Attorney Docket No: JCR-002WO1 RECOMBINANT AAV VECTORS AND METHODS FOR TREATMENT OF DISEASES WITH CENTRAL NERVOUS SYSTEM DISORDERS CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63/468,775, filed on May 24, 2023; the contents of which are incorporated herein by reference in entirety for all purpose. INCORPORATION BY REFERENCE OF SEQUENCE LISTING [0002] The present application contains a Sequence Listing which is submitted electronically in XML format and is herein incorporated by reference in its entirety. The sequence listing file entitled JCR-002WO1.XML, was created on May 14, 2024, which is 91,327 bytes in size. BACKGROUND [0003] There are many diseases accompanied by a central nervous system disorder. Mucopolysaccharidosis III or Sanfilippo syndrome is one of such diseases, which is a lysosomal storage disorder caused by mutations in genes responsible for the degradation of heparan sulfate, a glycosaminoglycan located in the extracellular membrane. Undegraded heparan sulfate molecules accumulate within lysosomes leading to cellular dysfunction and pathology in several organs, with severe central nervous system degeneration as the main phenotypical feature. Patients are characterized by severe neurological pathology leading to childhood dementia. There are four different subtypes of Sanfilippo syndrome based on the mutated gene and the consequent enzyme deficiency: types A-D. [0004] Sanfilippo syndrome type A is considered the most aggressive form, with patients surviving until 15–18 years old on average. Sanfilippo syndrome type A or MPS IIIA is caused by mutations in the SGSH gene, coding for sulfamidase (also known as heparan sulfate sulfatase or N-sulfoglucosamine sulfohydrolase, EC 3.10.1.1), which releases sulfate groups linked to the amino group of glucosamine. [0005] Currently, there is no treatment to effectively slow down or reverse Sanfilippo syndrome patients’ neurodegeneration, and their management consists only of palliative measures to alleviate the symptomatology. The main approaches consist of enzyme replacement
Attorney Docket No: JCR-002WO1 therapy (ERT), substrate reduction therapy (SRT), pharmacological chaperones, stem cell transplantation, and gene therapy. Gene therapy consists of the delivery of the correct copy of the gene to affected cells in order to recover enzyme activity. Gene therapy is the most promising therapeutic option for LSDs since, as already referred, it has been proposed that only 5–15% of enzyme activity is required to maintain a healthy condition in affected patients. SUMMARY OF THE INVENTION [0006] The present invention provides highly efficient gene therapy agents for treating diseases accompanied by a central nervous system disorder including lysosomal storage diseases (LSDs), such as Sanfilippo syndrome or Mucopolysaccharidosis III (MPS III) (e.g., type A). The gene therapy agent comprises a recombinant adeno-associated virus (rAAV) vector with increased ability to let a therapeutic protein cross the blood-brain barrier (BBB), thereby increasing an amount of the therapeutic protein in the central nervus system, the therapeutic protein including a human lysosomal protein, a human cytokine, and human neurotrophic factor. A human N-sulfoglucosamine sulfohydrolase (SGSH) is one of the human lysosomal proteins. [0007] In one aspect of the present disclosure, provided herein is a recombinant adeno- associated viral vector (rAAV) comprising: a transgene polynucleotide encoding a fusion protein comprising at least one anti-human transferrin receptor (hTfR1) VHH antibody fused to a human lysosomal enzyme, for example, a human N-sulfoglucosamine sulfohydrolase (SGSH), a human cytokine, or a human neurotrophic factor. In particular, the vectors described herein result in increased ability of the lysosomal enzyme, for example, N-sulfoglucosamine sulfohydrolase (SGSH), the cytokine, or the neurotrophic factor 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 transgene is flanked by a viral inverted terminal repeat at both ends, i.e., a 5’ inverted terminal repeat (ITR) and a 3’ ITR. [0008] In some embodiments, 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. 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 any one of SEQ ID NOs.8-14. [0009] In some embodiments, the human N-sulfoglucosamine sulfohydrolase (SGSH) of the fusion protein is a human wild type N-sulfoglucosamine sulfohydrolase (SGSH) or a functional
Attorney Docket No: JCR-002WO1 variant thereof. In some embodiments, the human N-sulfoglucosamine sulfohydrolase comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the human N- sulfoglucosamine sulfohydrolase is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 21. [0010] In some embodiments, the fusion protein encoded by the vector described herein comprises a peptide linker sequence that links the anti-hTfR1 VHH antibody and one of the human lysosomal enzymes, the human cytokines, and the human neurotrophic factors. The peptide linker may be about 4-100 amino acid residues, e.g., 4-50 amino acid residues, 4-20 amino acid residues, 5-100 amino acid residues, 5-50 amino acid residues, 5-20 amino acid residues or 10-50 amino acid residues. In one preferred embodiment, the peptide linker is a GGGGS (G4S) peptide linker. As a non-limiting example, the peptide linker comprises GGGGSGGGGSGGGGS (SEQ ID NO: 18). [0011] In some embodiments, the fusion protein further comprises a signal peptide at the N- terminus of the fusion protein. As a non-limiting example, the signal peptide of the fusion protein comprises SEQ ID NO: 19. [0012] In some embodiments, the anti-hTfR1 VHH antibody is fused to the N-terminus of one of the human lysosomal enzymes, the human cytokines, or the human neurotrophic factors. In other embodiments, the anti-hTfR1 VHH antibody is fused to the C-terminus of one of the human lysosomal enzymes, the human cytokines, or the human neurotrophic factors. [0013] In some embodiments, the vector described herein encodes a fusion protein of the anti-hTfR1 VHH antibody and the human SGSH, the fusion protein comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the vector described herein encodes a fusion protein comprising the amino acid sequence of SEQ ID NO: 48. In other 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 any one of SEQ ID NO: 46 and SEQ ID NO: 48. [0014] In some embodiments, the vector described herein encodes a fusion protein of the anti-hTfR1 VHH antibody and the human SGSH, the fusion protein comprises the nucleotide sequence of SEQ ID NO: 47. In some embodiments, the vector encoding a fusion protein described herein comprises the nucleotide sequence of SEQ ID NO: 49. In other embodiments, the vector encoding a fusion protein described herein comprises a nucleotide sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 47 and SEQ ID NO: 49.
Attorney Docket No: JCR-002WO1 [0015] In some embodiments, the promoter of the vector described herein is a ubiquitous promoter. For example, the ubiquitous promoter is a CBh promoter. As non-limiting example, the CBh promoter comprises the sequence of SEQ ID NO.25. [0016] In some embodiments, the promoter of the vector described herein is a liver specific promoter. For example, the liver specific promoter is transthyretin (TTR) promoter. As non- limiting example, the TTR promoter comprises the sequence of SEQ ID NO.23. [0017] In some embodiments, the vector described herein further comprises a liver specific enhancer. In some embodiments, the liver specific enhancer comprises one or more copies of CRM8. As non-limiting example, the vector comprises three copies of CRM8 (SEQ ID NO: 22). [0018] In some embodiments, the vector described herein comprises a Woodchuck Hepatitis Virus posttranscriptional regulatory element (WPRE) and a polyA sequence. In some embodiment, the WPRE is a WPRE3 or a variant thereof. As a non-limiting example, the WPRE is a WPRE3 variant, WPRE3mut5delATG, comprising SEQ ID NO.28. [0019] In some embodiments, the vector described herein comprises a polyA sequence. In some embodiments, the polyA sequence is an in-silicon synthetic polyA sequence. As non- limiting example, the polyA comprises the sequence of SEQ ID NO: 29. [0020] In some embodiments. The vector comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR flanking the transgene encoding the fusion protein. As a non-limiting example, the vector comprises a 5’ITR having the sequence of SEQ ID NO: 32 and a 3’ITR having the sequence of SEQ ID NO: 31. [0021] In some embodiments, the recombinant vector described herein further comprises an IRES. As a non-limiting example, the IRES comprises the sequence of SEQ ID NO: 33. [0022] In some embodiments, wherein the fusion protein comprises the anti-hTfR1 VHH antibody and the human SGSH, the recombinant vector described herein comprises a nucleic acid sequence presented by SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 44. [0023] In other embodiments, wherein the fusion protein comprises the anti-hTfR1 VHH antibody and the human SGSH, 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 any one of SEQ ID NOs.34-44. [0024] In another aspect of the present invention, provided is a recombinant AAV viral particle comprising a transgene vector described herein.
