WO2024105638A1 - Vecteurs aav recombinants et méthodes de traitement de la maladie de hunter - Google Patents

Vecteurs aav recombinants et méthodes de traitement de la maladie de hunter Download PDF

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WO2024105638A1
WO2024105638A1 PCT/IB2023/061655 IB2023061655W WO2024105638A1 WO 2024105638 A1 WO2024105638 A1 WO 2024105638A1 IB 2023061655 W IB2023061655 W IB 2023061655W WO 2024105638 A1 WO2024105638 A1 WO 2024105638A1
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Prior art keywords
raav
seq
vector
htfr1
sulfatase
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PCT/IB2023/061655
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English (en)
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Sarah Melissa JACOBO
Vivian CHOI
Wanida RUANGSIRILUK
Rizwana ISLAM
Takashi Onouchi
Kenichi Takahashi
Haruna TAKAGI
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Jcr Pharmaceuticals Co., Ltd.
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Publication of WO2024105638A1 publication Critical patent/WO2024105638A1/fr

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    • 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
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2881—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD71
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Hunter Syndrome is a clinically heterogeneous disease regarding the severity and rate of progression of organ manifestation. The severe form is characterized by hernia in early life, frequent infections of the upper and lower airways, skeletal deformities, mental retardation, and early death. [0004] The management of Hunter Syndrome consists of supportive care and treatment of complications.
  • the present invention provides highly efficient gene therapy agents for treating Hunter Syndrome.
  • the gene therapy agent comprises a recombinant adeno-associated virus (rAAV) vector with increased ability to cross the blood-brain barrier (BBB), thereby increasing expression of a transgene encoding a human iduronate-2-sulfatase (I2S).
  • rAAV recombinant adeno-associated virus
  • a recombinant adeno- associated viral vector comprising: a transgene polynucleotide encoding a fusion protein comprising at least one anti-human transferrin receptor (hTfR1) VHH antibody fused to a human iduronate-2-sulfatase.
  • hTfR1 anti-human transferrin receptor
  • the vectors described herein result in increased ability of iduronate-2-sulfatase (I2S) to cross BBB via the transcytosis mediated by TfR.
  • the vector encoding the fusion protein transgene product further comprises a promoter and one or more other regulatory sequences.
  • the vector comprises the anti-hTfR1 VHH antibody having an amino acid sequence selected from SEQ ID NOs.1-7.
  • the anti-hTfR1 VHH antibody may be encoded by a codon optimized nucleic acid sequence.
  • the codon-optimized nucleic acid sequence for encoding the anti-hTfR1 VHH antibody comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs.18-24.
  • the codon-optimized nucleic acid sequence for encoding the anti-hTfR1 VHH antibody comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.18. In some embodiments, the codon-optimized nucleic acid sequence for encoding the anti-hTfR1 VHH antibody comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.19.
  • the codon-optimized nucleic acid sequence for encoding the anti- hTfR1 VHH antibody comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.20.
  • the codon- optimized nucleic acid sequence for encoding the anti-hTfR1 VHH antibody comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.21.
  • the codon-optimized nucleic acid sequence for encoding the anti-hTfR1 VHH antibody comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.22. In some embodiments, the codon-optimized nucleic acid sequence for encoding the anti-hTfR1 VHH antibody comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.23.
  • the codon- optimized nucleic acid sequence for encoding the anti-hTfR1 VHH antibody comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.24.
  • the human iduronate-2-sulfatase of the fusion protein is a human wild type iduronate-2-sulfatase (I2S) or a functional variant thereof.
  • the human iduronate-2-sulfatase is encoded by a wild type nucleic acid sequence (e.g., SEQ ID NO.25).
  • the human iduronate-2-sulfatase is encoded by a codon optimized nucleic acid sequence.
  • the codon optimized nucleic acid sequence encoding the human iduronate 2-sulfatase comprising a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO.26, or SEQ ID NO.27.
  • the fusion protein encoded the vector described herein comprises a peptide linker sequence that links the anti-hTfR1 VHH antibody and the human iduronate 2-sulfatase.
  • the peptide linker may be about 4-100 amino acid residues, e.g., 4-50 amino acid residues, 4-20 amino acid residues, 4-10 amino acid residues, 5-100 amino acid residues, 5-50 amino acid residues, 5-20 amino acid residues, 10-100 amino acid residues, 10-50 amino acid residues, or 10-25 amino acid residues.
  • the peptide linker is a GGGGS (G4S) peptide linker.
  • the peptide linker comprises GGGGSGGGGSGGGGS (SEQ ID NO.17).
  • the fusion protein further comprises a signal peptide at the N-terminus of the fusion protein.
  • the signal peptide is an IgG Attorney Docket No: MIL-025WO1 signal peptide.
  • the signal peptide of the fusion protein comprises SEQ ID NO.13.
  • the anti-hTfR1 VHH antibody is fused to the N- terminus of the human iduronate 2-sulfatase. In other embodiments, the anti-hTfR1 VHH antibody is fused to the C-terminus of the human iduronate 2-sulfatase.
  • the vector described herein encodes a fusion protein comprising the amino acid sequence of SEQ ID NO.10.
  • the vector described herein encodes a fusion protein comprising the amino acid sequence of SEQ ID NO.11. In some embodiments, the vector described herein encodes a fusion protein comprising the amino acid sequence of SEQ ID NO.12. In some embodiments, the vector described herein encodes a fusion polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.10. In some embodiments, the vector described herein encodes a fusion polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.11.
  • the vector described herein encodes a fusion polypeptide comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO.12.
  • the promoter of the vector described herein is a ubiquitous promoter.
  • the ubiquitous promoter is a CBh promoter.
  • the CBh promoter comprises the sequence of SEQ ID NO.31.
  • the promoter of the vector described herein is a liver specific promoter.
  • the liver specific promoter is transthyretin (TTR) promoter.
  • the TTR promoter comprises the sequence of SEQ ID NO.29.
  • the vector described herein further comprises a liver specific enhancer.
  • the liver specific enhancer comprises one or more copies of CRM8.
  • the vector comprises three copies of CRM8 (SEQ ID NO.28).
  • the vector described herein comprises a Woodchuck Hepatitis Virus posttranscriptional regulatory element (WPRE) and a polyA sequence.
  • WPRE Woodchuck Hepatitis Virus posttranscriptional regulatory element
  • the WPRE is a WPRE3 or a variant thereof.
  • the WPRE is a WPRE3 variant, WPRE3mut5delATG, comprising SEQ ID NO. 34.
  • the vector described herein comprises a polyA sequence.
  • the polyA sequence is an in-silicon synthetic polyA sequence.
  • the polyA comprises the sequence of SEQ ID NO.35.
  • the vector comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR flanking the transgene encoding the fusion protein.
  • the vector comprises a 5’ITR having the sequence of SEQ ID NO.37 and a 3’ITR having the sequence of SEQ ID NO.38.
  • the recombinant vector described herein further comprises an IRES.
  • the IRES comprises the sequence of SEQ ID NO.39.
  • the recombinant vector described herein comprises a nucleic acid sequence presented by SEQ ID NO.40.
  • the recombinant vector described herein comprises a nucleic acid sequence presented by SEQ ID NO.41.
  • the recombinant vector described herein comprises a nucleic acid sequence presented by SEQ ID NO.42.
  • the recombinant vector described herein comprises a nucleic acid sequence presented by SEQ ID NO.43. In some embodiments, the recombinant vector described herein comprises a nucleic acid sequence presented by SEQ ID NO.44. In some embodiments, the recombinant vector described herein comprises a nucleic acid sequence presented by SEQ ID NO.45. [0022] In some embodiments, the recombinant vector described herein comprises a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO.40.
  • the recombinant vector described herein comprises a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO.41. In some embodiments, the recombinant vector described herein comprises a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO.42.
  • the recombinant vector described herein comprises a nucleic acid sequence at least 75%, at Attorney Docket No: MIL-025WO1 least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO.43. In some embodiments, the recombinant vector described herein comprises a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO.44.
  • the recombinant vector described herein comprises a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO.45.
  • a recombinant AAV viral particle comprising a transgene vector described herein.
  • the recombinant vector described herein is packaged with an AAV9 capsid.
  • a rAAV viral particle comprising an AAV9 capsid and a recombinant vector encoding a fusion protein comprising an anti-human transferrin receptor (anti-hTfR1) VHH antibody fused to a human iduronate-2-sulfatase described in the present disclosure is provided.
  • the vector, the transgene encoding a fusion protein described herein is packaged with a delivery vehicle.
  • the delivery vehicle is a virus, such as an adeno-associated-virus, an adenovirus and a lentivirus.
  • the vector, the transgene encoding a fusion protein described herein is packaged with a non-viral based delivery vehicle, such as liposome and nanoparticles (e.g., lipid nanoparticles (LNPs)).
  • a non-viral based delivery vehicle such as liposome and nanoparticles (e.g., lipid nanoparticles (LNPs)).
  • LNPs lipid nanoparticles
  • the present disclosure provides a cell that is transformed with the recombinant vector, the rAAV viral particle, or the transgene that encodes a fusion protein described herein.
  • a pharmaceutical composition comprising the recombinant vector, the rAAV viral particle, or the transgene that encodes a fusion protein described herein, and at least one pharmaceutically acceptable carrier.
  • the present disclosure provides a method for treating Hunter Syndrome (MPSII) in a subject in need; the method may comprise administering to the subject the recombinant vector, the rAAV viral particle, the delivery vehicle or the transgene described herein.
  • the administration results in an increase of an enzymatic activity of iduronate 2-sulfatase (I2S) in various tissues in the subject, such as in the serum, liver, central nervous system, brain, cerebrospinal fluid (CSF), lung, kidney, spleen, quadriceps, heart and/or bone marrow.
  • I2S iduronate 2-sulfatase
  • the administration reduces the level of glycosaminoglycan (GAG) in the subject, such as HS-GAG and DS-GAG levels in the subject. In some embodiments, the administration reduces the level of GAG in the serum, liver, central nervous system, brain, cerebrospinal fluid (CSF), lung, kidney, spleen, quadriceps, heart and/or bone marrow of the subject.
  • GAG glycosaminoglycan
  • the administration reduces the level of GAG in the serum, liver, central nervous system, brain, cerebrospinal fluid (CSF), lung, kidney, spleen, quadriceps, heart and/or bone marrow of the subject.
  • CSF cerebrospinal fluid
  • lung kidney, spleen, quadriceps, heart and/or bone marrow of the subject.
  • the levels of heparan sulfate (HS), and dermatan sulfate (DS) are reduced in the serum, liver, brain, lung, kidney,
  • the levels of HS and DS are reduced about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more.
  • the administration reduces the lysosomal storage burden, wherein the lysosomal stress is determined by the level of LAMP1.
  • the administration reduces neuro-inflammation, wherein the neuroinflammation is determined by the expression level of GFAP.
  • the rAAV vector, the viral particle, the delivery vehicle or the transgene is administered to the subject intravenously, intramuscularly, subcutaneously, by infusion, by perfusion, by intrathecal (IT) injection, or by Intracerebroventricular (ICV) administration.
  • IVT Intracerebroventricular
  • provided also includes a method for inducing or increasing the enzymatic activity of iduronate 2-sulfatase in a subject in need using the rAAV vector, the viral particle, the delivery vehicle or the transgene described herein.
  • FIG. 1 shows the schematics of the rAAV.VHH-I2S vector constructs.
  • Figure 2 is a western blot that shows the transgene product expression of rAAV-VHH-I2S vector constructs to confirm the integrity of the transgene product produced and secreted into circulation of C57Bl6 male mice post injection.
  • Figure 3 comprises different graphs which show the I2S protein levels (nM, or nmol/g total protein/) detected in serum ( Figures 3A and 3I)) and different peripheral tissues of rAAV-VHH-I2S vector constructs treated hTfR1KI / IdsKO mice after 6 weeks post injection:
  • Figures 3B and 3J liver
  • Figures 3C and 3K spleen
  • Figures 3D and 3L lung
  • Figures 3E and 3M heart
  • Figures 3F and 3N kidney
  • Figures 3G and 3O quadriceps
  • Figures 3H and 3P bone marrow.
  • Figures 4A and 4B show human I2S activity in brain of hTfR1KI / IdsKO mice treated with the vector constructs as a percentage (%) of the WT (wildtype) control.
