventors evaluated the therapeutic efficacy by measuring GALNS activity and GAG levels in blood and tissues, examining pathology, and conducting micro-CT analysis. The inventors determined that MED could provide enough therapeutic effects for MPS IVA mice.
[00206] GALNS Enzyme Assay: GALNS activity in plasma and tissue extracts was performed using a 4-methylumbelliferone (4-MU) assay (Melford Laboratories Ltd, Suffolk, UK). Briefly, tissues were dissected and immediately homogenized for 30 sec on ice (Omni homogenizer; Kennesaw GA) in 5 vols of homogenization buffer (25 mM Tris-HCl, pH 7.2, 1 mM PMSF). Activity was expressed as nanomoles of 4-methylumbelliferone released per milligram of protein per hr. Protein concentrations were determined using a BCA kit (Thermo Fisher Scientific). Enzyme activity in samples treated with a dose of 1x1011 TU/kg. As shown in FIG. 20A, the enzyme activity fluctuated in the plasma of all groups treated with lentiviruses under CBh and C0L2A1 promoters. In a comparison of LV-CBh-hGALNS and LV-CBh- hGALNSco, LV-CBh-hGALNSco vector had a stable expression over time starting at 6 weeks when compared to other groups. Furthermore, LV-COL2 Al -hGALNS treated group had an increased trend over time while starting to drop after 12 weeks. Overall, the secreted enzyme activity in plasma was elevated over time (FIG. 20A); however, it was reduced in LV-CBh- hGALNSco and LV-COL2Al-hGALNS groups at 16 weeks old in liver (FIG 20B), heart (FIG. 20C), and bone (FIG. 20D). hGALNS enzyme activity was detected in all visceral organs including brain, heart, lung, liver, kidney, spleen, and muscle at variable levels. The trachea, arm and bone had undetectable enzyme levels in all lentivirus-treated groups. The highest enzyme activity was detected in liver of mice treated with LV-CBh-hGALNS, which was followed by LV-CBh-hGALNSco and LV-COL2A1 -hGALNS. In the group of mice treated with LV-CBh- hGALNS, the enzyme activity was 5-fold higher in the wild-type level. Moreover, the inventors analyzed lymph nodes and thymus to identify the enzyme expression levels, which would guide us in analyzing immune reactions due to the enzyme levels.
[00207] GAG and Other Biomarkers: KS in plasma (FIG. 21A-C) and tissues (FIG. 22A-C) from untreated and treated mice were measured by LC-MS/MS to evaluate the reduction of accumulated GAG (Hintze et al., 2011; Kubaski et al., 2016; Kubaski et aL, 2017; Oguma et al., 2007; Rowan et al., 2013; Shimada et al., 2014; Shimada et al., 2015; Tomatsu et al., 2010a). Tissue GAGs were extracted by the acetone precipitation method (Long et al., 2016). Crude GAGs were digested by chondroitinase ABC (1 mU per sample) and keratanase II (1 mU per sample) incubated at 37°C for 15 h overnight. Then, the sample solution was injected into LC- MS/MS. LC-MS/MS to measure keratan sulfate (KS) levels in the liver, muscle, and bone of wild type, untreated MPS IVA and lentivirus-treated MPS IVA mice at 16 weeks old. KS has previously been reported to be a critical biomarker of disease pathology in MPS IVA mice. Plasma KS levels had variations among the groups (Fig 22A). In all treated groups, KS levels were normalized to wild-type mice KS levels, except the group treated with LV-CBh-hGALNS. Furthermore, KS levels were found to be reduced to wild-type levels in the livers of MPS IVA- affected mice when compared to untreated (Fig 22B). Muscle KS levels had similar data with liver KS levels (Fig 22C).
[00208] Vector Copy Number: Viral genome and mRNA were extracted at autopsy from brain, heart, lung, liver, kidney, muscle, and spleen to identify tissues transduced by the vectors expressing human GALNS. Quantification of vectors was done by digital PCR using the primers specific to the LV vector, 5'-CGACTGGTGAGTACGCCAAA-3' and 5'- CCCGCTTAATACTGACGCTCTC-3' and produced an 82-bp PCR-product (Probe;