Attorney Docket No: JCR-002WO1 [0025] In some embodiments, the recombinant vector described herein is packaged with an AAV9 capsid. Accordingly, 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 lysosomal enzyme, a human cytokine, or a human neurotrophic factor described in the present disclosure is provided. [0026] In some embodiments, the vector, the transgene encoding a fusion protein described herein is packaged with a delivery vehicle. In some embodiments, the delivery vehicle is a virus, such as an adeno-associated-virus, an adenovirus, a retrovirus and a lentivirus. [0027] In some embodiments, 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)). [0028] In yet another aspect, 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. [0029] In yet another aspect of the present disclosure, provides is 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. [0030] In yet another aspect, the present disclosure provides a method for treating Sanfilippo syndrome type A (also known as Mucopolysaccharidosis IIIA, MPS IIIA) 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. [0031] In some embodiments, the administration results in an increase of an enzymatic activity of N-sulfoglucosamine sulfohydrolase 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. In one example, the increase of an enzymatic activity of N-sulfoglucosamine sulfohydrolase is observed in the central nervous system of the subject (e.g., the brain). [0032] In some embodiments, the administration reduces the level of glycosaminoglycan (GAG) in the subject, such as HS-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.
Attorney Docket No: JCR-002WO1 [0033] In some embodiments, the levels of heparan sulfate (HS) are reduced in the serum, liver, brain, lung, kidney, spleen, quadriceps, heart and/or bone marrow of the subject. [0034] In some embodiments, the levels of HS are reduced about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. [0035] In some embodiments, the administration reduces the lysosomal storage burden, wherein the lysosomal stress is determined by the level of LAMP1. [0036] In some embodiments, the administration reduces neuro-inflammation, wherein the neuroinflammation is determined by the expression level of GFAP. [0037] In some embodiments, 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. [0038] In another aspect of the present disclosure, provided also includes a method for inducing or increasing the enzymatic activity of the lysosomal enzyme, for example, N- sulfoglucosamine sulfohydrolase in a subject in need using the rAAV vector, the viral particle, the delivery vehicle or the transgene described herein. Also provided herein includes a method for increasing the expression level of the lysosomal enzyme, for example, N-sulfoglucosamine sulfohydrolase in a subject using the rAAV vector, the viral particle, the delivery vehicle or the transgene described herein. BRIEF DESCRIPTION OF THE DRAWINGS [0039] Figure 1 shows the schematics of the rAAV.VHH-SGSH or rAAV. SGSH-VHH vectors. Typically, a viral vector consists of a 5’ inverted terminal repeat (5’ ITR) and 3’‘ITR flanking the coding sequence (CDS). A liver-specific enhancer-promoter combination is used, with or without an MVM intron, to drive the expression of VHH fused to SGSH CDS at the N- terminus (VHH-SGSH) or C-terminus (SGSH-VHH). The fusion is separated by a linker peptide. The expression cassette has a signal peptide. A 3’ regulatory element, WPRE, for enhancing mRNA transcript stability is appended between the stop codon and a synthetic polyA sequence. [0040] Figure 2 shows that systemic delivery of rAAV9-VHH-SGSH resulted in significant exposure in the brain. Using AAV vectors that drive transgene protein product expression via a liver-specific promoter, SGSH exposure was significantly detected in the CNS compartment of
Attorney Docket No: JCR-002WO1 mice that intravenously received vectors encoding VHH-SGSH or SGSH-VHH, but not untagged SGSH. [0041] Figure 3 shows serum SGSH levels attained after systemic delivery of rAAV9- VHH-SGSH. Intravenous delivery of AAV vectors that encode liver-specific promoter-to drive the expression of untagged SGSH, VHH-SGSH, or SGSH-VHH resulted in robust expression of transgene products in circulation. [0042] Figure 4 shows that systemic delivery of rAAV9 vectors encoding liver promoter to drive the expression of VHH-SGSH and SGSH-VHH, resulted in robust clearance of toxic HS- GAG in the CSF of an MPSIIIA mouse model, as compared to the mice that received vectors encoding untagged SGSH. [0043] Figure 5 shows that systemic delivery of vectors encoding liver promoter to drive the expression of VHH-SGSH and SGSH-VHH, resulted in robust clearance of toxic HS-GAG in the brain of an MPSIIIA mouse model, as compared to the mice that received vectors encoding untagged SGSH. DETAILED DESCRIPTION Definitions [0044] Unless defined otherwise in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application. [0045] The term “a” or “an” refers to one or more. As such, the terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein. [0046] The words “comprise”, “comprises”, and “comprising” are to be interpreted inclusively rather than exclusively. The words “consist”, “consisting”, and its variants, are to be interpreted exclusively, rather than inclusively. While various embodiments in the specification are presented using “comprising” language, under other circumstances, a related embodiment is also intended to be interpreted and described using “consisting of” or “consisting essentially of” language. [0047] The term “about” encompasses a variation within and including 10%, unless otherwise specified. [0048] Antibody: As used herein, the term “antibody” refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or antigen binding fragments
Attorney Docket No: JCR-002WO1 thereof, which specifically binds and recognizes an antigen. In the context of the present disclosure, 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. The term “human antibody”, however, also includes an 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. The term “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. As far as it originates from a non-human mammal, there is no particular limitation as to the biological species from which those CDRs originate that are grafted into a proper position of the human antibody, though preferred are mouse, rat, rabbit, horse or non-human primate, more preferred are mouse and rat, and mouse, for example.