  • Figures 5A and 5C show the remaining level of HS ( ⁇ g/mL) in the CSF (cerebrospinal fluid) of hTfR1KI / IdsKO mice treated with the vector constructs after 6 weeks post injection.
  • Figures 5B and 5D show the remaining level of HS ( ⁇ g/mg of tissue) in the brain of hTfR1KI / IdsKO mice treated with the vector constructs after 6 weeks post injection.
  • Figure 6 shows the remaining level of HS ( ⁇ g/mg tissue) in peripheral tissues of hTfR1KI / IdsKO mice treated with the vector constructs after 6 weeks post injection.
  • Figures 6A and 6H liver;
  • Figures 6B and 6I spleen;
  • Figures 6C and 6J lung;
  • Figures 6D and 6K heart;
  • Figures 6E and 6L kidney;
  • Figures 6F and 6M quadriceps;
  • Figures 6G and 6N bone marrow.
  • Figure 7 shows the remaining level of DS ( ⁇ g/mg tissue) in peripheral tissues of hTfR1KI / IdsKO mice treated with the vector constructs after 6 weeks post injection.
  • Figures 7A and 7H liver;
  • Figures 7B and 7I spleen;
  • Figure 7C and 7J lung;
  • Figure 7D and 7K heart;
  • Figure 7E and 7L kidney;
  • Figure 7F and 7M quadriceps;
  • Figure 7G and 7N bone marrow.
  • Figure 8 shows the vector genome copy number (copy/ ⁇ g DNA normalized to Actin) in brain ( Figure 8A) and liver ( Figure 8B) of hTfR1KI / IdsKO mice treated with the vector constructs after 6 weeks post injection; and Figures 8C and 8D show the mRNA copy number (copy/ ⁇ g total RNA) in brain and liver, respectively.
  • Antibody refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or antigen binding fragments thereof, which specifically binds and recognizes an antigen.
  • the antibody refers mainly to a human antibody, mouse antibody, humanized antibody, as well as a chimeric antibody between human antibody and non-human mammalian antibody, and a chimeric antibody between mouse antibody and non-mouse mammalian antibody, but the meaning of the term is not limited to them insofar as a substance of interest has a property to specifically bind to a certain antigen, and there is no specific limitation as to the animal species of the antibody, either.
  • the term “human antibody” refers to an antibody whose entire protein is encoded by a gene originating from human.
  • human antibody also includes an Attorney Docket No: MIL-025WO1 antibody encoded by a gene obtained by introducing a mutation into an original human gene for a purpose of enhancing expression efficiency of the gene, for example, without modifying the original amino acid sequence.
  • human antibody also includes an antibody which is obtainable through combining two or more genes encoding human antibodies by replacing a certain part of a human antibody with a part of another human antibody.
  • a human antibody includes three complementarity determining regions (abbr. CDRs) in the light chain of the immunoglobulin and three complementarity determining regions (CDRs) in the heavy chain of the immunoglobulin.
  • the three CDRs in the light chain of the immunoglobulin are called, from the N-terminal side, CDR1, CDR2 and CDR3, respectively.
  • the three CDRs in the heavy chain of the immunoglobulin are also called, from the N-terminal side, CDR1, CDR2 and CDR3, respectively.
  • the team “human antibody” also includes a human antibody produced by replacing a CDR of a human antibody with a CDR of another human antibody to modify such properties as the antigen specificity and the affinity of the original human antibodies, etc.
  • the term human antibody broadly also comprises a humanized antibody.
  • a humanized antibody refers to an antibody in which part of the amino acid sequence of its variable region (e.g., especially the whole or part of its CDRs) originates from a non-human mammal while the rest originates from human.
  • An example of humanized antibody is an antibody produced by replacing the three complementarily determining regions (CDRs) of the light chain of the immunoglobulin and the three complementarity determining regions (CDRs) of the heavy chain of the immunoglobulin constituting a human antibody, with CDRs from a non-human mammal.
  • binding affinity refers to the affinity of an antibody, such as a VHH antibody, or antigen binding fragment thereof for an antigen.
  • affinity is calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol., 16:101-106, 1979.
  • binding affinity is measured by an antigen-antibody dissociation rate.
  • a high binding affinity is measured by a competition radioimmunoassay or by surface plasmon resonance in a BIACORE.
  • a high binding affinity is at least about 1 ⁇ 10 ⁇ 8 M.
  • a high binding affinity is at least about 1.5 ⁇ 10 ⁇ 8 , at least about 2.0 ⁇ 10 ⁇ 8 , at least about 2.5 ⁇ 10 ⁇ 8 , at least about 3.0 ⁇ 10 ⁇ 8 , at least about 3.5 ⁇ 10 ⁇ 8 , at least about 4.0 ⁇ 10 ⁇ 8 , at least about 4.5 ⁇ 10 ⁇ 8 , or at least about 5.0 ⁇ 10 ⁇ 8 M.
  • a recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
  • a recombinant protein is one that has an amino acid sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more separated proteins or segments of sequence.
  • a fusion protein comprising an anti-hTfR1 VHH antibody fused to a human I2S enzyme is a recombinant protein.
  • Recombinant AAV viral particle refers to nuclease-resistant particle (NRP) which has an AAV viral capsid, the capsid having packaged therein a heterologous nucleic acid molecule comprising an expression cassette for a desired gene product, i.e., a transgene encoding the desired gene product, e.g., a fusion protein described in the present disclosure.
  • a desired gene product i.e., a transgene encoding the desired gene product, e.g., a fusion protein described in the present disclosure.
  • Such an expression cassette typically contains an AAV 5′ and/or 3′ inverted terminal repeat sequence flanking a transgene sequence, in which the transgene sequence is operably linked to expression control sequences (e.g., promoters).
  • nucleic acid molecule may be used interchangeably, and refer to a polymer of nucleotides.
  • polymers of nucleotides may contain natural and/or non-natural nucleotides, and include, but are not limited to, DNA, RNA, and PNA.
  • Nucleic acid sequence refers to the linear sequence of nucleotides comprised in the nucleic acid molecule or polynucleotide.
  • Sequence identity The similarity between amino acid sequences is expressed in terms of the fidelity between the sequences, otherwise referred to as sequence identity.
  • Attorney Docket No: MIL-025WO1 Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.
  • Chemically related amino acid residues will have a high degree of similarity, Homologs or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods. Methods of alignment of polypeptide sequences for comparison are well known in the art.
  • the term “specifically binds” to an antigen or epitope is a term that is well understood in the art, and methods to determine such specific binding are also well known in the art.
  • a molecule is said to exhibit “specific binding” or “preferential binding” if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances.
  • a VHH- antibody “specifically binds” or “preferentially binds” to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances.
  • a VHH antibody that specifically or preferentially binds to human transferrin receptor 1 is a VHH-antibody that binds this epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to non-hTfR1 antigen.
  • a VHH antibody that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target.
  • “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding.
  • reference to binding means preferential binding.
  • “Specificity” refers to the ability of a binding protein to selectively bind an antigen.
  • beneficial or desired clinical results include, but are not limited to, any one or more of: alleviation of one or more symptoms, diminishment of extent of disease, preventing or delaying spread (for example, metastasis, for example metastasis to the lung or to the lymph node) of disease, preventing or delaying recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, inhibiting the disease or progression of the Attorney Docket No: MIL-025WO1 disease, inhibiting or slowing the disease or its progression, arresting its development, and remission (whether partial or total).
  • treatment is a reduction of pathological consequence and progression of a proliferative or degenerative disease.
  • VHH antibody refers to an antibody having a single monomeric domain antigen binding/recognition domain. Such antibodies include a camelid antibody.
  • a VHH comprises three CDRs and four framework regions, designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • a VHH may be truncated at the N-terminus or C-terminus such that it comprises only a partial FR1 and/or FR4, or lacks one or both of those framework regions, so long as the VHH substantially maintains antigen binding and specificity.
  • Vector is used to describe a polynucleotide that can be engineered to contain a cloned polynucleotide or polynucleotides that can be propagated in a host cell.
  • a vector can include one or more of the following elements: an origin of replication, one or more regulatory sequences (such as, for example, promoters and/or enhancers) that regulate the expression of the polypeptide of interest, and/or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, for example, ⁇ -galactosidase).
  • an origin of replication such as, for example, promoters and/or enhancers
  • selectable marker genes such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, for example, ⁇ -galactosidase.
  • expression vector refers to a vector that is used to express a polypeptide of interest in a host cell.
  • vector includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells.
  • the term includes cloning and expression vehicles, as well as viral vectors.
  • Subject As used herein, the terms “subject” and “patient” are used interchangeably.
  • Pharmaceutically acceptable carriers The pharmaceutically acceptable carriers of use are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the antibodies herein disclosed.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids, which include, but are not limited to, water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids include, but are not limited to, water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids include, but are not limited to, water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • solid compositions e.g., powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Therapeutic rAAV.VHH-I2S vectors [0062] The present disclosure relates to gene therapy vectors, compositions and methods useful for treating Hunter Syndrome and conditions associated thereof.
  • the present disclosure provides gene therapy vectors, such as recombinant AAV vectors, and viral particles, comprising a transgene encoding a fusion protein comprising at least one anti-hTfR1 VHH antibody fused to a human iduronate-2-sulfatase (I2S), or a functional variant thereof.
  • a schematic that illustrates exemplary rAAV vectors of the present disclosure is illustrated in Figure 1.
  • the recombinant vector in some embodiments, comprises a promoter, and one or more regulatory sequences that can drive expression of the transgene product (mRNA transcript or protein).
  • Fusing the iduronate-2- sulfatase (I2S) with the anti-hTfR1 antibody can increase the expression of I2S in the central nervous system (e.g., in the brain).
  • Use of the anti-hTfR1 VHH antibody having reduced affinity to the transferrin receptor 1 (TfR1) that is highly expressed by brain capillary endothelial cells (BCECs) forming the blood-brain barrier (BBB) can facilitate brain drug delivery.
  • the recombinant AAV vector, a viral particle and/or a delivery vehicle comprising the transgene may be used for gene therapy of Hunter Syndrome. 1.
  • transgene encoding VHH-I2S fusion proteins [0063]
  • a transgene encoding a fusion protein comprising at least one anti-human transferrin receptor (hTfR1) VHH antibody fused to a functional human iduronate-2-sulfatase (I2S), is provided.
  • the transgene polynucleotide Attorney Docket No: MIL-025WO1 in some embodiments, comprises codon optimized nucleic acid sequences that encode the anti-hTfR1 VHH antibody and/or the human iduronate-2-sulfatase (I2S).
  • the human anti-TfR1 VHH antibody specifically binds to human transferrin receptor 1.
  • the anti-TfR1 VHH antibody reduces its binding affinity to the human transferrin receptor 1.
  • the transferrin receptor 1 (TfR1) is highly expressed by brain capillary endothelial cells (BCECs) forming the blood-brain barrier (BBB) and is therefore considered as a potential target for brain drug delivery.
  • Antibodies binding to the TfR1, such as single chain antibodies VHH, have been shown to internalize into BCECs in vivo, thereby mediating updates of therapeutic agents at brain-barrier.
  • the anti-hTfR1 VHH antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs.1-7.
  • the anti-hTfR1 VHH antibody comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.1. In some examples, the anti-hTfR1 VHH antibody comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.2. In some examples, the anti-hTfR1 VHH antibody comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.3.
  • the anti-hTfR1 VHH antibody comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.4. In some examples, the anti-hTfR1 VHH antibody comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.5. In some examples, the anti-hTfR1 VHH antibody comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.6.
  • the anti-hTfR1 VHH antibody comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.7.
  • the variant of an anti-hTfR1 VHH antibody can bind to human transferrin receptor 1. In some embodiments, the binding affinity of the anti-hTfR1 VHH antibody to the transferrin receptor 1 is reduced.
  • a suitable iduronate-2-sulfatase (I2S) for the present invention is any protein or a portion of a protein that can substitute for at least partial activity of naturally-occurring Iduronate-2-sulfatase (I2S) protein or rescue one or more phenotypes or symptoms associated with I2S-deficiency.
  • I2S Iduronate-2-sulfatase
  • the terms “an I2S enzyme” and “an I2S Attorney Docket No: MIL-025WO1 protein”, and grammatical equivalents, are used inter-changeably.
  • the human I2S protein is produced as a precursor form.