AGCGGAGGCTAGAAGGAGAGAGATGGGT). Digital droplet PCR (ddPCR) detection of lentiviral-specific p24 protein was used to determine lentiviral copy numbers in liver samples (Fig. 23). 16 weeks after a single dose of the hGALNS or hGALNSco lentivirus, the highest level of vector was detected in the group of LV-CD1 lb*D8-hGALNS, while lower levels of vector were detected in the group of LV-CBh-hGALNS.
[00209] Assessment for immune responses to vectors and GALNS: Production of antibodies to LV vectors and enzymes is a crucial problem that can reduce the therapeutic effects of gene therapy. Plasma samples were assayed biweekly regarding anti-GALNS antibodies, cytokines and P24 viral protein assays by ELISA (MyBioSource, San Diego, CA) (Ahn et al., 2021; Debacker et al., 2020; Lin et al., 2021) and hGALNS (Milani et al., 2019; Schlimgen et al., 2016; Wang et al., 2011).
[00210] Pathology: Mice were euthanized with CO2 gas after treatment, and then the following tissues were collected: brain, heart, lung, liver, kidney, muscle, femur, tibia, and eye to evaluate the reduction of stored KS. To evaluate lysosomal storage by light microscopy, tissues collected was fixed in 2% paraformaldehyde, 4% glutaraldehyde, and toluidine blue-stained 0.5- Lim-thick sections prepared. Bone pathology was quantified by using Image J (NIH). To evaluate storage quantitatively, the inventors measured cell number, cell size, organization of the growth plate, and epiphyseal and articular cartilage thickness (Sawamoto et al., 2020b).
[00211] Immunohistochemistry: The inventors confirmed enzyme expression in bone and cartilage by performing immunohistochemistry (IHC) of GALNS. Additionally, the inventors analyzed anti-KS and anti-collagen levels following IHC. Tissues, including cartilage, were fixed in 10% formalin and sectioned with 5 pm-thickness for immunohistochemistry. The distribution and intensity patterns of KS and GALNS were investigated immunohistochemically to determine any correlation with therapeutic effects. Collagen, KS, and GALNS were stained by anti-KS (1 :50, sc-73518, Santa Cruz Biotechnology) anti-GALNS antibodies (Ghezzi et al., 2017).
[00212] MicroCT: A micro-CT scan was performed on the femur using SkyScan 1276 Micro- CT System (Bruker, Manning Park, MA). The bone was wrapped in salinated (0.9% saline) gauze in preparation for the micro-CT imaging, performed with high spatial resolution down to 2.8 .m pixel size, 528 projections, exposure time of 50 msec, photon energy of 80 keV, and current of 125 pA. Three-dimensional reconstruction of each bone was made (Azario et al., 2017; Pievani et al., 2015; Rowan et al., 2013). The inventors evaluated bone structure (trabecular and cortical bones) by measuring the following parameters: bone mineral density, total volume (TV), bone volume, thickness, BV/TV, etc. (Azario et al., 2017; Pievani et al., 2015).
[00213] Liver Toxicity Levels: To determine the potential side effect of the lentiviral gene therapy, levels of alanine transaminase (ALT, EALT- 100, BioAssay Systems, Hayward, CA, USA) and aspartate transaminase (ALT, EASTR-100, BioAssay Systems, Hayward, CA, USA) in liver cells were evaluated. All the reactions were conducted on 96-well plates and read in a FLUOstar Omega microplate reader (BMG LabTech, Weston Parkway, NC, USA) at the wavelength specified by the supplier. No significant differences between groups (T-test) suggest that treatment failed to induce chronic hepatic toxicity. T statical analysis was done according to normality distribution determined using the Shapiro test (Fig. 24A-B). Additionally, the inventors measured the liver toxicity in newborn-treated low-dose groups, in which there were no differences between groups.
TABLE OF SEQUENCES
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EQUIVALENTS AND INCORPORATIONS BY REFERENCE
[00214] Although the invention is described in detail with reference to specific embodiments thereof, it will be understood that variations which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
[00215] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference in their entireties.