Attorney Docket No: JCR-002WO1 [0049] Binding affinity: As used herein, the term “binding affinity” refers to the affinity of an antibody, such as a VHH antibody, or antigen binding fragment thereof for an antigen. In one embodiment, affinity is calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol., 16:101-106, 1979. In another embodiment, binding affinity is measured by an antigen-antibody dissociation rate. In yet another embodiment, a high binding affinity is measured by a competition radioimmunoassay or by surface plasmon resonance in a BIACORE. In several examples, a high binding affinity is at least about 1×10−8 M. In other embodiments, 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. [0050] Disease: As used herein, the terms "disease", "disorder" and "condition" refer to a disease accompanied by a central nervous system disorder, including neurodegenerative disease. Lysosomal storage diseases (LSDs) such as Mucopolysaccharidosis type IIIA (MPS IIIA, also known as Sanfilippo syndrome type A or Sanfilippo type A disease) is one of such diseases. As used herein, the term “MPS IIIA-related symptom(s)” or “symptom(s)” refers to symptom(s) found in MPS IIIA patients as well as in MPS IIIA animal models. Such symptoms include but not limited to delayed speech; difficulty with social interactions and communication; sleep disturbances; progressive intellectual disability and the loss of previously acquired skills (developmental regression); seizures and movement disorders; a large head; a slightly enlarged liver (mild hepatomegaly); a soft out-pouching around the belly-button (umbilical hernia) or lower abdomen (inguinal hernia); short stature, joint stiffness, mild dysostosis multiplex, multiple skeletal abnormalities; chronic diarrhea; recurrent upper respiratory infections; recurrent ear infections; hearing impairment; vision problems; Asymmetric septal hypertrophy; Coarse facial features; Coarse hair; Dense calvaria; Dysostosis multiplex; Growth abnormality; Heparan sulfate excretion in urine; and HS accumulation in the cerebrospinal fluid (CSF), serum, urine and/or any other biological samples, etc. [0051] Recombinant: 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. Similarly, 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. For
Attorney Docket No: JCR-002WO1 example, a fusion protein comprising an anti-hTfR1 VHH antibody fused to a human SGSH enzyme is a recombinant protein. [0052] Recombinant AAV viral particle: As used herein, “recombinant AAV (rAAV) viral particle” or “recombinant AAV (rAAV) 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. 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). These and other suitable elements of the expression cassette are described in more detail below. This may also be referred to as a “full” AAV capsid. Such a rAAV viral particle is termed “pharmacologically active” when it delivers the transgene to a host cell which is capable of expressing the desired gene product carried by the expression cassette. Further “rAAV viral particle 9” refers to a AAV viral particle comprising AAV9 capsid. Same can be applied to other serotypes of AAVs. [0053] The terms “nucleic acid molecule”, “nucleic acid” and “polynucleotide” may be used interchangeably, and refer to a polymer of nucleotides. Such 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. [0054] Sequence identity: The similarity between amino acid sequences is expressed in terms of the fidelity between the sequences, otherwise referred to as sequence identity. 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 (for example, Leu and Ile, Asn and Asp, Gln and Glu, Ser and Thr, to name a few) 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. [0055] 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
Attorney Docket No: JCR-002WO1 “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. For example, 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. It is also understood by reading this definition that; for example, a VHH antibody that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding. “Specificity” refers to the ability of a binding protein to selectively bind an antigen. [0056] As used herein, the terms “treating,” “treatment,” or “therapy” of a disease, disorder or condition is an approach for obtaining beneficial or desired clinical results. “Treatment” as used herein, covers any administration or application of a therapeutic for disease in a mammal, including a human. For purposes of this disclosure, 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 disease, inhibiting or slowing the disease or its progression, arresting its development, and remission (whether partial or total). Also encompassed by “treatment” is a reduction of pathological consequence and progression of a proliferative or degenerative disease. The methods provided herein contemplate any one or more of these aspects of treatment. In-line with the above, the term treatment does not require one-hundred percent removal of all aspects of the disorder. [0057] VHH antibody: The term “VHH” refers to an antibody having a single monomeric domain antigen binding/recognition domain. Such antibodies include a camelid antibody. In some embodiments, a VHH comprises three CDRs and four framework regions, designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, 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. [0058] Vector: The term “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
Attorney Docket No: JCR-002WO1 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, β-galactosidase). The term “expression vector” refers to a vector that is used to express a polypeptide of interest in a host cell. The term “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. Thus, the term includes cloning and expression vehicles, as well as viral vectors. [0059] Subject: As used herein, the terms “subject” and “patient” are used interchangeably. [0060] 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. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, 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. For 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. In addition to biologically neutral carriers, pharmaceutical 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. [0061] Lysosomal Storage Diseases (LSDs): Lysosomal storage diseases (LSDs) are a group of rare inherited metabolic disorders that result from defects in lysosomal function. LSDs include more than 70 different diseases depending on the defective gene, for example, Hurler syndrome, Hurler-Scheie syndrome, Scheie syndrome, Hunter syndrome, Gaucher’s disease, GM1 gangliosidosis Types 1-3, GM2-gangliosidosis AB variant, Sandhoffs disease, Tay-Sachs disease, I-cell disease, α-mannosidosis, β-mannosidosis, Krabbe disease, Gaucher’s disease-like storage disease, metachromatic white matter degeneration (metachromatic leukodystrophy), fucosidosis, aspartylglucosaminuria, Schindler disease, Kawasaki disease, Niemann-Pick
Attorney Docket No: JCR-002WO1 disease, Fabry disease, Sly syndrome, Sanfilippo syndrome (type A-D), Farber disease, Cori’s disease (Forbes-Cori’s disease), sialidase deficiency, neuronal ceroid lipofuscinosis, Santavuori- Haltia disease, neuronal ceroid lipofuscinosis, Jansky-Bielschowsky disease, hyaluronidase deficiency, Pompe’s disease, Batten disease, and GM1-gangliosidosis. [0062] Lysosomal enzymes: Lysosomal enzymes are a group of hydrolytic enzymes in the lysosome and are enzymes whose functions are weak or absent in patients with LSDs. Lysosomal enzymes include, for example, α-L-iduronidase (IDUA), iduronate 2-sulfatase (IDS), glucocerebrosidase (GBA), β-galactosidase, GM2 activator protein, β-hexosaminidase A, β- hexosaminidase B, N-acetylglucosamine-1-phosphotransferase, α-mannosidase (LAMAN), β- mannosidase, galactosylceramidase (GALC), saposin C, arylsulfatase A (ARSA), α-L- fucosidase (FUCA1), aspartylglucosaminidase, α-N-acetylgalactosaminidase, acidic sphingomyelinase (ASM), α-galactosidase A, β-glucuronidase (GUSB), heparan N-sulfatase (SGSH), α-N-acetylglucosaminidase (NAGLU), acetyl CoA:α-glucosaminide N- acetyltransferase, N-Acetylglucosamine-6-sulfate sulfatase, acid ceramidase (AC), amylo-1,6- glucosidase, sialidase, palmitoyl protein thioesterase 1 (PPT-1), tripeptidyl-peptidase 1 (TPP-1), hyaluronidase 1, acidic α-glucosidase (GAA), CLN1 and CLN2. [0063] Cytokines: Cytokines are low-molecular proteins and physiologically active substances secreted from cells, they