  • the precursor form of human I2S contains a signal peptide (amino acid residues 1-25 of the full length precursor), a pro-peptide (amino acid residues 26-33 of the full length precursor), and a chain (residues 34-550 of the full- length precursor) that may be further processed into the 42 kDa chain (residues 34-455 of the full-length precursor) and the 14 kDa chain (residues 446-550 of the full-length precursor).
  • the precursor form is also referred to as full-length precursor or full-length I2S protein, which contains 550 amino acids.
  • the amino acid sequences of the mature form (SEQ ID NO.8) having the signal peptide removed and full-length precursor (SEQ ID NO.9) of a typical wild-type or naturally-occurring human I2S protein are shown in Table 1.
  • the signal peptide is underlined.
  • the functional human iduronate-2-sulfatase (I2S) is a wild type enzyme comprising SEQ ID NO.8.
  • the functional human iduronate-2-sulfatase (I2S) is a variant but maintains the functionality of the enzyme.
  • an I2S protein within the fusion protein may comprise an amino acid sequence of SEQ ID NO.8.
  • a functional I2S variant may comprise an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO.8. Table 1.
  • the transgene encoding the fusion protein is codon optimized.
  • the codon optimized sequences increase transcript stability and efficiency of translation, and reduces immunogenicity.
  • Codon-optimized coding regions can be designed by various different methods.
  • This optimization may be performed using methods which are available on-line (e.g., GeneArt), published methods, or a company which provides codon optimizing services, e.g., as DNA2.0 (Menlo Park, Calif.).
  • GeneArt GeneArt
  • a company which provides codon optimizing services e.g., as DNA2.0 (Menlo Park, Calif.).
  • One codon optimizing approach is described, e.g., in Attorney Docket No: MIL-025WO1 International Patent Publication No. WO 2015/012924, which is incorporated by reference herein. See also, e.g., US Patent Publication No.2014/0032186 and US Patent Publication No.2006/0136184.
  • the entire length of the open reading frame (ORF) for the product is modified. However, in some embodiments, only a fragment of the ORF may be altered.
  • oligonucleotide pairs are synthesized such that upon annealing, they form double stranded fragments of 80-90 base pairs, containing cohesive ends, e.g., each oligonucleotide in the pair is synthesized to extend 3, 4, 5, 6, 7, 8, 9, 10, or more bases beyond the region that is complementary to the other oligonucleotide in the pair.
  • the single-stranded ends of each pair of oligonucleotides are designed to anneal with the single-stranded end of another pair of oligonucleotides.
  • the oligonucleotide pairs are allowed to anneal, and approximately five to six of these double-stranded fragments are then allowed to anneal together via the cohesive single stranded ends, and then they ligated together and cloned into a standard bacterial cloning vector, for example, a TOPO® vector available from Thermo Fisher Scientific Inc. [0071]
  • the construct is then sequenced by standard methods. Several of these constructs consisting of 5 to 6 fragments of 80 to 90 base pair fragments ligated together, i.e., fragments of about 500 base pairs, are prepared, such that the entire desired sequence is represented in a series of plasmid constructs.
  • the nucleic acid sequence encoding the anti-hTfR1 VHH antibody is codon optimized.
  • the codon optimized sequence encoding the anti-hTfR VHH antibody comprises a sequence selected from the group consisting of SEQ ID NOs.18-24.
  • the codon optimized sequence encoding the anti-hTfR1 VHH antibody comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.18. In some embodiments, the codon optimized sequence encoding the anti-hTfR1 VHH antibody comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.19.
  • the codon optimized sequence encoding the anti-hTfR1 VHH antibody comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.20. In some embodiments, the codon optimized sequence encoding the anti-hTfR1 VHH antibody comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.21.
  • the codon optimized sequence encoding the anti-hTfR1 VHH antibody comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.22. In some embodiments, the codon optimized sequence encoding the anti-hTfR1 VHH antibody comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.23.
  • the codon optimized sequence encoding the anti-hTfR1 VHH antibody comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.24.
  • the iduronate-2-sulfatse (I2S) of the transgene can be a wild-type or a codon- optimized variant.
  • the nucleic acid sequence encoding human I2S is a wild type sequence (i.e., SEQ ID NO.25).
  • the nucleic acid sequence encoding human I2S is codon optimized.
  • the codon optimized sequence encoding the human I2S comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.26. In some embodiments, the codon optimized sequence encoding the human I2S comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.27.
  • the nucleic acid sequence encoding the anti-hTfR1 VHH antibody and the nucleic acid sequence encoding the human iduronate-2-sulfatase (I2S) is codon optimized.
  • the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO.18 and the codon optimized nucleic acid sequence encoding the human iduronate-2-sulfatase (I2S) comprising SEQ ID NO.26.
  • the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO.19 and the codon optimized nucleic acid sequence encoding the human iduronate-2-sulfatase (I2S) comprising SEQ ID NO.26.
  • the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO.20 and the codon optimized nucleic acid sequence encoding the human iduronate-2-sulfatase (I2S) comprising SEQ ID NO.26.
  • the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO.21 and the codon optimized nucleic acid sequence encoding the human iduronate-2-sulfatase (I2S) comprising SEQ ID NO.26.
  • the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO.22 and the codon optimized nucleic acid sequence encoding the human iduronate-2-sulfatase (I2S) comprising SEQ ID NO.26.
  • the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO.23 and the codon optimized nucleic acid sequence encoding the human iduronate-2-sulfatase (I2S) comprising SEQ ID NO.26.
  • the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO.24 and the codon optimized nucleic acid sequence encoding the human iduronate-2-sulfatase (I2S) comprising SEQ ID NO.26.
  • the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO.18 and the codon optimized nucleic acid sequence encoding the human iduronate-2-sulfatase (I2S) comprising SEQ ID NO.27.
  • the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 Attorney Docket No: MIL-025WO1 VHH antibody comprising SEQ ID NO.19 and the codon optimized nucleic acid sequence encoding the human iduronate-2-sulfatase (I2S) comprising SEQ ID NO.27.
  • the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO.20 and the codon optimized nucleic acid sequence encoding the human iduronate-2-sulfatase (I2S) comprising SEQ ID NO.27.
  • the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO.21 and the codon optimized nucleic acid sequence encoding the human iduronate-2-sulfatase (I2S) comprising SEQ ID NO.27.
  • the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO.22 and the codon optimized nucleic acid sequence encoding the human iduronate-2-sulfatase (I2S) comprising SEQ ID NO.27.
  • the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO.23 and the codon optimized nucleic acid sequence encoding the human iduronate-2-sulfatase (I2S) comprising SEQ ID NO.27.
  • the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO.24 and the codon optimized nucleic acid sequence encoding the human iduronate-2-sulfatase (I2S) comprising SEQ ID NO.27.
  • the nucleic acid sequence encoding the anti-hTfR1 VHH antibody is codon-optimized and the nucleic acid sequence encoding the human iduronate-2-sulfatase (I2S) is a wild-type sequence.
  • the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO.18 and the nucleic acid sequence encoding the human iduronate-2-sulfatase (I2S) having a wild-type sequence.
  • the transgene encoding the fusion protein comprises the codon-optimized nucleic acid encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO.19 and the nucleic acid sequence encoding the human iduronate-2-sulfatase (I2S) having a wild-type sequence.
  • the transgene encoding the fusion protein comprises the codon-optimized nucleic acid encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO.20 and the nucleic acid sequence encoding the human iduronate-2-sulfatase (I2S) Attorney Docket No: MIL-025WO1 having a wild type sequence.
  • the transgene encoding the fusion protein comprises the codon-optimized nucleic acid encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO.21 and the nucleic acid sequence encoding the human iduronate- 2-sulfatase (I2S) having a wild-type sequence.
  • the transgene encoding the fusion protein comprises the codon-optimized nucleic acid encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO.22 and the nucleic acid sequence encoding the human iduronate-2-sulfatase (I2S) having a wildtype sequence.
  • the transgene encoding the fusion protein comprises the codon-optimized nucleic acid encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO.23 and the nucleic acid sequence encoding the human iduronate-2-sulfatase (I2S) having a wild- type sequence.
  • the transgene encoding the fusion protein comprises the codon-optimized nucleic acid encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO.24 and the nucleic acid sequence encoding the human iduronate-2-sulfatase (I2S) having a wildtype sequence.
  • the human anti-TfR1 VHH antibody is fused to the human I2S by a peptide linker.
  • the peptide linker comprises 4 to 100 amino acid residues, preferably 4 to 50 amino acid residues, more preferably 8 to 50 amino acid residues, more preferably 10 to 20 amino acid residues, or15 or 25 amino acid residues.
  • the peptide linker comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37. 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acid residues.
  • the linker peptide is preferably made of glycine only, or of glycine and serine: for example the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser (G2S), the amino acid sequence Gly-Gly-Gly (G3), the amino acid sequence Gly-Gly-Gly-Ser (G3S), the amino acid sequence Gly-Gly-Gly-Gly-Ser(G4S) (SEQ ID NO.14), the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (G5S) (SEQ ID NO.15), the amino acid sequence Ser-Gly-Gly- Gly-Gly (SG4) (SEQ ID NO.16), the amino acid sequence Ser-Gly-Gly-Gly-Gly-Gly (SG5) (SEQ ID NO: 48), or a sequence which includes 2 to 10, or 2 to 5 repeats of any of those
  • the linker peptide sequence is preferably a linker sequence comprising 15 amino acids corresponding to three copies of Attorney Docket No: MIL-025WO1 the amino acid sequence (Gly-Gly-Gly-Gly-Ser) (GGGGSGGGGSGGGGS; SEQ ID NO. 17) consecutively linked.
  • the transgene comprises two or more anti-hTfR1 VHH antibodies in tandem fused to the human I2S.
  • the two or more anti-hTfR1 VHH antibodies have the same sequence; alternatively, the two or more VHH antibodies have different sequences.
  • the human anti-TfR1 VHH antibody is fused to the N- terminus of the human I2S. In some embodiments, the human anti-TfR1 VHH antibody is fused to the C-terminus of the human I2S.
  • the fusion protein further comprises an IgG signal peptide.
  • signal peptide refers to a short tag of amino acids at the N- or C-terminal of a protein that predestinates the protein location extracellularly or within the cell to the organelles.
  • the polynucleotide described herein encodes a fusion protein comprises a signal peptide, at least one anti-hTfR1 VHH antibody and a human iduronate-2-sulfatase (I2S) or a functional variant thereof.
  • the VHH-I2S fusion protein comprises a signal peptide of SEQ ID NO.13.
  • the polynucleotide encoding a fusion protein having a signal peptide comprising SEQ ID NO.13, an anti-hTfR1 VHH antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOs.1- 7, and a human iduronate-2-sulfatase (I2S) having an amino acid sequence of SEQ ID NO. 8 or a functional variant thereof.
  • a signal peptide comprising SEQ ID NO.13
  • an anti-hTfR1 VHH antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOs.1- 7, and a human iduronate-2-sulfatase (I2S) having an amino acid sequence of SEQ ID NO. 8 or a functional variant thereof.
  • I2S human iduronate-2-sulfatase
  • the polynucleotide comprises a nucleic acid sequence encoding the signal peptide of SEQ ID NO.13, a codon optimized nucleic acid sequence encoding an anti-hTfR1 VHH antibody and a codon optimized nucleic acid sequence encoding a human iduronate-2-sulfatase (I2S) or a functional variant thereof.
  • the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody includes any one of SEQ ID NOs.18-24 and the codon optimized nucleic acid sequence encoding the human iduronate-2-sulfatase (I2S) includes SEQ ID NO.26 or SEQ ID NO.27.
  • the transgene described herein encodes a fusion protein having the amino acid sequence of SEQ ID NO.10.
  • the transgene encodes a fusion protein having the amino acid sequence of SEQ ID NO.11.
  • the transgene encodes a fusion protein having the amino acid sequence of SEQ ID NO.12.
  • the transgene described herein encodes a fusion protein having an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO. 10.
  • the transgene described herein encodes a fusion protein having an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO.11. In some embodiments, the transgene described herein encodes a fusion protein having an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO.12. 2.
  • the recombinant AAV (rAAV) vector for expressing a VHH-I2S fusion protein described herein comprises an expression cassette containing a transgene encoding a fusion protein of an anti-human transferrin receptor (hTfR1) VHH antibody and a functional human iduronate-2-sulfatase (I2S), a promoter, and one or more regulatory sequences.
  • Exemplary vectors, as shown in Figure 1, comprise a promoter, a 5’ and a 3’ inverted terminal repeat (ITR), a cis-acting regulatory module (CRM), an intron, a WPRE sequence, and a polyA tail.