Claims

WHAT IS CLAIMED IS:
1. A recombinant lentivirus (LV) comprising: a recombinant LV genome comprising a human N-acetylgalactosamine-6-sulfate sulfatase (hGALNS) expression cassette wherein said hGALNS expression cassette comprises a promoter operably linked to a nucleotide sequence encoding the hGALNS protein.
2. The LV of claim 1, wherein the promoter of the hGALNS expression cassette is a tissuespecific promoter.
3. The LV of claim 2, wherein the tissue-specific promoter: is a collagen-specific promoter; or comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90% identical, at least 95%, at least 98%, or at least 100% identical to SEQ ID NO: 6.
4. The LV of claim 1 or 3, wherein the expression cassette comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90% identical, at least 95%, at least 98%, or at least 100% identical to SEQ ID NO: 10.
5. The LV of claim 1, wherein the promoter of the hGALNS expression cassette is a ubiquitous promoter.
6. The LV of claim 1 or 6 where the ubiquitous promoter: comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90% identical, at least 95%, at least 98%, or at least 100% identical to SEQ ID NO: 5.
7. The LV of claim 1 or 3, wherein the expression cassette comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90% identical, at least 95%, at least 98%, or at least 100% identical to SEQ ID NO: 8.
8. The LV of any one of claims 1-7, wherein the nucleotide sequence encoding hGALNS or the nucleotide sequence encoding the fusion protein is codon-optimized.
9. A hematopoietic stem cell transduced with a LV of any one of claims 1-4.
10. A hematopoietic stem cell transduced with a LV of any one of claims 5-7
I L A pharmaceutical composition comprising the hematopoietic stem cell of claim 9.
12. A pharmaceutical composition comprising the hematopoietic stem cell of claim 10.
13. A method for treating a human subject diagnosed with mucopolysaccharidosis type IVA (MPS IVA), comprising administering to the human subject the LV of any one of claims 1-4 or the hematopoietic stem cell of claim 9 or pharmaceutical composition of claim 11.
14. A method for treating a human subject diagnosed with mucopolysaccharidosis type IVA (MPS IVA), comprising administering to the human subject the LV of any one of claims 5-7 or the hematopoietic stem cell of claim 10 or pharmaceutical composition of claim 12.
15. A method for treating a human subject diagnosed with MPS IVA, comprising delivering to the bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle, and/or heart valve of said human subject a therapeutically effective amount of a fusion protein that is hGALNS by administering to the human subject the LV of any one of claims 1-4 or the hematopoietic stem cell of claim 9 or pharmaceutical composition of claim 11.
16. A method for treating a human subject diagnosed with MPS IVA, comprising delivering to the bone, cartilage, ligament, meniscus, growth plate, liver, spleen, lung, kidney, trachea, heart muscle, and/or heart valve of said human subject a therapeutically effective amount of a fusion protein that is hGALNS by administering to the human subject the LV of any one of claims 5-7 or the hematopoietic stem cell of claim 10 or pharmaceutical composition of claim 12
PCT/US2024/056623 2023-11-20 2024-11-20 Lentiviral gene therapy of mucopolysaccharidosis iva Pending WO2025111316A1 (en)

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Citations (4)

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US20200157568A1 (en) * 2017-05-26 2020-05-21 Marshall University Research Corporation EXPRESSION VECTORS AND RELATED METHODS OF DELIVERY OF Na/K ATPASE/Src RECEPTOR COMPLEX ANTAGONISTS
US20210214695A1 (en) * 2018-05-30 2021-07-15 Esteve Pharmaceuticals, S.A. Adenoassociated virus vectors for the treatment of mucopolysaccharidoses type iv a
US20220290131A1 (en) * 2018-12-19 2022-09-15 Institut National De La Sante Et De La Recherche Medicale (Inserm) Hybrid promoters and their uses in therapy, notably for treating type ii collagenopathies
US20230123802A1 (en) * 2019-03-27 2023-04-20 Sigilon Therapeutics, Inc. Compositions, devices, and methods for factor vii therapy

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200157568A1 (en) * 2017-05-26 2020-05-21 Marshall University Research Corporation EXPRESSION VECTORS AND RELATED METHODS OF DELIVERY OF Na/K ATPASE/Src RECEPTOR COMPLEX ANTAGONISTS
US20210214695A1 (en) * 2018-05-30 2021-07-15 Esteve Pharmaceuticals, S.A. Adenoassociated virus vectors for the treatment of mucopolysaccharidoses type iv a
US20220290131A1 (en) * 2018-12-19 2022-09-15 Institut National De La Sante Et De La Recherche Medicale (Inserm) Hybrid promoters and their uses in therapy, notably for treating type ii collagenopathies
US20230123802A1 (en) * 2019-03-27 2023-04-20 Sigilon Therapeutics, Inc. Compositions, devices, and methods for factor vii therapy

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