are involved in cell-cell interactions and influence surrounding cells. Cytokines include interleukins, hematopoietic factors, interferons, tumor necrosis factors, growth factors, and chemokines. In particular, interleukins include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL- 12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, and IL-19 to IL-36. [0064] Neurotrophic factors: Neurotrophic factors are factors required for neuronal survival, development, and function. Neurotrophic factors include brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), NT-3, NT-4, NT-5, and glial cell line-derived neurotrophic factor (GDNF). [0065] Fusion proteins: Fusion proteins are a protein wherein a human anti-TfR VHH antibody is fused with a physiologically active protein directly or via a linker, wherein the physiologically active protein is any one of lysosomal enzymes, cytokines, or neurotrophic factors. [0066] Pharmaceutical composition: Pharmaceutical composition is a pharmaceutical composition comprising the recombinant vector, the rAAV viral particle, or the transgene that encodes a fusion protein described herein, i.e. a fusion protein comprising a human anti-TfR
Attorney Docket No: JCR-002WO1 VHH antibody and a physiologically active protein, and at least one pharmaceutically acceptable carrier. A pharmaceutical composition comprising SGSH or a gene encoding SGSH can be used as a therapeutic agent for Sanfilippo syndrome type A. A pharmaceutical composition comprising IDUA or a gene encoding IDUA can be used as a therapeutic agent for Hurler syndrome or Hurler-Scheie syndrome. A pharmaceutical composition comprising IDS or a gene encoding IDS can be used as a therapeutic agent for Hunter syndrome. A pharmaceutical composition comprising GBA or a gene encoding GBA can be used as a therapeutic agent for Gaucher's disease. A pharmaceutical composition comprising β-galactosidase or a gene encoding β-galactosidase can be used as a therapeutic agent for GM1 gangliosidosis Types 1 to 3. A pharmaceutical composition comprising GM2 activator protein or a gene encoding GM2 activator protein can be used as a therapeutic agent for GM2-gangliosidosis, AB variant. A pharmaceutical composition comprising β-hexosaminidase A or a gene encoding β- hexosaminidase A can be used as a therapeutic agent for Sandhoff's disease and Tay-Sachs disease. A pharmaceutical composition comprising β-hexosaminidase B or a gene encoding β- hexosaminidase B can be used as a therapeutic agent for Sandhoff's disease. A pharmaceutical composition comprising N-acetylglucosamine-1-phosphotransferase or a gene encoding N-acetylglucosamine-1-phosphotransferase can be used as a therapeutic agent for I- cell disease. A pharmaceutical composition comprising α-mannosidase or a gene encoding α- mannosidase can be used as a therapeutic agent for α-mannosidosis. A pharmaceutical composition comprising β-mannosidase or a gene encoding β-mannosidase can be used as a therapeutic agent for β-mannosidosis. A pharmaceutical composition comprising galactosylceramidase or a gene encoding galactosylceramidase can be used as a therapeutic agent for Krabbe disease. A pharmaceutical composition comprising saposin C or a gene encoding saposin C can be used as a therapeutic agent for Gaucher's disease-like storage disease. A pharmaceutical composition comprising arylsulfatase A or a gene encoding arylsulfatase A can be used as a therapeutic agent for metachromatic white matter degeneration (metachromatic leukodystrophy). A pharmaceutical composition comprising α-L-fucosidase or a gene encoding α-L-fucosidase can be used as a therapeutic agent for fucosidosis. A pharmaceutical composition comprising aspartylglucosaminidase or a gene encoding aspartylglucosaminidase can be used as a therapeutic agent for aspartylglucosaminuria. A pharmaceutical composition comprising α-N- acetylgalactosaminidase or a gene encoding α-N-acetylgalactosaminidase can be used as a therapeutic agent for Schindler disease and Kawasaki disease. A pharmaceutical composition comprising ASM or a gene encoding ASM can be used as a therapeutic agent for Niemann-Pick
Attorney Docket No: JCR-002WO1 disease. A pharmaceutical composition comprising α-galactosidase A or a gene encoding α- galactosidase A can be used as a therapeutic agent for Fabry disease. A pharmaceutical composition comprising GUSB or a gene encoding GUSB can be used as a therapeutic agent for Sly syndrome. A pharmaceutical composition comprising NAGLU or a gene encoding NAGLU can be used as a therapeutic agent for Sanfilippo syndrome B. A pharmaceutical composition comprising N-Acetylglucosamine-6-sulfate sulfatase or a gene encoding N- Acetylglucosamine-6-sulfate sulfatase can be used as a therapeutic agent for Sanfilippo syndrome. A pharmaceutical composition comprising AC or a gene encoding AC can be used as a therapeutic agent for Farber disease. A pharmaceutical composition comprising amylo-1,6- glucosidase or a gene encoding amylo-1,6-glucosidase can be used as a therapeutic agent for Cori's disease (Forbes-Cori's disease) A pharmaceutical composition comprising sialidase or a gene encoding sialidase can be used as a therapeutic agent for sialidase deficiency. A pharmaceutical composition comprising aspartylglucosaminidase or a gene encoding aspartylglucosaminidase can be used as a therapeutic agent for aspartylglucosaminuria. A pharmaceutical composition comprising PPT-1 or a gene encoding PPT-1 can be used as a therapeutic agent for neuronal ceroid lipofuscinosis and Santavuori-Haltia disease. A pharmaceutical composition comprising TPP-1 or a gene encoding TPP-1 can be used as a therapeutic agent for ceroid lipofuscinosis or Jansky-Bielschowsky disease. A pharmaceutical composition comprising hyaluronidase 1 or a gene encoding hyaluronidase 1 can be used as a therapeutic agent for hyaluronidase deficiency. A pharmaceutical composition comprising CLN1 or a gene encoding CLN1 can be used as a therapeutic agent for Batten disease. A pharmaceutical composition comprising CLN2 or a gene encoding CLN2 can be used as a therapeutic agent for Batten disease. A pharmaceutical composition comprising CLN2 or a gene encoding CLN2 can be used as a therapeutic agent for Batten disease. A pharmaceutical composition comprising NGF or a gene encoding NGF can be used as a therapeutic agent for neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. A pharmaceutical composition comprising BDNF or a gene encoding BDNF can be used as a therapeutic agent for neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, spinal cord degenerative disease such as amyotrophic lateral sclerosis, diabetic neuropathy, cerebral ischemia-related disease, developmental disorder such as Rett syndrome, schizophrenia, and depression. A pharmaceutical composition comprising CTNF or a gene encoding CTNF can be used as a therapeutic agent for