  • the rAAV vector comprises a cis-acting regulatory module (CRM).
  • CRM cis-acting regulatory module
  • Various kinds of CRM are suitable for use in the vectors described herein and include tissue-specific enhancers, for example hepatocyte-specific CRM, neuronal- specific CRM.
  • the vectors described herein include a hepatocyte- specific CRM, for example, CRM8.
  • the vector includes more than one CRM.
  • the vector comprises two, three, four, five or six CRMs.
  • the vector comprises three CRM, for example three CRM8 (e.g., SEQ ID NO.28).
  • the recombinant vector comprises a promoter.
  • the rAAV vector comprises a ubiquitous promoter.
  • ubiquitous promoter has been described, for example, in Steven Gray et al, Hum Gene Ther.2011 Sep; 22(9): 1143–1153, the contents of which are incorporated herein for reference.
  • the ubiquitous promoter is a CBh promoter, a novel version of the CBA promoter, which provides long-term transgene expression in all cell types.
  • This hybrid version comprises of a CMV enhancer, a shorter CBA promoter, and an MVM intron (SEQ ID NO.30).
  • the CBh promoter comprises the sequence of SEQ ID NO.31.
  • Other ubiquitous promoters may also be used in the present rAAV vector, including but not limited to, a chicken beta actin (CBA) promoter, human cytomegalovirus (CMV) promoter, phosphoglycerate kinase (PGK) promoter, or elongation factor 1 ⁇ promoter.
  • CBA chicken beta actin
  • CMV human cytomegalovirus
  • PGK phosphoglycerate kinase
  • the rAAV vector comprise a tissue-specific promoter, such as a liver-specific promoter.
  • liver-specific promoters examples include, for example, human transthyrethin promoter (TTR), ⁇ 1-Antitrypsin promoter, factor IX pro/liver transcription factor-responsive oligomers, and the basic albumin promoter. Liver specific promoters are described, for example, in Zhijian Wu et al., Molecular Therapy vol 16, no 2, February 2008, the contents of which are incorporated herein by reference. [0090] In some embodiments, the liver specific promoter is TTR promoter comprising SEQ ID NO.29. [0091] In some embodiments, the rAAV vector comprises one or more small elements, such as an intron. Various introns are known in the art.
  • Suitable introns for the rAAV vector described herein include for example an MVM intron, a truncated FIX intron, a chimeric ⁇ globin Splice Donor/immunoglobulin heavy chain Splice Acceptor intron, SV40 and/or an alpha globin 1 st intron.
  • the rAAV vector comprises an SV40 intron.
  • the rAAV vector comprises an MVM intron (e.g., SEQ ID NO.30).
  • the MVM intron is grafted to a novel chimeric promoter, such as a CBh promoter (SEQ ID NO.31).
  • an expression cassette and/or a vector may contain other appropriate transcription initiation, termination, enhancer sequences, and efficient RNA processing signals.
  • sequences include splicing and polyadenylation (polyA) signals; regulatory elements that enhance expression (e.g., WPRE); sequences that Attorney Docket No: MIL-025WO1 stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • a KOZAK sequence is included.
  • a polyadenylation (polyA) signal is included to mediate efficient termination of the fusion protein mRNA transcripts.
  • a polyA signal may be an artificial polyA.
  • suitable polyA sequences include, e.g., bovine growth hormone, SV40, rabbit beta globin, and TK polyA, amongst others.
  • the polyA signal is an in silico designed synthetic polyA sequence.
  • the polyA sequence comprises SEQ ID NO. 35.
  • the polyA signal comprises the sequence of SEQ ID NO.36.
  • the rAAV vector described herein comprises a posttranscriptional response element.
  • posttranscriptional response element refers to a nucleic acid sequence that, when transcribed, adopts a tertiary structure that enhances expression of the transgene product, which can be the mRNA transcript or the protein.
  • posttranscriptional regulatory elements include, but are not limited to, woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), mouse RNA transport element (RTE), constitutive transport element (CTE) of the simian retrovirus type 1 (SRV-1), the CTE from the Mason-Pfizer monkey virus (MPMV), and the 5′ untranslated region of the human heat shock protein 70 (Hsp70 5′UTR).
  • the rAAV vector comprises a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).
  • WPRE woodchuck hepatitis virus posttranscriptional regulatory element
  • WPRE3mut5delATG a woodchuck hepatitis virus posttranscriptional regulatory element
  • WPRE2 WPRE_wt (GenBank accession no. J04514); WPRE_wt (GenBank accession no. J02442) and WPREmut6.
  • the WPRE is WPRE3 or a variant thereof, e.g., WPRE3mut5delATG.
  • the rAAV vector comprises a WPRE having a sequence of SEQ ID NO.32, SEQ ID NO.33 or SEQ ID NO.34.
  • the rAAV vector comprises a WPRE3mut5delATG having SEQ ID NO.34.
  • the vector comprises a 5’ and a 3’ inverted terminal repeat (ITR).
  • ITR inverted terminal repeat
  • the rAAV vector described herein comprises an AAV 5’ ITR and a 3’ ITR, which may be of the same AAV origin as the capsid, or which of a different AAV origin (to produce an AAV pseudotype).
  • the ITR sequences from AAV2, or the deleted version thereof ( ⁇ ITR) are used.
  • ITRs from other AAV sources may be selected. Where the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be termed pseudotyped.
  • an expression cassette for a rAAV vector comprises an AAV 5′ ITR, a transgene encoding a recombinant protein (e.g., a fusion protein described herein) and any regulatory sequences, and an AAV 3′ ITR.
  • a shortened version of the 5′ ITR termed ⁇ ITR, has been described in which the D-sequence and terminal resolution site (trs) are deleted.
  • trs terminal resolution site
  • the full-length AAV 5′ and 3′ ITRs are used.
  • the rAAV comprises a 5’ITR having SEQ ID NO.38 and a 3’ITR having SEQ ID NO.37.
  • the vector comprises an internal ribosome entry site (IRES), e.g., an IRES having SEQ ID NO.39.
  • IRES internal ribosome entry site
  • the rAAV vector comprises a rAAV vector element comprising a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with a vector element sequence shown in Table 2.
  • the rAAV vector comprises a vector element nucleotide sequence identical to a vector element nucleotide sequence shown in Table 2. Table 2.
  • a recombinant AAV vector for expressing a human iduronate-2-sulfatase comprises a transgene encoding a fusion protein of an anti- human transferrin receptor (hTfR1) VHH antibody and a functional human iduronate-2- sulfatase (I2S), a ubiquitous promoter CBh, a WPRE3mut5 ⁇ ATG and a polyA sequence.
  • a recombinant AAV vector for expressing a human iduronate-2-sulfatase comprises a transgene encoding a fusion protein of an anti- human transferrin receptor (hTfR1) VHH antibody and a functional human iduronate-2- sulfatase (I2S), a liver specific promoter TTR, a liver specific enhancer CRM8, a WPRE3mut5 ⁇ ATG and a polyA sequence.
  • a recombinant AAV vector for expressing a human iduronate-2-sulfatase comprises a transgene encoding a fusion protein of a tandem of anti-human transferrin receptor (hTfR1) VHH antibodies and a functional human Attorney Docket No: MIL-025WO1 iduronate-2-sulfatase (I2S), a liver specific promoter TTR, a liver specific enhancer CRM8, a WPRE3mut5 ⁇ ATG and a polyA sequence.
  • a recombinant AAV vector for expressing a human iduronate-2-sulfatase comprises a transgene encoding a fusion protein of a tandem of anti-human transferrin receptor (hTfR1) VHH antibodies and a functional human iduronate-2-sulfatase (I2S), a ubiquitous promoter CBh, a WPRE3mut5 ⁇ ATG and a polyA sequence.
  • Exemplary rAAV.VHH-I2S vectors are shown in Table 3.
  • the rAAV vector for expressing a human iduronate-2- sulfatase comprises the nucleic acid sequence of SEQ ID NO.40.
  • the rAAV vector for expressing a human iduronate-2-sulfatase comprises a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO.40.
  • the rAAV vector for expressing a human iduronate-2- sulfatase (I2S) comprises the nucleic acid sequence of SEQ ID NO.41.
  • the rAAV vector for expressing a human iduronate-2-sulfatase comprises a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO.41.
  • the rAAV vector for expressing a human iduronate-2- sulfatase (I2S) comprises the nucleic acid sequence of SEQ ID NO.42.
  • the rAAV vector for expressing a human iduronate-2-sulfatase comprises a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO.42.
  • the rAAV vector for expressing a human iduronate-2- sulfatase (I2S) comprises the nucleic acid sequence of SEQ ID NO.43.
  • the rAAV vector for expressing a human iduronate-2-sulfatase comprises a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID Attorney Docket No: MIL-025WO1 [0110]
  • the rAAV vector for expressing a human iduronate-2- sulfatase (I2S) comprises the nucleic acid sequence of SEQ ID NO.44.
  • the rAAV vector for expressing a human iduronate-2-sulfatase comprises a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO.44.
  • the rAAV vector for expressing a human iduronate-2- sulfatase (I2S) comprises the nucleic acid sequence of SEQ ID NO.45.
  • the rAAV vector for expressing a human iduronate-2-sulfatase comprises a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO.45. 4.
  • Recombinant AAV.VHH-I2S viral particles [0112]
  • the present disclosure provides isolated adeno- associated viruses (AAVs) comprising an AAV capsid and a transgene encoding the fusion protein described herein.
  • the term “isolated” refers to an AAV that has been artificially produced or obtained. Isolated AAVs may be produced using recombinant methods. Such AAVs are referred to herein as “recombinant AAVs”.
  • Recombinant AAVs preferably have tissue-specific targeting capabilities, such that a nuclease and/or transgene of the rAAV will be delivered specifically to one or more predetermined tissue(s).
  • the AAV capsid is an important element in determining these tissue-specific targeting capabilities. Thus, an rAAV having a capsid appropriate for the tissue being targeted can be selected.
  • capsid proteins are structural proteins encoded by the cap gene of an AAV.
  • AAVs comprise three capsid proteins, virion proteins 1 to 3 (named VP1, VP2 and VP3), Attorney Docket No: MIL-025WO1 all of which are transcribed from a single cap gene via alternative splicing.
  • the molecular weights of VP1, VP2 and VP3 are respectively about 87 kDa, about 72 kDa and about 62 kDa.
  • capsid proteins upon translation, form a spherical 60-mer protein shell around the viral genome.
  • the functions of the capsid proteins are to protect the viral genome, deliver the genome and interact with the host.
  • capsid proteins deliver the viral genome to a host in a tissue specific manner.
  • an AAV capsid protein is of an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10 and AAV 11.
  • the capsid is an AAV9 capsid protein or a variant thereof.
  • a rAAV viral particle of the present invention comprises a) an AAV9 capsid and b) a polynucleotide encoding a fusion protein comprising an anti-hTfR1 VHH antibody fused to a human I2S.
  • the gene therapy vector is an AAV9 vector expressing a fusion protein transgene under control of a liver specific promoter referred to as rAAV9.
  • LSP.-anti-hTfR1 VHH-I2S-WPRE e.g., Figure 1
  • the external AAV vector component is a serotype 9; the AAV9 capsid contains a single- stranded DNA rAAV vector genome.
  • a rAAV viral particle comprises an AAV9 capsid and the transgene polynucleotide of SEQ ID NO.40.
  • a rAAV viral particle comprises an AAV9 capsid and the transgene polynucleotide of SEQ ID NO.41. In another example, a rAAV viral particle comprises an AAV9 capsid and the transgene polynucleotide of SEQ ID NO.42. In another example, a rAAV viral particle comprises an AAV9 capsid and the transgene polynucleotide of SEQ ID NO.43. In another example, a rAAV viral particle comprises an AAV9 capsid and the transgene polynucleotide of SEQ ID NO.44.
  • a rAAV viral particle comprises an AAV9 capsid and the transgene polynucleotide of SEQ ID NO.45.
  • the components to be cultured in the host cell to package a rAAV vector in an AAV capsid may be provided to the host cell in trans.
  • any one or more of the required components e.g., recombinant AAV vector, rep sequences, cap sequences, and/or helper functions
  • MIL-025WO1 may be provided by a stable host cell which has been engineered Attorney Docket No: MIL-025WO1 to contain one or more of the required components using methods known to those of skill in the art.