Attorney Docket No: JCR-002WO1 amyotrophic lateral sclerosis. A pharmaceutical composition comprising GDNF or a gene encoding GDNF can be used as a therapeutic agent for brain ischemia. A pharmaceutical composition comprising NT-3 or a gene encoding NT-3 can be used as a therapeutic agent for neurodegenerative diseases and brain ischemia. A pharmaceutical composition comprising NT-4 or a gene encoding NT-4 can be used as a therapeutic agent for neurodegenerative diseases and brain ischemia. A pharmaceutical composition comprising NT -5 or a gene encoding NT -5 can be used as a therapeutic agent for brain ischemia. A pharmaceutical composition comprising IL- 10 or a gene encoding IL-10 can be used as a therapeutic agent for brain tumor or brain inflammation. Therapeutic rAAV.VHH-SGSH vectors [0067] The present disclosure relates to gene therapy constructs, vectors, compositions and methods useful for treating Sanfilippo syndrome type A (Mucopolysaccharidosis IIIA, MPS IIIA) and conditions associated thereof. Accordingly, the present disclosure provides gene therapy constructs, 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 N-sulfoglucosamine sulfohydrolase (SGSH), or a functional variant thereof. In one embodiment, the compositions and methods described herein involve nucleic acid sequences, expression cassettes, vectors, recombinant viruses, other compositions and methods for expression of a functional human SGSH. A schematic that illustrates exemplary rAAV vectors of the present disclosure is illustrated in Figure 1. The recombinant AAV 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 N- sulfoglucosamine sulfohydrolase (SGSH) with the anti-hTfR1 antibody can increase the expression of SGSH 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 Sanfilippo Syndrome type A. 1. Transgene encoding VHH-SGSH fusion proteins [0068] In one aspect of the present disclosure, a transgene encoding a fusion protein comprising at least one anti-human transferrin receptor (hTfR1) VHH antibody fused to a human
Attorney Docket No: JCR-002WO1 N-sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof, is provided. The transgene polynucleotide, in some embodiments, comprises codon optimized nucleic acid sequences that encode the anti-hTfR1 VHH antibody and/or the human N-sulfoglucosamine sulfohydrolase (SGSH) or functional variant thereof. [0069] The human anti-TfR1 VHH antibody specifically binds to human transferrin receptor 1. In some embodiments, 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. [0070] In some embodiments, the anti-hTfR1 VHH antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7. In some examples, the anti- hTfR1 VHH antibody comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 95%, 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%, 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%, 99% or 100% identical to SEQ ID NO: 3. In some examples, the anti-hTfR1 VHH antibody comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 95%, 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%, 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%, 99% or 100% identical to SEQ ID NO: 6. In some examples, the anti-hTfR1 VHH antibody comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 95%, 99% or 100% identical to SEQ ID NO: 7. [0071] 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. [0072] A suitable human N-sulfoglucosamine sulfohydrolase (SGSH) for the present invention is any protein or a portion of a protein that can substitute for at least partial activity of naturally-occurring N-sulfoglucosamine sulfohydrolase (SGSH) protein or rescue one or more phenotypes or symptoms associated with SGSH-deficiency. As used herein, the terms “N- sulfoglycosamine sulfohydrolase” and “SGSH” are used interchangeably with heparan-N-
Attorney Docket No: JCR-002WO1 sulfatase, HNS. The invention includes any variant of SGSH protein expressed from the nucleic acid sequences provided herein, or a functional fragment thereof, which restores a desired function, ameliorates a symptom, improves symptoms associated with a MPS IIIA -related biomarker, or facilitate other treatment(s) for MPS IIIA when delivered in a composition or by a method as provided herein. As used herein, the term “functional SGSH variant” means an enzyme having the amino acid sequence of the full-length wild-type (native) human SGSH (as shown in SEQ ID NO: 20), a variant thereof, a mutant thereof with a conservative amino acid replacement, a fragment thereof, a full-length or a fragment of any combination of the variant and the mutant with a conservative amino acid replacement. [0073] In some embodiments, a human SGSH protein is a naturally occurring wild type SGSH protein. In other embodiments, the SGSH protein may be an engineered functional variant of the enzyme. For Example, a functional variant of SGSH may comprise one or more modifications that can enhance stability, expression and/or catalytic activity of the enzyme as compared to wild type human SGSH. In some embodiments, a functional SGSH variant provides at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, or about the same, or greater than 100% of the biological activity level of normal human SGSH in a healthy subject. [0074] For example, a SGSH protein within the fusion protein may comprise the amino acid sequence of SEQ ID NO: 20. In some examples, a functional SGSH 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: 20. [0075] A SGSH protein or a functional variant thereof may further comprise a signal peptide. In some embodiment, the signal peptide is the native human SGSH signal peptide. In other embodiments, the signal peptide is a non-native signal peptide, such as a human immunoglobulin signal peptide. In one embodiment, the signal peptide is an engineered signal peptide comprising SEQ ID NO: 19. Table 1: Amino acid and nucleotide sequences of VHH and Human SGSH fusion protein VHH 1 QVQLVESGGGLVQPGGSLRLSCAASGSIFDIYVMRWYRQAPGKGLEWVASIYD GGRNDYDNVVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCNVLTYAGRPY WGQGTQVTVSS (SEQ ID NO.1)
Attorney Docket No: JCR-002WO1 VHH 1 (optimized CAAGTGCAGCTGGTGGAGAGTGGAGGTGGCCTGGTCCAGCCTGGAGGCTC nucleotide CCTGCGGCTGAGCTGTGCTGCCTCTGGCAGCATCTTTGACATCTACGTGATG sequence 1) AGATGGTACAGACAAGCTCCTGGGAAGGGGCTGGAGTGGGTGGCCTCCAT CTATGATGGGGGCAGAAATGACTATGACAATGTGGTGAAGGGCCGCTTCA CCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCC TCAGAGCTGAGGACACCGCTGTGTACTTCTGCAATGTCCTGACCTATGCTGG AAGACCCTACTGGGGCCAAGGCACCCAAGTCACTGTGTCCAGC (SEQ ID NO: 8) VHH 1 (optimized CAAGTGCAGCTGGTGGAGTCTGGAGGTGGCCTGGTCCAGCCTGGAGGCTC nucleotide CCTGCGGCTGAGCTGTGCTGCCTCTGGCAGCATCTTTGACATCTACGTGATG sequence 2) AGATGGTACAGACAAGCACCTGGGAAGGGGCTGGAGTGGGTGGCCTCCAT CTATGATGGAGGAAGAAATGACTATGACAATGTGGTGAAGGGCCGCTTCAC CATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCT CAGAGCTGAGGACACCGCTGTGTACTTCTGCAATGTCCTGACCTATGCTGG AAGGCCCTACTGGGGCCAAGGCACCCAAGTCACTGTGTCCAGC (SEQ ID NO: 9) VHH 1 (optimized CAAGTGCAGCTGGTGGAGAGTGGAGGTGGCCTGGTCCAGCCTGGAGGCAG nucleotide CCTGAGGCTGAGCTGTGCTGCCTCTGGCTCCATCTTTGACATCTATGTGATG sequence 3) AGATGGTACAGACAAGCTCCTGGGAAGGGGCTGGAGTGGGTGGCCTCCAT TTATGATGGAGGAAGAAATGACTATGACAATGTGGTGAAGGGCAGATTCA CCATCAGCAGAGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCC TGAGAGCTGAGGACACAGCTGTGTACTTCTGCAATGTCCTGACCTATGCTG GAAGGCCCTACTGGGGCCAAGGCACCCAAGTCACTGTGTCCAGC (SEQ ID NO: 10) VHH 2 QVQLVESGGGLVQPGGSLRLSCAASGSIFDIYVMRWYRQAPGKGLEWVASIYA GGRNDYDNVVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCNVLTYAGRPY WGQGTQVTVSS (SEQ ID NO: 2) VHH 2 (optimized CAAGTGCAGCTGGTGGAGAGTGGAGGTGGCCTGGTCCAGCCTGGAGGCTC nucleotide sequence CCTGCGGCTGAGCTGTGCTGCCTCTGGCAGCATCTTTGACATCTACGTGATG 1) AGATGGTACAGACAAGCTCCTGGGAAGGGGCTGGAGTGGGTGGCCTCCAT CTATGcTGGGGGCAGAAATGACTATGACAATGTGGTGAAGGGCCGCTTCAC CATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCT CAGAGCTGAGGACACCGCTGTGTACTTCTGCAATGTCCTGACCTATGCTGG AAGACCCTACTGGGGCCAAGGCACCCAAGTCACTGTGTCCAGC (SEQ ID NO: 11) VHH 2 (optimized CAAGTGCAGCTGGTGGAGTCTGGAGGTGGCCTGGTCCAGCCTGGAGGCTC nucleotide sequence CCTGCGGCTGAGCTGTGCTGCCTCTGGCAGCATCTTTGACATCTACGTGATG 2) AGATGGTACAGACAAGCACCTGGGAAGGGGCTGGAGTGGGTGGCCTCCAT CTATGCTGGAGGAAGAAATGACTATGACAATGTGGTGAAGGGCCGCTTCAC CATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCT CAGAGCTGAGGACACCGCTGTGTACTTCTGCAATGTCCTGACCTATGCTGG AAGGCCCTACTGGGGCCAAGGCACCCAAGTCACTGTGTCCAGC (SEQ ID NO: 12)