  • the host cell is a mammalian cell (e.g., HEK293 cell, or MPNST cells) or an insect cell (e.g., SF9 cell).
  • the disclosure relates to a composition comprising the host cell described above.
  • the composition comprising the host cell above further comprises a cryopreservative.
  • the recombinant AAV vector, rep sequences, cap sequences, and helper functions required for producing the rAAV of the disclosure may be delivered to the packaging host cell using any appropriate genetic element (vector).
  • the selected genetic element may be delivered by any suitable method, including those described herein.
  • any embodiment of this disclosure are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods of generating rAAV virions are well known and the selection of a suitable method is not a limitation on the present disclosure. See, e.g., K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Pat. No.5,478,745.
  • recombinant AAV viral particles may be produced using the triple transfection method (described in detail in U.S. Pat. No.6,001,650).
  • the recombinant AAVs are produced by transfecting a host cell with a recombinant AAV vector (comprising a transgene encoding the fusion protein described herein) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector.
  • An AAV helper function vector encodes the “AAV helper function” sequences (i.e., rep and cap), which function in trans for productive AAV replication and encapsidation.
  • the AAV helper function vector supports efficient AAV vector production without generating any detectable wild-type AAV virions (i.e., AAV virions containing functional rep and cap genes).
  • vectors suitable for use with the present disclosure include pHLP19, described in U.S. Pat. No.6,001,650 and pRep6cap6 vector, described in U.S. Pat. No.6,156,303, the entirety of both incorporated by reference herein.
  • the accessory function vector encodes nucleotide sequences for non-AAV derived viral and/or cellular functions upon which AAV is dependent for replication (i.e., “accessory functions”).
  • the accessory functions include those functions required for AAV replication, including, without limitation, those moieties Attorney Docket No: MIL-025WO1 involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly.
  • Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1), and vaccinia virus.
  • a “host cell” refers to any cell that harbors, or is capable of harboring, a substance of interest. Often a host cell is a mammalian cell.
  • a host cell may be used as a recipient of an AAV helper construct, an AAV plasmid (e.g., AAV vectors encoding fusion proteins described herein), an accessory function vector, or other transfer DNA associated with the production of recombinant AAVs.
  • the term includes the progeny of the original cell which has been transfected.
  • a “host cell” as used herein may also refer to a cell which has been transfected with an exogenous DNA sequence.
  • the rAAV. VHH-I2S vector comprises a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with a nucleotide sequence shown in Table 3.
  • the rAAV. VHH-I2S vector comprises a sequence identical to a nucleotide sequence shown in Table 3.
  • Methods for generating and isolating AAV viral vectors suitable for delivery to a subject are known in the art. See, e.g., US Patent 7790449; US Patent 7282199; WO 2003/042397; WO 2005/033321, WO 2006/110689; and US 7588772 B2.
  • a producer cell line is transiently transfected with a construct that encodes the transgene flanked by ITRs and a construct(s) that encodes rep and cap.
  • a packaging cell line that stably supplies rep and cap is transiently transfected with a construct encoding the transgene flanked by ITRs.
  • AAV virions are produced in response to infection with helper adenovirus or herpesvirus, requiring the separation of the rAAVs from contaminating virus.
  • systems have been developed that do not require infection with helper virus to recover the AAV (i.e., adenovirus El, E2a, VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase) are also supplied, in trans, by the system.
  • the helper functions can be supplied by transient transfection of the cells with constructs that encode the required helper functions, or the cells can be engineered to stably contain genes encoding the helper functions, the expression of which can be controlled at the transcriptional or posttranscriptional level.
  • Attorney Docket No: MIL-025WO1 [0124]
  • the expression cassette flanked by ITRs and rep/cap genes are introduced into a desired cell or cell line by infection with baculovirus-based vectors.
  • the expression cassette flanked by ITRs and rep/cap genes are introduced into insect cells by infection with baculovirus-based vectors.
  • the production plasmids are cultured in the host cells which express the AAV cap and/or rep proteins. In the host cells, each rAAV genome is rescued and packaged into the capsid protein or envelope protein to form an infectious viral particle.
  • the rAAV expression cassette, the vector (such as rAAV vector), the virus (such as rAAV), the production plasmid comprises AAV inverted terminal repeat sequences, a codon optimized nucleic acid sequence that encodes IDS and/or SUMF-1, and expression control sequences that direct expression of the encoded proteins in a host cell.
  • the rAAV expression cassette, the virus, the vector (such as rAAV vector), the production plasmid further comprise one or more of an intron, a Kozak sequence, a polyA, posttranscriptional regulatory elements and others.
  • the post-transcriptional regulatory element is Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE).
  • a plasmid comprising a gene of interest may be combined with one or more helper plasmids, e.g., that contain a rep gene (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and a cap gene (encoding VP1, VP2, and VP3, including a modified VP2 region as described herein), and transfected into a recombinant cells such that the rAAV can be packaged and subsequently purified.
  • the packaging is performed in a helper cell or producer cell, such as a mammalian cell or an insect cell.
  • Exemplary mammalian cells include, but are not limited to, HEK293 cells, COS cells, HeLa cells, BHK cells, or CHO cells (see, e.g., ATCC® CRL-1573TM, ATCC® CRL-1651TM, ATCC® CRL-1650TM, ATCC® CCL- 2, ATCC® CCL-10TM, or ATCC® CCL-61TM).
  • Exemplary insect cells include, but are not limited to Sf9 cells (see, e.g., ATCC® CRL-1711TM).
  • the helper cell may comprise rep and/or cap genes that encode the Rep protein and/or Cap proteins for use in a method described herein.
  • the packaging is performed in vitro.
  • a plasmid containing comprising the gene of interest is combined with one or more helper plasmids, e.g., that contain a rep gene of a first serotype and a cap gene of the same serotype or a different serotype, and transfected into helper cells such that the rAAV is packaged.
  • the one or more helper plasmids include a first helper plasmid comprising a rep gene and a cap gene, and a second helper plasmid comprising one or more of the following helper genes: Ela gene, Elb gene, E4 gene, E2a gene, and VA gene.
  • helper genes are genes that encode helper proteins Ela, Elb, E4, E2a, and VA.
  • the cap gene is modified such that one or more of the proteins VP1, VP2 and VP3 do not get expressed.
  • the cap gene is modified such that VP2 does not get expressed. Methods for making such modifications are known in the art (Lux et al. (2005), J Virology, 79: 11776-87).
  • Helper plasmids and methods of making such plasmids, are generally known in the art and generally commercially available (see, e.g., pDF6, pRep, pDM, pDG, pDPlrs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E/R585E), and pDP8.ape plasmids from PlasmidFactory, Bielefeld, Germany; other products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, Ca; and Addgene, Cambridge, MA; pxx6; Grimm et al.
  • the rAAV.VHH-I2S gene therapy virus is provided in a pharmaceutical composition which comprises an aqueous carrier, excipient, diluent or buffer.
  • the rAAV.VHH-I2S gene therapy virus formulation is a suspension containing an effective amount of a rAAV.VHH-I2S vector described herein.
  • suitable solutions are known including those which include one or more of: buffering saline, a surfactant, and a physiologically compatible salt or mixture of salts.
  • a suspension comprising a rAAV.VHH-I2S viral particle may contain NaCl and/or KCl.
  • the pH may be in the range of 6.5 to 8.5, or 7 to 8.5, or 7.5 to 8.
  • a suitable surfactant, or combination of surfactants may be selected from among Poloxamers, i.e., nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (Macrogol-15 Hydroxystearate), LABRASOL (Polyoxy capryllic glyceride), polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol.
  • the formulation contains a poloxamer.
  • a composition further comprises a pharmaceutically acceptable carrier.
  • Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the rAAV is directed.
  • one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline).
  • Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present disclosure.
  • the compositions of the disclosure may contain, in addition to the rAAV particles and carrier(s), other pharmaceutical ingredients, such as preservatives, or chemical stabilizers.
  • Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.
  • Suitable chemical stabilizers include gelatin and albumin.
  • these formulations may contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation.
  • the amount of active compound in each therapeutically-useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound.
  • Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as Attorney Docket No: MIL-025WO1 well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form is sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture, storage, and transport, and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • polyol e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., vegetable oils
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion
  • isotonic agents for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • a suitable sterile aqueous medium may be employed.
  • one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. Attorney Docket No: MIL-025WO1 The person responsible for administration will, in any event, determine the appropriate dose for the individual host. [0143] Sterile injectable solutions are prepared by incorporating the active rAAV in the required amount in the appropriate solvent with various of the other ingredients enumerated herein, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the rAAV compositions disclosed herein may also be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium,
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • Supplementary active ingredients can also be incorporated into the compositions.
  • pharmaceutically-acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.
  • some recombinant retroviruses may also be used to package the transgene encoding the fusion protein described herein, or the Attorney Docket No: MIL-025WO1 vector for expressing the fusion protein.
  • the recombinant retroviruses may adenoviruses and lentiviruses.
  • the transgene polynucleotide and the vector comprising the expression cassette for expressing the fusion protein described herein may be formulated to non-viral based delivery vehicles.
  • the non-viral based delivery vehicles may be used to introduce the gene therapy product of the present disclosure into suitable host cells.
  • Such delivery vehicles may include but are not limited to, liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like.
  • the transgenes encoding the fusion protein described herein, or the vectors for expressing transgenes of the fusion proteins described herein may be formulated for delivery either encapsulated in a lipid particle (e.g., a lipid nanoparticle (LNP)), a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
  • lipid particle e.g., a lipid nanoparticle (LNP)
  • LNP lipid nanoparticle
  • Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids or the fusion protein constructs disclosed herein.
  • liposomes The formation and use of liposomes is generally known to those of skill in the art. Recently, liposomes were developed with improved serum stability and circulation half- times (U.S. Pat. No.5,741,516). Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (U.S. Pat. Nos.5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587). [0150] Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures. In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems.
  • Liposomes have been used effectively to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors and allosteric effectors into a variety of cultured cell lines and animals. In addition, several successful clinical trials examining the effectiveness of liposome-mediated drug delivery have been completed. [0151] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 ⁇ m.
  • Nanocapsule formulations of the AAV vectors and constructs may be used. Nanocapsules can generally entrap substances in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 ⁇ m) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use.
  • the formulation is suitable for use in human subjects and is administered intravenously.
  • the formulation is delivered via a peripheral vein by bolus injection.
  • the formulation is delivered via a peripheral vein by infusion.
  • Any suitable method or route can be used to administer a rAAV-containing composition as described herein, and optionally, to co-administer other active drugs or therapies in conjunction with the rAAV-mediated delivery of the fusion protein described herein.
  • Routes of administration include, for example, systemic, oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parental routes of administration.
  • rAAV.VHH-I2S vectors and viral particles described herein are provided.
  • the rAAV vectors described herein are suitable for treating a subject that has an I2S deficiency, such as Hunter Syndrome (also known as mucopolysaccharidosis type II (MPSII)).
  • the method of treating Hunter Syndrome includes administering to the subject in need thereof a recombinant adeno-associated virus (rAAV) vector as described herein.
  • rAAV recombinant adeno-associated virus
  • Methods for treating Hunter Syndrome in a subject are provided herein.
  • the methods typically involve administering to a subject in need thereof an effective amount of a rAAV comprising a transgene encoding a fusion protein described herein, in the subject.
  • An effective amount of a rAAV vector may be an amount sufficient to have a therapeutic benefit in a subject.
  • the rAAV vector remains episomal following administration to a subject in need thereof.
  • the rAAV vector does not remain episomal following administration to a subject in need thereof.
  • Attorney Docket No: MIL-025WO1 in some embodiments, the rAAV vector integrates into the genome of the subject.
  • a rAAV vector for expressing a VHH-I2S fusion protein described herein is administered to a subject in need thereof via a suitable route.
  • the rAAV vector is administered by intravenous, intraperitoneal, subcutaneous, intradermal, intrathecal injection, or Intracerebroventricular (ICV) administration.
  • the rAAV vector is administered intravenously (IV).
  • the rAAV vector is administered by intrathecal (IT) injection. In embodiments, the rAAV vector is administered by ICV injection. In embodiments, the intradermal administration comprises administration by use of a “gene gun” or biolistic particle delivery system. In some embodiments, the rAAV vector is administered via a non-viral lipid nanoparticle.
  • a composition comprising the rAAV vector may comprise one or more diluents, buffers, liposomes, a lipid, a lipid complex. In some embodiments, the rAAV vector is comprised within a microsphere or a nanoparticle, such as a lipid nanoparticle.