Attorney Docket No: JCR-002WO1 VHH 3 (tandem QVQLVESGGGLVQPGGSLRLSCAASGSIFDIYVMRWYRQAPGKGLEWVASIY amino acid DGGRNDYDNVVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCNVLTYAGRP sequence) YWGQGTQVTVSSGSQVQLVESGGGLVQPGGSLRLSCAASGSIFDIYVMRWY RQAPGKGLEWVASIYDGGRNDYDNVVKGRFTISRDNSKNTLYLQMNSLRAE DTAVYFCNVLTYAGRPYWGQGTQVTVSS (SEQ ID NO: 3) VHH 3 (tandem CAAGTGCAGCTGGTGGAGAGTGGAGGTGGCCTGGTCCAGCCTGGAGGCT nucleotide CCCTGCGGCTGAGCTGTGCTGCCTCTGGCAGCATCTTTGACATCTACGTGA sequence optimized TGAGATGGTACAGACAAGCTCCTGGGAAGGGGCTGGAGTGGGTGGCCTC 1) CATCTATGATGGGGGCAGAAATGACTATGACAATGTGGTGAAGGGCCGCT TCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAAC TCCCTCAGAGCTGAGGACACCGCTGTGTACTTCTGCAATGTCCTGACCTAT GCTGGAAGACCCTACTGGGGCCAAGGCACCCAAGTCACTGTGTCCAGCgga tccCAAGTGCAGCTGGTGGAGAGTGGAGGTGGCCTGGTCCAGCCTGGAGG CTCCCTGCGGCTGAGCTGTGCTGCCTCTGGCAGCATCTTTGACATCTACGT GATGAGATGGTACAGACAAGCTCCTGGGAAGGGGCTGGAGTGGGTGGCC TCCATCTATGATGGGGGCAGAAATGACTATGACAATGTGGTGAAGGGCCG CTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGA ACTCCCTCAGAGCTGAGGACACCGCTGTGTACTTCTGCAATGTCCTGACCT ATGCTGGAAGACCCTACTGGGGCCAAGGCACCCAAGTCACTGTGTCCAGC (SEQ ID NO: 13) VHH 3 (tandem CAAGTGCAGCTGGTGGAGAGTGGAGGTGGCCTGGTCCAGCCTGGAGGCA nucleotide GCCTGAGGCTGAGCTGTGCTGCCTCTGGCTCCATCTTTGACATCTATGTGA sequence optimized TGAGATGGTACAGACAAGCTCCTGGGAAGGGGCTGGAGTGGGTGGCCTC 2) CATTTATGATGGAGGAAGAAATGACTATGACAATGTGGTGAAGGGCAGAT TCACCATCAGCAGAGACAACAGCAAGAACACCCTGTACCTGCAGATGAAC TCCCTGAGAGCTGAGGACACAGCTGTGTACTTCTGCAATGTCCTGACCTAT GCTGGAAGGCCCTACTGGGGCCAAGGCACCCAAGTCACTGTGTCCAGCgg atccCAAGTGCAGCTGGTGGAGAGTGGAGGTGGCCTGGTCCAGCCTGGAG GCAGCCTGAGGCTGAGCTGTGCTGCCTCTGGCTCCATCTTTGACATCTATG TGATGAGATGGTACAGACAAGCTCCTGGGAAGGGGCTGGAGTGGGTGGC CTCCATTTATGATGGAGGAAGAAATGACTATGACAATGTGGTGAAGGGCA GATTCACCATCAGCAGAGACAACAGCAAGAACACCCTGTACCTGCAGATG AACTCCCTGAGAGCTGAGGACACAGCTGTGTACTTCTGCAATGTCCTGACC TATGCTGGAAGGCCCTACTGGGGCCAAGGCACCCAAGTCACTGTGTCCAG C (SEQ ID NO: 14) VHH 4 QVQLVESGGGLVHPGGSLRLSCVASGTSFSLNVVTWYRQALGKQREWVATI NQSEAPTYADSVKGRFTISRDGAKNTVYLQMNSLVTEDTALYYCYDRYNEIRG QGTQVTVSS (SEQ ID NO: 4) VHH 5 QVQLVESGGGLVHPGGSLSLSCAASGTSFSFNTVTWYRQALGKQREWVATIN GSGAPTYADSVKGRFTISRDGAKNTVYLLMNSLVTEDTALYYCYDRYNEIRGQ GTQVTVSS
Attorney Docket No: JCR-002WO1 (SEQ ID NO: 5) VHH 6 QVQLVESGGGLVQPGESLRLSCAASGSIFDIYVMRWYRQAPGNQREWVASIY DGGRNDYDNVVKGRFTISRDNAKNTVHLQMNSLKPEDTAVYFCNVLTYAGR PYWGQGTQVTVSS (SEQ ID NO: 6) VHH 7 QVQLVESGGGLVQAGASLRLSCAASGRTDGIDRMAWFHQAPGEGREFVAGI NKRGGGVDYAPGVKGRFTLSLDSDKNTLYLQMNNLRLEDTGVYSCAVGSGRII VKSDTWGQGTQVTVSS (SEQ ID NO: 7) Linker 1 GGGGS (SEQ ID NO: 15) Linker 2 GGGGGS (SEQ ID NO: 16) Linker 3 SGGGG (SEQ ID NO: 17) Linker 4 GGGGSGGGGSGGGGS (SEQ ID NO: 18) Signal peptide MGWSCIILFLVATATGAHS (SEQ ID NO: 19) (amino acid sequence) Signal peptide ATGGGATGGTCCTGCATCATCCTGTTTCTGGTGGCCACAGCCACAGGCGCT (nucleotide CACTCC (SEQ ID NO: 50) sequence) SGSH amino acid MSCPVPACCALLLVLGLCRARPRNALLLLADDGGFESGAYNNSAIATPHLDAL sequence ARRSLLFRNAFTSVSSCSPSRASLLTGLPQHQNGMYGLHQDVHHFNSFDKVR SLPLLLSQAGVRTGIIGKKHVGPETVYPFDFAYTEENGSVLQVGRNITRIKLLVR KFLQTQDDRPFFLYVAFHDPHRCGHSQPQYGTFCEKFGNGESGMGRIPDWT PQAYDPLDVLVPYFVPNTPAARADLAAQYTTVGRMDQGVGLVLQELRDAGV LNDTLVIFTSDNGIPFPSGRTNLYWPGTAEPLLVSSPEHPKRWGQVSEAYVSLL DLTPTILDWFSIPYPSYAIFGSKTIHLTGRSLLPALEAEPLWATVFGSQSHHEVT MSYPMRSVQHRHFRLVHNLNFKMPFPIDQDFYVSPTFQDLLNRTTAGQPTG WYKDLRHYYYRARWELYDRSRDPHETQNLATDPRFAQLLEMLRDQLAKWQ WETHDPWVCAPDGVLEEKLSPQCQPLHNEL (SEQ ID NO: 20) SGSH nucleotide ATGAGCTGCCCCGTGCCCGCCTGCTGCGCGCTGCTGCTAGTCCTGGGGCTC sequence TGCCGGGCGCGTCCCCGGAACGCACTGCTGCTCCTCGCGGATGACGGAGG CTTTGAGAGTGGCGCGTACAACAACAGCGCCATCGCCACCCCGCACCTGG ACGCCTTGGCCCGCCGCAGCCTCCTCTTTCGCAATGCCTTCACCTCGGTCAG CAGCTGCTCTCCCAGCCGCGCCAGCCTCCTCACTGGCCTGCCCCAGCATCA GAATGGGATGTACGGGCTGCACCAGGACGTGCACCACTTCAACTCCTTCG ACAAGGTGCGGAGCCTGCCGCTGCTGCTCAGCCAAGCTGGTGTGCGCACA GGCATCATCGGGAAGAAGCACGTGGGGCCGGAGACCGTGTACCCGTTTG ACTTTGCGTACACGGAGGAGAATGGCTCCGTCCTCCAGGTGGGGCGGAAC ATCACTAGAATTAAGCTGCTCGTCCGGAAATTCCTGCAGACTCAGGATGAC CGGCCTTTCTTCCTCTACGTCGCCTTCCACGACCCCCACCGCTGTGGGCACT CCCAGCCCCAGTACGGAACCTTCTGTGAGAAGTTTGGCAACGGAGAGAGC
Attorney Docket No: JCR-002WO1 GGCATGGGTCGTATCCCAGACTGGACCCCCCAGGCCTACGACCCACTGGA CGTGCTGGTGCCTTACTTCGTCCCCAACACCCCGGCAGCCCGAGCCGACCT GGCCGCTCAGTACACCACCGTCGGCCGCATGGACCAAGGAGTTGGACTGG TGCTCCAGGAGCTGCGTGACGCCGGTGTCCTGAACGACACACTGGTGATC TTCACGTCCGACAACGGGATCCCCTTCCCCAGCGGCAGGACCAACCTGTAC TGGCCGGGCACTGCTGAACCCTTACTGGTGTCATCCCCGGAGCACCCAAAA CGCTGGGGCCAAGTCAGCGAGGCCTACGTGAGCCTCCTAGACCTCACGCC CACCATCTTGGATTGGTTCTCGATCCCGTACCCCAGCTACGCCATCTTTGGC TCGAAGACCATCCACCTCACTGGCCGGTCCCTCCTGCCGGCGCTGGAGGCC GAGCCCCTCTGGGCCACCGTCTTTGGCAGCCAGAGCCACCACGAGGTCAC CATGTCCTACCCCATGCGCTCCGTGCAGCACCGGCACTTCCGCCTCGTGCA CAACCTCAACTTCAAGATGCCCTTTCCCATCGACCAGGACTTCTACGTCTCA CCCACCTTCCAGGACCTCCTGAACCGCACCACAGCTGGTCAGCCCACGGGC TGGTACAAGGACCTCCGTCATTACTACTACCGGGCGCGCTGGGAGCTCTAC GACCGGAGCCGGGACCCCCACGAGACCCAGAACCTGGCCACCGACCCGC GCTTTGCTCAGCTTCTGGAGATGCTTCGGGACCAGCTGGCCAAGTGGCAG TGGGAGACCCACGACCCCTGGGTGTGCGCCCCCGACGGCGTCCTGGAGGA GAAGCTCTCTCCCCAGTGCCAGCCCCTCCACAATGAGCT (SEQ ID NO: 21) VHH1 – Linker 4 – MGWSCIILFLVATATGAHSQVQLVESGGGLVQPGGSLRLSCAASGSIFDIYVM SGSH fusion protein RWYRQAPGKGLEWVASIYDGGRNDYDNVVKGRFTISRDNSKNTLYLQMNSL amino acid RAEDTAVYFCNVLTYAGRPYWGQGTQVTVSSGGGGSGGGGSGGGGSRPRN sequence ALLLLADDGGFESGAYNNSAIATPHLDALARRSLLFRNAFTSVSSCSPSRASLLT GLPQHQNGMYGLHQDVHHFNSFDKVRSLPLLLSQAGVRTGIIGKKHVGPETV YPFDFAYTEENGSVLQVGRNITRIKLLVRKFLQTQDDRPFFLYVAFHDPHRCGH SQPQYGTFCEKFGNGESGMGRIPDWTPQAYDPLDVLVPYFVPNTPAARADL AAQYTTVGRMDQGVGLVLQELRDAGVLNDTLVIFTSDNGIPFPSGRTNLYWP GTAEPLLVSSPEHPKRWGQVSEAYVSLLDLTPTILDWFSIPYPSYAIFGSKTIHLT GRSLLPALEAEPLWATVFGSQSHHEVTMSYPMRSVQHRHFRLVHNLNFKMP FPIDQDFYVSPTFQDLLNRTTAGQPTGWYKDLRHYYYRARWELYDRSRDPHE TQNLATDPRFAQLLEMLRDQLAKWQWETHDPWVCAPDGVLEEKLSPQCQP LHNEL (SEQ ID NO: 46) VHH1 – Linker 4 – ATGGGATGGTCCTGCATCATCCTGTTTCTGGTGGCCACAGCCACAGGCGCT SGSH fusion protein CACTCCCAAGTGCAGCTGGTGGAGAGTGGAGGTGGCCTGGTCCAGCCTGG nucleotide sequence AGGCTCCCTGCGGCTGAGCTGTGCTGCCTCTGGCAGCATCTTTGACATCTA CGTGATGAGATGGTACAGACAAGCTCCTGGGAAGGGGCTGGAGTGGGTG GCCTCCATCTATGATGGGGGCAGAAATGACTATGACAATGTGGTGAAGGG CCGCTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGAT GAACTCCCTCAGAGCTGAGGACACCGCTGTGTACTTCTGCAATGTCCTGAC CTATGCTGGAAGACCCTACTGGGGCCAAGGCACCCAAGTCACTGTGTCCA GCGGCGGTGGAGGCAGTGGTGGCGGAGGCAGTGGAGGTGGAGGCAGTC GTCCCCGGAACGCACTGCTGCTCCTCGCGGATGACGGAGGCTTTGAGAGT GGCGCGTACAACAACAGCGCCATCGCCACCCCGCACCTGGACGCCTTGGC