  • functional I2S is detectable in the serum, liver, brain, lung, kidney, spleen, quadriceps, heart and/or bone marrow of the subject.
  • functional I2S is detectable in the plasma of the subject.
  • functional I2S is detectable in the liver of the subject.
  • functional I2S is detectable in the central nervous system of the subject. Particularly, functional I2S is detectable in the brain of the subject.
  • functional I2S is detectable in various tissues and organs of the subject at 2 weeks, at 6 weeks, at least 3 months, 6 months, 12 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, or longer after administration of the rAAV vector. In some embodiments, functional I2S is detectable in various tissues and organs of the subject for the remainder of the subject’s life following administration of the rAAV vector.
  • the administered rAAV vector expressing a VHH- I2S fusion protein results in the production of active I2S to the same extent as found following Attorney Docket No: MIL-025WO1 administration of purified I2S protein delivered intravenously.
  • the administered rAAV comprising I2S results in production of a greater amount of active I2S as compared to administration of purified I2S protein delivered intravenously.
  • the administered rAAV vector expressing a VHH- I2S fusion protein results in the production of active I2S to the same extent as found in a normal subject, i.e., a subject having a normal functional I2S.
  • the levels of functional I2S detectable in the circulation are between about 2 and 20 times greater than the amount of functional I2S detectable in the subject before administration of the rAAV comprising I2S.
  • the levels of functional I2S detectable in the cerebrospinal fluid (CSF) and brain are between 2 to 20 times greater than the amount of functional I2S detectable in the subject before administration of the rAAV comprising I2S.
  • the levels of functional I2S detectable in the affected tissues are between 2 to 20 times greater than the amount of functional I2S detectable in the subject before administration of the rAAV comprising I2S.
  • the enzyme iduronate-2-sulfatase (I2S) removes the sulfate group from the glycosaminoglycans (GAGs), dermatan and heparan sulfate, and its absence or inactivity results in accumulation of GAGs resulting in Hunter Syndrome, a lysosomal storage disorder.
  • the administration of a rAAV vector expressing a VHH- I2S fusion protein described herein results in the reduction of glycosaminoglycan (GAG) in the subject.
  • the administered rAAV vector expressing a VHH- I2S fusion protein described herein reduces GAG in the subject by about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or about 10% in comparison to the subject’s baseline GAG level prior to administering the rAAV vector.
  • the administered rAAV vector expressing a VHH- I2S fusion protein described herein reduces GAG in the subject by about 95%.
  • the administered rAAV vector expressing a VHH- I2S fusion protein Attorney Docket No: MIL-025WO1 described herein reduces GAG in the subject by about 90%.
  • the administered rAAV vector expressing a VHH- I2S fusion protein described herein reduces GAG in the subject by about 85%.
  • the administered rAAV vector expressing a VHH- I2S fusion protein described herein reduces GAG in the subject by about 80%.
  • the administered rAAV vector expressing a VHH- I2S fusion protein described herein reduces GAG in the subject by about 75%. In some embodiments, the administered rAAV vector expressing a VHH- I2S fusion protein described herein reduces GAG in the subject by about 70%. In some embodiments, the administered rAAV vector expressing a VHH- I2S fusion protein described herein reduces GAG in the subject by about 65%. In some embodiments, the administered rAAV vector expressing a VHH- I2S fusion protein described herein reduces GAG in the subject by about 60%.
  • the administered rAAV vector expressing a VHH- I2S fusion protein described herein reduces GAG in the subject by about 55%. In some embodiments, the administered rAAV vector expressing a VHH- I2S fusion protein described herein reduces GAG in the subject by about 50%. In some embodiments, the administered rAAV vector expressing a VHH- I2S fusion protein described herein reduces GAG in the subject by about 45%. In some embodiments, the administered rAAV vector expressing a VHH- I2S fusion protein described herein reduces GAG in the subject by about 40%.
  • the administered rAAV vector expressing a VHH- I2S fusion protein described herein reduces GAG in the subject by about 35%. In some embodiments, the administered rAAV vector expressing a VHH- I2S fusion protein described herein reduces GAG in the subject by about 30%. In some embodiments, the administered rAAV vector expressing a VHH- I2S fusion protein described herein reduces GAG in the subject by about 25%. In some embodiments, the administered rAAV vector expressing a VHH- I2S fusion protein described herein reduces GAG in the subject by about 20%.
  • the administered rAAV vector expressing a VHH- I2S fusion protein described herein reduces GAG in the subject by about 15%. In some embodiments, the administered rAAV vector expressing a VHH- I2S fusion protein described herein reduces GAG in the subject by about 10%. [0165] In some embodiments, the level of GAG is reduced in the serum, liver, brain, lung, kidney, spleen, quadriceps, heart and/or bone marrow of the subject. In some embodiments, the level of GAG is reduced in the serum of the subject. In some Attorney Docket No: MIL-025WO1 embodiments, the level of GAG is reduced in the liver of the subject.
  • the level of GAG is reduced in the central nervous system of the subject. Particularly, the level of GAG is reduced in the brain of the subject.
  • the administration of a rAAV vector expressing a VHH- I2S fusion protein described herein results in the reduction of heparan sulfate (HS), and dermatan sulfate (DS) in the serum, liver, brain, lung, kidney, spleen, quadriceps, heart and/or bone marrow of the subject.
  • HS heparan sulfate
  • DS dermatan sulfate
  • the administered rAAV vector reduces HS and DS by about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or about 10% in comparison to the subject’s baseline HS and DS levels prior to administering the rAAV vector. In some embodiments, the administered rAAV vector reduces HS and DS by about 95% in comparison to the subject’s baseline HS and DS levels prior to administering the rAAV vector. In some embodiments, the administered rAAV vector reduces HS and DS by about 90% in comparison to the subject’s baseline HS and DS levels prior to administering the rAAV vector.
  • the administered rAAV vector reduces HS and DS by about 85% in comparison to the subject’s baseline HS and DS levels prior to administering the rAAV vector. In some embodiments, the administered rAAV vector reduces HS and DS by about 80% in comparison to the subject’s baseline HS and DS levels prior to administering the rAAV vector. In some embodiments, the administered rAAV vector reduces HS and DS by about 75% in comparison to the subject’s baseline HS and DS levels prior to administering the rAAV vector. In some embodiments, the administered rAAV vector reduces HS and DS by about 70% in comparison to the subject’s baseline HS and DS levels prior to administering the rAAV vector.
  • the administered rAAV vector reduces HS and DS by about 65% in comparison to the subject’s baseline HS and DS levels prior to administering the rAAV vector. In some embodiments, the administered rAAV vector reduces HS and DS by about 60% in comparison to the subject’s baseline HS and DS levels prior to administering the rAAV vector. In some embodiments, the administered rAAV vector reduces HS and DS by about 55% in comparison to the subject’s baseline HS and DS levels prior to administering the rAAV vector. In some embodiments, the administered rAAV vector reduces HS and DS by about 50% in comparison to the subject’s baseline HS and DS levels prior to administering the rAAV vector.
  • the administered rAAV vector Attorney Docket No: MIL-025WO1 reduces HS and DS by about 45% in comparison to the subject’s baseline HS and DS levels prior to administering the rAAV vector. In some embodiments, the administered rAAV vector reduces HS and DS by about 35% in comparison to the subject’s baseline HS and DS levels prior to administering the rAAV vector. In some embodiments, the administered rAAV vector reduces HS and DS by about 25% in comparison to the subject’s baseline HS and DS levels prior to administering the rAAV vector.
  • the administered rAAV vector reduces HS and DS by about 10% in comparison to the subject’s baseline HS and DS levels prior to administering the rAAV vector.
  • the administration of a rAAV vector expressing a VHH- I2S fusion protein described herein results in reduction of lysosomal storage stress in the subject.
  • the administration of a rAAV vector expressing a VHH- I2S fusion protein described herein results in reduction of neuro-inflammation in the subject.
  • the administration of a rAAV vector expressing a VHH- I2S fusion protein described herein improves one or more symptoms of Hunter Syndrome in the subject.
  • Example 1 Design and generation of rAAV therapeutic vectors
  • This example relates to the design of a recombinant adeno-associated virus (rAAV) vector encoding an iduronate-2-sulfatase (I2S) protein described herein.
  • rAAV VHH-I2S vector design A schematic that illustrates exemplary rAAV vectors of the present disclosure is illustrated in Figure 1.
  • the key components of a rAAV vector of Attorney Docket No: MIL-025WO1 comprises a promoter, a transgene encoding a fusion protein comprising an anti-human transferrin receptor (anti-TfR1) VHH antibody fused to a human iduronate-2-sulfatse (I2S), a WPRE sequence and a 5’ and a 3’ inverted terminal repeat (ITR) sequences flanking the transgene coding sequence (CDS).
  • anti-TfR1 anti-human transferrin receptor
  • I2S human iduronate-2-sulfatse
  • WPRE sequence a 5’ and a 3’ inverted terminal repeat (ITR) sequences flanking the transgene coding sequence (CDS).
  • the transgene CDS encoding the anti-hTfR1 VHH-I2S fusion protein comprises a nucleic acid sequence encoding the anti-TfR1 VHH antibody and a nucleic acid sequence encoding the human I2S.
  • the nucleic acid sequence encoding the human iduronate-2-sulfatse (I2S) can be a wild-type or a codon-optimized variant.
  • the rAAV vector comprises a wild-type I2S nucleic acid sequence.
  • the rAAV vector comprises a codon-optimized I2S nucleic acid sequence.
  • rAAV vectors comprising coding sequences for a fusion protein comprising at least one anti-hTfR1 VHH antibody fused to a human iduronate 2-sulfatase (I2S) and variations of the same are provided in this Example.
  • I2S human iduronate 2-sulfatase
  • rAAV9 recombinant AAV vector
  • the basic design of a rAAV vector comprises an expression cassette flanked by inverted terminal repeats (ITRs): a 5’-ITR and a 3’-ITR. These ITRs mediate the replication and packaging of the vector genome by the AAV replication protein Rep and associated factors in vector producer cells.
  • an expression cassette contains a promoter, a coding sequence, a polyA tail and/or a tag, as shown in Figure 1A.
  • An expression construct encoding human I2S (hI2S) was designed and prepared using standard molecular biology techniques. The coding sequence for the hI2S was inserted downstream of a promoter, which can be ubiquitous or liver-specific. Additionally, liver- specific enhancers or cis-acting regulatory module (CRM) was inserted upstream of the promoter, and a minute virus of mice (MVM) intron sequence was grafted downstream of the promoter. This regulatory element and promoter combination was tested for ability to promote high transgene product expression, as shown in the examples that follow.
  • CCM liver- specific enhancers or cis-acting regulatory module
  • FIG. 1 shows schematic representations of the expression constructs described above.
  • the expression construct Attorney Docket No: MIL-025WO1 was then cloned into an AAV plasmid backbone and confirmed by sequencing. Vectors were packaged in viral particles and stored. Any number of variations of the above scheme can be performed.
  • Codon Optimization [0174] Additionally, the coding sequences for the VHH and I2S were codon-optimized based on multiple parameters, such as codon adaptation index (CAI), individual codon usage and codon context (ICU-CC) CpG site count, GC content, and repetitious base sequences. High CAI was preferred to utilize more frequently used codons and to potentially increase transgene product expression level from the vector. CpG sites, which can elicit immune response, were reduced. Repetitious bases were also removed. A web- based multi-objective optimization platform for synthetic gene design called COOL (Codon Optimization Online) and internal codon usage frequency table were used for this purpose. Additionally, potential splicing sites were manually removed.
  • COOL Codon Optimization Online
  • VHH and hI2S coding sequences are summarized in Table 4, and the schematics for the representative constructs of VHH-hI2S are shown in Figure 1, respectively. Any number of variations of the above scheme can be performed. For example, more than one promoter may be used, and/or an intron or IRES sequence may be introduced upstream of the coding region. Additionally, different combinations of regulatory regions, promoter, and intron can be contemplated. Table 4: Characteristics of optimized VHH1 coding sequences
  • Example 2 In vivo expression of rAAV.
  • VHH-I2S vectors in mice [0175] This study evaluates recombinant AAV vectors described herein for expressing recombinant protein in mice. The following vectors were tested.
  • Circulating transgene products expressed by injected vectors showed robustly higher I2S activity than vehicle control group A, after 2 weeks post injection, with ubiquitously expressed vectors resulting in more circulating I2S activity in serum than with liver-specific vectors (Table 6).
  • Table 6 Terminal serum I2S activity data Attorney Docket No: MIL-025WO1 Western blot against human I2S protein in serum [0178] Serum samples from each group were also analyzed through western blot using antibody against human I2S protein. Briefly, mouse serum was denatured by adding a reducing agent and heated at 95 degrees Celsius, then separated through SDS-PAGE gel electrophoresis.
  • the gel was subjected to protein transfer to a nitrocellulose membrane, blocked and incubated with a goat polyclonal anti-human I2S. Specific signal was detected through a detection antibody with infrared fluorescent conjugation. The blot showed a strong band at corresponding molecular weight of the VHH-I2S fusion protein ( Figure 2). The results confirm the integrity of the transgene product produced and secreted in the circulation.
  • Example 3 Pharmacokinetics and pharmacodynamics of rAAV therapeutic vectors in mice [0179] This study evaluated the pharmacokinetics and pharmacodynamics (PK/PD) of rAAV therapeutic vectors encoding anti-TfR1 VHH-hI2S fusion protein in human transferrin receptor 1 knock-in (hTfR1KI)/Iduronate-2-Sulfase knock-out (IdsKO) mice. [0180] Vectors were intravenously injected into hTfR1KI / IdsKO male mice at age of 8-9 weeks as listed in Table 7.
  • the hTfR1KI / IdsKO mouse model includes a knock-in of human transferrin receptor 1 (hTfR1) sequence into a single, endogenous mouse locus at Tfrc1 gene and a knockout of the Ids gene in the mouse genome (Sonoda et al., 2018. Mol Thr 26:1366-1374), which were used to evaluate the exposure and efficacy of a human specific anti-TfR1 antibody in the IdsKO background.
  • Table 7 rAAV vectors Attorney Docket No: MIL-025WO1 [0181] Mice were divided into groups and administered a dose of rAAV vectors according to the study design in Table 8.
  • test articles were injected into hTfR1KI / IdsKO mice.
  • Four of the test articles constituted the ubiquitous promoter driven expression cassettes that encoded VHH 1-I2S, VHH 2-I2S, VHH 3-I2S, or I2S.
  • the other four test articles constituted the liver specific promoter driven expression cassettes of the same transgenes as the ubiquitous promoter.
  • the AAVs consisting of ubiquitous promoter panel were single injected at two dosages, 2.5e11 and 2.5e12 vg/kg, and the AAVs harboring of liver specific promoter were injected at one dosage of 2.5e12 vg/kg. There were six to eight animals per group and all the mice were sacrificed at six weeks post injection.
  • Table 8 Study design [0182] Serum, cerebrospinal fluid (CSF) and other tissue samples were collected at the end of the study. Tissue samples collected were brain, liver, lung, quadriceps muscle, kidney, spleen, heart and bone marrow.
  • CSF cerebrospinal fluid
  • the analyses of the tissue samples included determination of the level of heparan sulfate and dermatan sulfate glycosaminoglycan (HS-GAG and DS-GAG, respectively), I2S enzymatic activity, I2S protein concentration by enhanced chemiluminescence (ECL) assay, protein quantification by bi-cinchonicic acid (BCA) assay, Immunohistochemistry (IHC) analysis by LAMP1 antibody, vector genome and mRNA analysis by droplet digital polymerase chain reaction (ddPCR).
  • ECL enhanced chemiluminescence
  • BCA bi-cinchonicic acid
  • IHC Immunohistochemistry analysis by LAMP1 antibody
  • ddPCR droplet digital polymerase chain reaction
  • ECL reagent is utilized to release luminescence signals to provide quantitative measurement of I2S against a standard of each purified VHH-I2S protein.
  • Transgene products were detected in serum and all evaluated tissues of rAAV test articles treated mice after 6 weeks post injection, in a dose-response manner for the corresponding groups with 2 doses ( Figure 3 and Table 9).
  • ubiquitous promoter test articles Figures 3I-3P
  • Figures 3A-H except rAAV9- SJ075 and rAAV9-SJ077 encoding untagged I2S protein that resulted in comparable serum and tissue exposure.
  • I2S level in serum and all tissues from liver-specific promoter driven transgene I2S yielded more transgene exposure than any of VHH tagged I2S test articles (TJ016, TJ031 or TJ032).
  • Table 9 I2S concentration in serum and tissues (nM or nmol/g of total protein)
  • Enzyme activity of I2S in the collected serum, brain, liver, lung, quadriceps muscle, kidney, spleen, heart and bone marrow was measured using a fluorometric assay for I2S enzymatic activity. This is a two-step reaction under acidic conditions (Azadeh et al.2018).
  • I2S hydrolyzes 4-methylumbellifery ⁇ -L-iduronide-2- sulfate (4-MUS) to 4-methylumbelliferyl ⁇ -L-iduronide (MUBI).
  • another enzyme IDUA hydrolyzes MUBI to the final product, 4-methylumbelliferone (4-MU) that emits fluorescence at 365nM (Ex) and 450nM (Em), and the signal can be quantified against 4-MU standard curve.
  • the activity was normalized by protein Attorney Docket No: MIL-025WO1 concentration measured by BCA, then calculated and reported as percentage of WT endogenous I2S activity set at 100%.
  • rAAV9-TJ027 CBh-VHH 3-I2S
  • Table 10 I2S activity in brain tissue in percentage of WT HS/DS concentrations
  • Glycosaminoglycans (GAGs) clearance was measured to evaluate the efficacy of anti-TfR1VHH-hI2S fusion protein expressed by the rAAV constructs delivered intravenously.
  • GAGs were assessed using a liquid chromatography - mass spectrometry (LC-MS/MS) assay, detecting the heparan sulfate (HS) and dermatan sulfate (DS) levels in Attorney Docket No: MIL-025WO1 mice.
  • LC-MS/MS liquid chromatography - mass spectrometry
  • HS heparan sulfate
  • DS dermatan sulfate
  • Figure 5 showed remaining level of HS substrates in CNS of mice treated with test articles.
  • All VHH fused I2S test articles TJ025, TJ026, TJ027, TJ016, TJ031, TJ032
  • TJ075 and SJ076 showed significant HS substrates reduction, almost or had normalized to WT level, compared to the untagged test articles (SJ075 and SJ076).
  • the VHH 2-I2S transgene product from TJ031 and TJ026 was the least efficacious of all VHH types.
  • VHH-I2S transgene from vectorized test articles were able to cross the blood brain barrier and cleared the GAG substrates from CNS.
  • the liver-producing VHH-I2S transgene (TJ016, TJ031 and TJ032) ensured a brain substrate crossed correction, since there would not be any local brain production.
  • Figure 6 demonstrated the level of HS substrates remaining in peripheral tissues that demonstrated statistically significantly near, if not normalized to WT control with all test articles evaluated at both doses with lung tissue being the least robust tissue.
  • Table 11 reported the percentage of HS remaining in CSF and all tissues, calculated using WT as 100% and hTfR1 KI Hetero/ Ids KO as 0%.
  • Figure 7 and Table 12 demonstrated the level of DS substrates in all collected peripheral tissues. All test articles significantly reduced DS substrates compared to untreated control (hTfR1 KI Hetero/ Ids KO). At 2.5e12 vg/kg, DS substrates of mice treated with any test article, were near if not normalized, regardless of promoter type and Attorney Docket No: MIL-025WO1 VHH tag. At a 10 folds lower dose, lung tissue was the least reduced for TJ025 and TJ027 test articles, especially in lung and kidney tissues.
  • Table 11 Percentage of HS remaining in CNS and peripheral tissues Attorney Docket No: MIL-025WO1
  • Table 12 Percentage of DS remaining in peripheral tissues Attorney Docket No: MIL-025WO1 Immunohistochemistry [0191] Brain, liver, lung, kidney, spleen, and quadricep sections are collected in formalin-fixed paraffin-embedded blocks, and tissue sections are subjected to immunostaining using anti-LAMP1 or anti-GFAP antibodies.
  • Vector genome titer and mRNA concentration [0192] Copies of vector genome DNA (vg) and mRNA in hTfR1 KI / Ids KO mice were assayed from tissues after systemic delivery of test articles.
  • RNAlater Thermofisher Cat#AM7020
  • Oligonucleotide primer and probe sets against the AAV vector genome DNA and the mRNA transcript were designed to specifically recognize the target sequences with minimum background signal against no template control.
  • Figures 8B and 8D demonstrated liver vector genome transduction, detected robustly, in a dose-response matter for all CBh promoter driven test articles (TJ025, TJ026, TJ027 and SJ075).
  • Test articles with the hTTR liver specific promoter (TJ016, TJ031, TJ032 and SJ077) also transduced in liver with significantly high vector genome copy number and elevated mRNA copy number.
  • EQUIVALENTS AND SCOPE [0194] Those skilled in the art would recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the Attorney Docket No: MIL-025WO1 invention described herein. The scope of the invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

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Abstract

La présente invention concerne des vecteurs de thérapie génique pour le traitement de la maladie de Hunter. Le vecteur de thérapie génique exprime une protéine de fusion comprenant au moins un anticorps anti-hTfR1 VHH fusionné à une iduronate-2-sulfatase humaine.