Attorney Docket No: JCR-002WO1 CCGCCGCAGCCTCCTCTTTCGCAATGCCTTCACCTCGGTCAGCAGCTGCTCT CCCAGCCGCGCCAGCCTCCTCACTGGCCTGCCCCAGCATCAGAATGGGATG TACGGGCTGCACCAGGACGTGCACCACTTCAACTCCTTCGACAAGGTGCG GAGCCTGCCGCTGCTGCTCAGCCAAGCTGGTGTGCGCACAGGCATCATCG GGAAGAAGCACGTGGGGCCGGAGACCGTGTACCCGTTTGACTTTGCGTAC ACGGAGGAGAATGGCTCCGTCCTCCAGGTGGGGCGGAACATCACTAGAAT TAAGCTGCTCGTCCGGAAATTCCTGCAGACTCAGGATGACCGGCCTTTCTT CCTCTACGTCGCCTTCCACGACCCCCACCGCTGTGGGCACTCCCAGCCCCA GTACGGAACCTTCTGTGAGAAGTTTGGCAACGGAGAGAGCGGCATGGGT CGTATCCCAGACTGGACCCCCCAGGCCTACGACCCACTGGACGTGCTGGT GCCTTACTTCGTCCCCAACACCCCGGCAGCCCGAGCCGACCTGGCCGCTCA GTACACCACCGTCGGCCGCATGGACCAAGGAGTTGGACTGGTGCTCCAGG AGCTGCGTGACGCCGGTGTCCTGAACGACACACTGGTGATCTTCACGTCCG ACAACGGGATCCCCTTCCCCAGCGGCAGGACCAACCTGTACTGGCCGGGC ACTGCTGAACCCTTACTGGTGTCATCCCCGGAGCACCCAAAACGCTGGGGC CAAGTCAGCGAGGCCTACGTGAGCCTCCTAGACCTCACGCCCACCATCTTG GATTGGTTCTCGATCCCGTACCCCAGCTACGCCATCTTTGGCTCGAAGACC ATCCACCTCACTGGCCGGTCCCTCCTGCCGGCGCTGGAGGCCGAGCCCCTC TGGGCCACCGTCTTTGGCAGCCAGAGCCACCACGAGGTCACCATGTCCTAC CCCATGCGCTCCGTGCAGCACCGGCACTTCCGCCTCGTGCACAACCTCAAC TTCAAGATGCCCTTTCCCATCGACCAGGACTTCTACGTCTCACCCACCTTCC AGGACCTCCTGAACCGCACCACAGCTGGTCAGCCCACGGGCTGGTACAAG GACCTCCGTCATTACTACTACCGGGCGCGCTGGGAGCTCTACGACCGGAG CCGGGACCCCCACGAGACCCAGAACCTGGCCACCGACCCGCGCTTTGCTC AGCTTCTGGAGATGCTTCGGGACCAGCTGGCCAAGTGGCAGTGGGAGACC CACGACCCCTGGGTGTGCGCCCCCGACGGCGTCCTGGAGGAGAAGCTCTC TCCCCAGTGCCAGCCCCTCCACAATGAGCTGTGA (SEQ ID NO: 47) SGSH – Linker 4 – MSCPVPACCALLLVLGLCRARPRNALLLLADDGGFESGAYNNSAIATPHLDAL VHH1 fusion ARRSLLFRNAFTSVSSCSPSRASLLTGLPQHQNGMYGLHQDVHHFNSFDKVR protein amino acid SLPLLLSQAGVRTGIIGKKHVGPETVYPFDFAYTEENGSVLQVGRNITRIKLLVR seqeunce KFLQTQDDRPFFLYVAFHDPHRCGHSQPQYGTFCEKFGNGESGMGRIPDWT PQAYDPLDVLVPYFVPNTPAARADLAAQYTTVGRMDQGVGLVLQELRDAGV LNDTLVIFTSDNGIPFPSGRTNLYWPGTAEPLLVSSPEHPKRWGQVSEAYVSLL DLTPTILDWFSIPYPSYAIFGSKTIHLTGRSLLPALEAEPLWATVFGSQSHHEVT MSYPMRSVQHRHFRLVHNLNFKMPFPIDQDFYVSPTFQDLLNRTTAGQPTG WYKDLRHYYYRARWELYDRSRDPHETQNLATDPRFAQLLEMLRDQLAKWQ WETHDPWVCAPDGVLEEKLSPQCQPLHNELGGGGSGGGGSGGGGSQVQL VESGGGLVQPGGSLRLSCAASGSIFDIYVMRWYRQAPGKGLEWVASIYDGGR NDYDNVVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCNVLTYAGRPYWG QGTQVTVSS (SEQ ID NO: 48) SGSH – Linker 4 – ATGAGCTGCCCCGTGCCCGCCTGCTGCGCGCTGCTGCTAGTCCTGGGGCTC VHH1 fusion TGCCGGGCGCGTCCCCGGAACGCACTGCTGCTCCTCGCGGATGACGGAGG
Attorney Docket No: JCR-002WO1 protein nucleotide CTTTGAGAGTGGCGCGTACAACAACAGCGCCATCGCCACCCCGCACCTGG seqeunce ACGCCTTGGCCCGCCGCAGCCTCCTCTTTCGCAATGCCTTCACCTCGGTCAG CAGCTGCTCTCCCAGCCGCGCCAGCCTCCTCACTGGCCTGCCCCAGCATCA GAATGGGATGTACGGGCTGCACCAGGACGTGCACCACTTCAACTCCTTCG ACAAGGTGCGGAGCCTGCCGCTGCTGCTCAGCCAAGCTGGTGTGCGCACA GGCATCATCGGGAAGAAGCACGTGGGGCCGGAGACCGTGTACCCGTTTG ACTTTGCGTACACGGAGGAGAATGGCTCCGTCCTCCAGGTGGGGCGGAAC ATCACTAGAATTAAGCTGCTCGTCCGGAAATTCCTGCAGACTCAGGATGAC CGGCCTTTCTTCCTCTACGTCGCCTTCCACGACCCCCACCGCTGTGGGCACT CCCAGCCCCAGTACGGAACCTTCTGTGAGAAGTTTGGCAACGGAGAGAGC GGCATGGGTCGTATCCCAGACTGGACCCCCCAGGCCTACGACCCACTGGA CGTGCTGGTGCCTTACTTCGTCCCCAACACCCCGGCAGCCCGAGCCGACCT GGCCGCTCAGTACACCACCGTCGGCCGCATGGACCAAGGAGTTGGACTGG TGCTCCAGGAGCTGCGTGACGCCGGTGTCCTGAACGACACACTGGTGATC TTCACGTCCGACAACGGGATCCCCTTCCCCAGCGGCAGGACCAACCTGTAC TGGCCGGGCACTGCTGAACCCTTACTGGTGTCATCCCCGGAGCACCCAAAA CGCTGGGGCCAAGTCAGCGAGGCCTACGTGAGCCTCCTAGACCTCACGCC CACCATCTTGGATTGGTTCTCGATCCCGTACCCCAGCTACGCCATCTTTGGC TCGAAGACCATCCACCTCACTGGCCGGTCCCTCCTGCCGGCGCTGGAGGCC GAGCCCCTCTGGGCCACCGTCTTTGGCAGCCAGAGCCACCACGAGGTCAC CATGTCCTACCCCATGCGCTCCGTGCAGCACCGGCACTTCCGCCTCGTGCA CAACCTCAACTTCAAGATGCCCTTTCCCATCGACCAGGACTTCTACGTCTCA CCCACCTTCCAGGACCTCCTGAACCGCACCACAGCTGGTCAGCCCACGGGC TGGTACAAGGACCTCCGTCATTACTACTACCGGGCGCGCTGGGAGCTCTAC GACCGGAGCCGGGACCCCCACGAGACCCAGAACCTGGCCACCGACCCGC GCTTTGCTCAGCTTCTGGAGATGCTTCGGGACCAGCTGGCCAAGTGGCAG TGGGAGACCCACGACCCCTGGGTGTGCGCCCCCGACGGCGTCCTGGAGGA GAAGCTCTCTCCCCAGTGCCAGCCCCTCCACAATGAGCTGGGCGGTGGAG GCAGTGGTGGCGGAGGCAGTGGAGGTGGAGGCAGTCAAGTGCAGCTGG TGGAGAGTGGAGGTGGCCTGGTCCAGCCTGGAGGCTCCCTGCGGCTGAG CTGTGCTGCCTCTGGCAGCATCTTTGACATCTACGTGATGAGATGGTACAG ACAAGCTCCTGGGAAGGGGCTGGAGTGGGTGGCCTCCATCTATGATGGG GGCAGAAATGACTATGACAATGTGGTGAAGGGCCGCTTCACCATCAGCCG GGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTCAGAGCTG AGGACACCGCTGTGTACTTCTGCAATGTCCTGACCTATGCTGGAAGACCCT ACTGGGGCCAAGGCACCCAAGTCACTGTGTCCAGCTGA (SEQ ID NO: 49) [0076] In some embodiments, the transgene encoding the fusion protein is codon optimized. The codon optimized sequences increase transcript stability and efficiency of translation, and reduces immunogenicity. [0077] 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),
Attorney Docket No: JCR-002WO1 published methods, or a company which provides codon optimizing services, e.g., as DNA2.0 (Menlo Park, Calif.). One codon optimizing approach is described, e.g., in 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. Suitably, 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. By using one of these methods, one can apply the frequencies to any given polypeptide sequence, and produce a nucleic acid fragment of a codon-optimized coding region which encodes the polypeptide. [0078] A number of options are available for performing the actual changes to the codons or for synthesizing the codon-optimized coding regions designed as described herein. Such modifications or synthesis can be performed using standard and routine molecular biological manipulations well known to those of ordinary skill in the art. In one approach, a series of complementary oligonucleotide pairs of 80-90 nucleotides each in length and spanning the length of the desired sequence are synthesized by standard methods. These 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. [0079] 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 inserts of these plasmids are then cut with appropriate restriction enzymes and ligated together to form the final construct. The final construct is then cloned into a standard bacterial cloning vector, and sequenced. Additional methods would be immediately apparent to the skilled artisan. In addition, gene synthesis is readily available commercially. [0080] In some embodiments, the nucleic acid sequence encoding the anti-hTfR1 VHH antibody is codon optimized. As non-limiting examples, the codon optimized sequence encoding
Attorney Docket No: JCR-002WO1 the anti-hTfR VHH antibody comprises a sequence selected from the group consisting of SEQ ID NOs: 8-14. [0081] In some embodiments, the fusion protein comprises the anti-hTfR1 VHH antibody of SEQ ID NO: 1. In some embodiments, the codon optimized sequence encoding the anti-hTfR1 VHH antibody of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 8, or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8. In some embodiments, the codon optimized sequence encoding the anti-hTfR1 VHH antibody of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 9, or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9. 1. In some embodiments, the codon optimized sequence encoding the anti-hTfR1 VHH antibody of SEQ ID NO: 1 comprises the sequence of SEQ ID NO: 10, or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10. [0082] In some embodiments, the fusion protein comprises the anti-hTfR1 VHH antibody of SEQ ID NO: 2. In some embodiments, the codon optimized sequence encoding the anti-hTfR1 VHH antibody of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 11, or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11. In some embodiments, the codon optimized sequence encoding the anti-hTfR1 VHH antibody of SEQ ID NO: 2 comprises the sequence of SEQ ID NO: 12, or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12. [0083] In some embodiments, the fusion protein comprises the anti-hTfR1 VHH antibody of SEQ ID NO: 3. In some embodiments, the codon optimized sequence encoding the anti-hTfR1 VHH antibody of SEQ ID NO: 3 comprises the sequence of SEQ ID NO: 13, or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13. In some embodiments, the codon optimized sequence encoding the anti-hTfR1 VHH antibody of SEQ ID NO: 3 comprises the sequence of SEQ ID NO: 14, or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14. [0084] In some embodiments, the fusion protein comprises the anti-hTfR1 VHH antibody of SEQ ID NO: 4. In some embodiments, the sequence encoding the anti-hTfR1 VHH antibody of SEQ ID NO: 4 is codon optimized. [0085] In some embodiments, the fusion protein comprises the anti-hTfR1 VHH antibody of SEQ ID NO: 5. In some embodiments, the sequence encoding the anti-hTfR1 VHH antibody of SEQ ID NO: 5 is codon optimized.