PCT/IB2023/061655 2022-11-18 2023-11-17 Vecteurs aav recombinants et méthodes de traitement de la maladie de hunter WO2024105638A1 (fr)

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Citations (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5139941A (en) 1985-10-31 1992-08-18 University Of Florida Research Foundation, Inc. AAV transduction vectors
US5478745A (en) 1992-12-04 1995-12-26 University Of Pittsburgh Recombinant viral vector system
US5552157A (en) 1990-08-27 1996-09-03 Kabushiki Kaisha Vitamin Kenkyusya Liposome for entrapping gene, liposomal preparation and process for the manufacture of the preparation
US5565213A (en) 1990-07-26 1996-10-15 Taisho Pharmaceutical Co., Ltd. Stable liposome aqueous suspension
US5567434A (en) 1989-03-31 1996-10-22 The Regents Of The University Of California Preparation of liposome and lipid complex compositions
US5738868A (en) 1995-07-18 1998-04-14 Lipogenics Ltd. Liposome compositions and kits therefor
US5741683A (en) 1995-06-07 1998-04-21 The Research Foundation Of State University Of New York In vitro packaging of adeno-associated virus DNA
US5741516A (en) 1994-06-20 1998-04-21 Inex Pharmaceuticals Corporation Sphingosomes for enhanced drug delivery
US5795587A (en) 1995-01-23 1998-08-18 University Of Pittsburgh Stable lipid-comprising drug delivery complexes and methods for their production
US6001650A (en) 1995-08-03 1999-12-14 Avigen, Inc. High-efficiency wild-type-free AAV helper functions
US6057152A (en) 1992-12-04 2000-05-02 University Of Pittsburgh Recombinant viral vector system
US6156303A (en) 1997-06-11 2000-12-05 University Of Washington Adeno-associated virus (AAV) isolates and AAV vectors derived therefrom
US6204059B1 (en) 1994-06-30 2001-03-20 University Of Pittsburgh AAV capsid vehicles for molecular transfer
US6268213B1 (en) 1992-06-03 2001-07-31 Richard Jude Samulski Adeno-associated virus vector and cis-acting regulatory and promoter elements capable of expressing at least one gene and method of using same for gene therapy
US6491907B1 (en) 1998-11-10 2002-12-10 The University Of North Carolina At Chapel Hill Recombinant parvovirus vectors and method of making
WO2003042397A2 (fr) 2001-11-13 2003-05-22 The Trustees Of The University Of Pennsylvania Methode de detection et/ou d'identification de sequences de virus associes aux adenovirus (aav) et d'isolation de nouvelles sequences ainsi identifiees
US6660514B1 (en) 1998-05-27 2003-12-09 University Of Florida Research Foundation Method of preparing recombinant adeno-associated virus compositions
WO2005033321A2 (fr) 2003-09-30 2005-04-14 The Trustees Of The University Of Pennsylvania Variantes des virus associes aux adenovirus (aav), sequences, vecteurs les contenant, et leur utilisation
US6951753B2 (en) 1989-09-07 2005-10-04 The 501 Trustees Of Princeton University Helper-free stocks of recombinant adeno-associated virus vectors
US20060136184A1 (en) 2003-08-01 2006-06-22 Dna Twopointo Inc. Systems and methods for antibody engineering
US7094604B2 (en) 2002-06-05 2006-08-22 University Of Florida Research Foundation, Inc. Production of pseudotyped recombinant AAV virions
WO2006110689A2 (fr) 2005-04-07 2006-10-19 The Trustees Of The University Of Pennsylvania Procede d'augmentation de la fonction d'un vecteur aav
US7201898B2 (en) 2000-06-01 2007-04-10 The University Of North Carolina At Chapel Hill Methods and compounds for controlled release of recombinant parvovirus vectors
US7229823B2 (en) 1997-04-14 2007-06-12 Richard Jude Samulski Methods for increasing the efficiency of recombinant AAV product
US7282199B2 (en) 2001-12-17 2007-10-16 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) serotype 8 sequences, vectors containing same, and uses therefor
US7439065B2 (en) 1995-06-07 2008-10-21 The University Of North Carolina At Chapel Hill Helper virus-free AAV production
US7588772B2 (en) 2006-03-30 2009-09-15 Board Of Trustees Of The Leland Stamford Junior University AAV capsid library and AAV capsid proteins
WO2012158757A1 (fr) 2011-05-16 2012-11-22 The Trustees Of The University Of Pennsylvania Plasmides pro-viraux pour la production de virus adéno-associé recombinant
US20120322861A1 (en) 2007-02-23 2012-12-20 Barry John Byrne Compositions and Methods for Treating Diseases
US20140032186A1 (en) 2003-08-01 2014-01-30 Dna Twopointo, Inc. Systems and methods for antibody engineering
WO2015012924A2 (fr) 2013-04-29 2015-01-29 The Trustees Of The University Of Pennsylvania Cassettes d'expression préférentielle pour tissus modifiées par un codon, vecteurs les contenant, et utilisation

Patent Citations (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5139941A (en) 1985-10-31 1992-08-18 University Of Florida Research Foundation, Inc. AAV transduction vectors
US5567434A (en) 1989-03-31 1996-10-22 The Regents Of The University Of California Preparation of liposome and lipid complex compositions
US6951753B2 (en) 1989-09-07 2005-10-04 The 501 Trustees Of Princeton University Helper-free stocks of recombinant adeno-associated virus vectors
US5565213A (en) 1990-07-26 1996-10-15 Taisho Pharmaceutical Co., Ltd. Stable liposome aqueous suspension
US5552157A (en) 1990-08-27 1996-09-03 Kabushiki Kaisha Vitamin Kenkyusya Liposome for entrapping gene, liposomal preparation and process for the manufacture of the preparation
US6268213B1 (en) 1992-06-03 2001-07-31 Richard Jude Samulski Adeno-associated virus vector and cis-acting regulatory and promoter elements capable of expressing at least one gene and method of using same for gene therapy
US6057152A (en) 1992-12-04 2000-05-02 University Of Pittsburgh Recombinant viral vector system
US5478745A (en) 1992-12-04 1995-12-26 University Of Pittsburgh Recombinant viral vector system
US5741516A (en) 1994-06-20 1998-04-21 Inex Pharmaceuticals Corporation Sphingosomes for enhanced drug delivery
US6204059B1 (en) 1994-06-30 2001-03-20 University Of Pittsburgh AAV capsid vehicles for molecular transfer
US5795587A (en) 1995-01-23 1998-08-18 University Of Pittsburgh Stable lipid-comprising drug delivery complexes and methods for their production
US5741683A (en) 1995-06-07 1998-04-21 The Research Foundation Of State University Of New York In vitro packaging of adeno-associated virus DNA
US7439065B2 (en) 1995-06-07 2008-10-21 The University Of North Carolina At Chapel Hill Helper virus-free AAV production
US5738868A (en) 1995-07-18 1998-04-14 Lipogenics Ltd. Liposome compositions and kits therefor
US6001650A (en) 1995-08-03 1999-12-14 Avigen, Inc. High-efficiency wild-type-free AAV helper functions
US7229823B2 (en) 1997-04-14 2007-06-12 Richard Jude Samulski Methods for increasing the efficiency of recombinant AAV product
US6156303A (en) 1997-06-11 2000-12-05 University Of Washington Adeno-associated virus (AAV) isolates and AAV vectors derived therefrom
US6660514B1 (en) 1998-05-27 2003-12-09 University Of Florida Research Foundation Method of preparing recombinant adeno-associated virus compositions
US7172893B2 (en) 1998-11-10 2007-02-06 University Of North Carolina At Chapel Hill Virus vectors and methods of making and administering the same
US6491907B1 (en) 1998-11-10 2002-12-10 The University Of North Carolina At Chapel Hill Recombinant parvovirus vectors and method of making
US7201898B2 (en) 2000-06-01 2007-04-10 The University Of North Carolina At Chapel Hill Methods and compounds for controlled release of recombinant parvovirus vectors
WO2003042397A2 (fr) 2001-11-13 2003-05-22 The Trustees Of The University Of Pennsylvania Methode de detection et/ou d'identification de sequences de virus associes aux adenovirus (aav) et d'isolation de nouvelles sequences ainsi identifiees
US20030138772A1 (en) 2001-11-13 2003-07-24 Guangping Gao Method of detecting and/or identifying adeno-associated virus (AAV) sequences and isolating novel sequences identified thereby
US7790449B2 (en) 2001-12-17 2010-09-07 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) serotype 8 sequences, vectors containing the same, and uses therefor
US7282199B2 (en) 2001-12-17 2007-10-16 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) serotype 8 sequences, vectors containing same, and uses therefor
US20070015238A1 (en) 2002-06-05 2007-01-18 Snyder Richard O Production of pseudotyped recombinant AAV virions
US7094604B2 (en) 2002-06-05 2006-08-22 University Of Florida Research Foundation, Inc. Production of pseudotyped recombinant AAV virions
US20060136184A1 (en) 2003-08-01 2006-06-22 Dna Twopointo Inc. Systems and methods for antibody engineering
US20140032186A1 (en) 2003-08-01 2014-01-30 Dna Twopointo, Inc. Systems and methods for antibody engineering
WO2005033321A2 (fr) 2003-09-30 2005-04-14 The Trustees Of The University Of Pennsylvania Variantes des virus associes aux adenovirus (aav), sequences, vecteurs les contenant, et leur utilisation
WO2006110689A2 (fr) 2005-04-07 2006-10-19 The Trustees Of The University Of Pennsylvania Procede d'augmentation de la fonction d'un vecteur aav
US7588772B2 (en) 2006-03-30 2009-09-15 Board Of Trustees Of The Leland Stamford Junior University AAV capsid library and AAV capsid proteins
US20120322861A1 (en) 2007-02-23 2012-12-20 Barry John Byrne Compositions and Methods for Treating Diseases
WO2012158757A1 (fr) 2011-05-16 2012-11-22 The Trustees Of The University Of Pennsylvania Plasmides pro-viraux pour la production de virus adéno-associé recombinant
WO2015012924A2 (fr) 2013-04-29 2015-01-29 The Trustees Of The University Of Pennsylvania Cassettes d'expression préférentielle pour tissus modifiées par un codon, vecteurs les contenant, et utilisation

Non-Patent Citations (24)

* Cited by examiner, † Cited by third party
Title
AZADEH ET AL., JIMD REP., vol. 38, 2018, pages 89 - 95
BUNING ET AL.: "Recent developments in adeno-associated virus vector technology", J. GENE MED, vol. 10, 2008, pages 717 - 733
E. W. MARTIN: "Remington's Pharmaceutical Sciences", 1995, MACK PUBLISHING CO., pages: 1035 - 1038,1570-1580
FRANKEL ET AL., MOL. IMMUNOL., vol. 16, 1979, pages 101 - 106
GREENSAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2012, COLD SPRING HARBOR PRESS
GRIEGERSAMULSKI: "Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications", ADV. BIOCHEM. ENGINLBIOTECHNOL., vol. 99, 2005, pages 119 - 145, XP009125595
GRIMM ET AL.: "Helper Virus-Free, Optically Controllable, and Two-Plasmid-Based Production of Adeno-associated Virus Vectors of Serotypes 1 to 6", MOLECULAR THERAPY, vol. 7, 2003, pages 839 - 850, XP002256403, DOI: 10.1016/S1525-0016(03)00095-9
GRIMM ET AL.: "Novel Tools for Production and Purification of Recombinant Adeno associated Virus Vectors", HUMAN GENE THERAPY, vol. 9, 1998, pages 2745 - 2760
HIROYUKI SONODA ET AL: "A Blood-Brain-Barrier-Penetrating Anti-human Transferrin Receptor Antibody Fusion Protein for Neuronopathic Mucopolysaccharidosis II", MOLECULAR THERAPY, vol. 26, no. 5, 1 May 2018 (2018-05-01), US, pages 1366 - 1374, XP055530922, ISSN: 1525-0016, DOI: 10.1016/j.ymthe.2018.02.032 *
HUANG QIN: "An AAV capsid reprogrammed to bind human Transferrin Receptor mediates brain-wide gene delivery", BIORXIV, 22 December 2023 (2023-12-22), pages 1 - 34, XP093128498, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2023.12.20.572615v1.full.pdf> [retrieved on 20240207], DOI: 10.1101/2023.12.20.572615 *
K. FISHER ET AL., J. VIROL, vol. 70, 1993, pages 520 - 532
K. FISHER ET AL., J. VIROL., vol. 70, 1993, pages 520 - 532
KANUT LAOHARAWEE ET AL: "Prevention of Neurocognitive Deficiency in Mucopolysaccharidosis Type II Mice by Central Nervous System-Directed, AAV9-Mediated Iduronate Sulfatase Gene Transfer", HUMAN GENE THERAPY, vol. 28, no. 8, 1 August 2017 (2017-08-01), GB, pages 626 - 638, XP055401129, ISSN: 1043-0342, DOI: 10.1089/hum.2016.184 *
KEM, A. ET AL.: "Identification of a Heparin-Binding Motif on Adeno- Associated Virus Type 2 Capsids", JOURNAL OF VIROLOGY, vol. 77, 2003, pages 11072 - 11081
KIDA SACHIHO: "Non-clinical evaluation of a blood-brain barrier-penetrating enzyme for the treatment of mucopolysaccharidosis type I", MOLECULAR GENETICS AND METABOLISM, vol. 126, no. 2, 1 February 2019 (2019-02-01), AMSTERDAM, NL, pages S83 - S84, XP093127944, ISSN: 1096-7192, DOI: 10.1016/j.ymgme.2018.12.202 *
LUX ET AL., J VIROLOGY, vol. 79, 2005, pages 11776 - 87
MIZUKAMI, HIROAKI ET AL., A PROTOCOL FOR AAV VECTOR PRODUCTION AND PURIFICATION
SONODA ET AL., MOL THR, vol. 26, 2018, pages 1366 - 1374
STEVEN GRAY ET AL., HUM GENE THER., vol. 22, no. 9, September 2011 (2011-09-01), pages 1143 - 1153
TANAKA ET AL.: "Evaluation of cerebrospinal fluid heparan sulfate as a biomarker of neuropathology in a murine model of mucopolysaccharidosis type II using high-sensitivity LC/MS/MS", MOLECULAR GENETICS AND METABOLISM, vol. 125, 2018, pages 53 - 58, XP085496517, DOI: 10.1016/j.ymgme.2018.07.013
WOUTERS YESSICA ET AL: "VHHs as tools for therapeutic protein delivery to the central nervous system", FLUIDS AND BARRIERS OF THE CNS, vol. 19, no. 1, 3 October 2022 (2022-10-03), XP093065073, Retrieved from the Internet <URL:https://link.springer.com/article/10.1186/s12987-022-00374-4/fulltext.html> DOI: 10.1186/s12987-022-00374-4 *
YAMAMOTO RYUJI ET AL: "Nonclinical safety evaluation of pabinafusp alfa, an anti-human transferrin receptor antibody and iduronate-2-sulfatase fusion protein, for the treatment of neuronopathic mucopolysaccharidosis type II", MOLECULAR GENETICS AND METABOLISM REPORTS, vol. 27, 1 June 2021 (2021-06-01), pages 100758, XP093127774, ISSN: 2214-4269, DOI: 10.1016/j.ymgmr.2021.100758 *
ZHANG ET AL.: "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production", HUMAN GENE THERAPY, vol. 20, 2009, pages 922 - 929, XP055873883
ZHIJIAN WU ET AL., MOLECULAR THERAPY, vol. 16, no. 2, February 2008 (2008-02-01)

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