Attorney Docket No: JCR-002WO1 [0086] In some embodiments, the fusion protein comprises the anti-hTfR1 VHH antibody of SEQ ID NO: 6. In some embodiments, the sequence encoding the anti-hTfR1 VHH antibody of SEQ ID NO: 6 is codon optimized. [0087] In some embodiments, the fusion protein comprises the anti-hTfR1 VHH antibody of SEQ ID NO: 7. In some embodiments, the sequence encoding the anti-hTfR1 VHH antibody of SEQ ID NO: 7 is codon optimized. [0088] The N-sulfoglucosamine sulfohydrolase (SGSH) of the fusion protein can be a wild- type or a functional variant thereof. In some embodiments, the fusion protein comprises a wild type SGSH protein having the amino acid sequence of SEQ ID NO: 20. In some embodiments, the transgene comprises a nucleic acid sequence encoding the wild type SGSH protein, which is a wild type sequence (i.e., SEQ ID NO: 21). In other embodiments, the sequence encoding the SGSH protein is codon-optimized. [0089] In some embodiments, the transgene encodes a fusion protein comprising the anti- hTfR1 VHH antibody having the amino acid sequence of SEQ ID NO: 1 and the human N- sulfoglucosamine sulfohydrolase (SGSH) protein having the amino acid sequence of SEQ ID NO: 20. In some embodiments, the transgene encodes a fusion protein comprising the anti- hTfR1 VHH antibody having the amino acid sequence of SEQ ID NO: 2 and the human N- sulfoglucosamine sulfohydrolase (SGSH) protein having the amino acid sequence of SEQ ID NO: 20. In some embodiments, the transgene encodes a fusion protein comprising the anti- hTfR1 VHH antibody having the amino acid sequence of SEQ ID NO: 3 and the human N- sulfoglucosamine sulfohydrolase (SGSH) protein having the amino acid sequence of SEQ ID NO: 20. In some embodiments, the transgene encodes a fusion protein comprising the anti- hTfR1 VHH antibody having the amino acid sequence of SEQ ID NO: 4 and the human N- sulfoglucosamine sulfohydrolase (SGSH) protein having the amino acid sequence of SEQ ID NO: 20. In some embodiments, the transgene encodes a fusion protein comprising the anti- hTfR1 VHH antibody having the amino acid sequence of SEQ ID NO: 5 and the human N- sulfoglucosamine sulfohydrolase (SGSH) protein having the amino acid sequence of SEQ ID NO: 20. In some embodiments, the transgene encodes a fusion protein comprising the anti- hTfR1 VHH antibody having the amino acid sequence of SEQ ID NO: 6 and the human N- sulfoglucosamine sulfohydrolase (SGSH) protein having the amino acid sequence of SEQ ID NO: 20. In some embodiments, the transgene encodes a fusion protein comprising the anti- hTfR1 VHH antibody having the amino acid sequence of SEQ ID NO: 7 and the human N-
Attorney Docket No: JCR-002WO1 sulfoglucosamine sulfohydrolase (SGSH) protein having the amino acid sequence of SEQ ID NO: 20. [0090] In some embodiments, the nucleic acid sequence encoding the anti-hTfR1 VHH antibody and the nucleic acid sequence encoding the human N-sulfoglucosamine sulfohydrolase (SGSH) is codon optimized. In some embodiments, the nucleic acid sequence encoding the anti-hTfR1 VHH antibody is codon-optimized and the nucleic acid sequence encoding the human N-sulfoglucosamine sulfohydrolase (SGSH) is a wild type sequence. [0091] In some embodiments, the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO: 8 and the nucleic acid sequence encoding the human N-sulfoglucosamine sulfohydrolase (SGSH) having a wild type sequence. In some embodiments, the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO: 9 and the nucleic acid sequence encoding the human N- sulfoglucosamine sulfohydrolase (SGSH) having a wild type sequence. In some embodiments, the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO: 10 and the nucleic acid sequence encoding the human N-sulfoglucosamine sulfohydrolase (SGSH) having a wild type sequence. In some embodiments, the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO: 11 and the nucleic acid sequence encoding the human N-sulfoglucosamine sulfohydrolase (SGSH) having a wild type sequence. In some embodiments, the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO: 12 and the nucleic acid sequence encoding the human N- sulfoglucosamine sulfohydrolase (SGSH) having a wild type sequence. In some embodiments, the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO: 13 and the nucleic acid sequence encoding the human N-sulfoglucosamine sulfohydrolase (SGSH) having a wild type sequence. In some embodiments, the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO: 14 and the nucleic acid sequence encoding the human N-sulfoglucosamine sulfohydrolase (SGSH) having a wild type sequence. [0092] In some embodiments, the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ
Attorney Docket No: JCR-002WO1 ID NO: 8 and the nucleic acid sequence encoding the human N-sulfoglucosamine sulfohydrolase (SGSH) comprising SEQ ID NO.21. In some embodiments, the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO: 9 and the nucleic acid sequence encoding the human N- sulfoglucosamine sulfohydrolase (SGSH) comprising SEQ ID NO.21. In some embodiments, the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO: 10 and the nucleic acid sequence encoding the human N-sulfoglucosamine sulfohydrolase (SGSH) comprising SEQ ID NO.21. In some embodiments, the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO: 11 and the nucleic acid sequence encoding the human N-sulfoglucosamine sulfohydrolase (SGSH) comprising SEQ ID NO.21. In some embodiments, the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO: 12 and the nucleic acid sequence encoding the human N- sulfoglucosamine sulfohydrolase (SGSH) comprising SEQ ID NO.21. In some embodiments, the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO: 13 and the nucleic acid sequence encoding the human N-sulfoglucosamine sulfohydrolase (SGSH) comprising SEQ ID NO.21. In some embodiments, the transgene encoding the fusion protein comprises the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody comprising SEQ ID NO: 14 and the nucleic acid sequence encoding the human N-sulfoglucosamine sulfohydrolase (SGSH) comprising SEQ ID NO.21. [0093] In some embodiments, the human anti-TfR1 VHH antibody is fused to the human SGSH protein by a peptide linker. In some embodiments, 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. While there is no particular limitation as to the specific amino acid sequence of a linker sequence, 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 (GGS), the amino acid sequence Gly-Gly-Gly (GGG), the amino acid sequence Gly-Gly-Gly-Ser (GGGS), the amino acid sequence Gly-Gly-Gly-Gly-Ser(GGGGS) (SEQ ID NO: 15), the amino acid sequence Gly- Gly-Gly-Gly-Gly-Ser (GGGGGS) (SEQ ID NO: 16), the amino acid sequence Ser-Gly-Gly-Gly- Gly (SGGGG) (SEQ ID NO:17), the amino acid sequence Ser-Gly-Gly-Gly-Gly-Gly
Attorney Docket No: JCR-002WO1 (SGGGGG) (SEQ ID NO: 45), or a sequence which includes 2 to 10, or 2 to 5 repeats of any of those amino acid sequences. In one non-limiting example, the linker peptide sequence is preferably a linker sequence comprising 15 amino acids corresponding to three copies of the amino acid sequence (Gly-Gly-Gly-Gly-Ser) (GGGGSGGGGSGGGGS; SEQ ID NO: 18) consecutively linked. [0094] In some embodiments, the transgene comprises two or more anti-hTfR1 VHH antibodies in tandem fused to the human SGSH. The two or more anti-hTfR1 VHH antibodies have the same sequence; alternatively, the two or more VHH antibodies have different sequences. [0095] In some embodiments, the human anti-TfR1 VHH antibody is fused to the N- terminus of the human SGSH. In some embodiments, the human anti-TfR1 VHH antibody is fused to the C-terminus of the human SGSH. [0096] In some embodiment, the fusion protein further comprises a signal peptide. As used herein, the term “signal peptide (SP)” 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. In this regard, the polynucleotide described herein encodes a fusion protein comprises a signal peptide, at least one anti-hTfR1 VHH antibody and a human N- sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof. In some embodiments, the signal peptide is the signal peptide of a wild type SGSH protein. In some embodiments, the signal peptide is an IgG signal peptide. In one embodiment, the signal peptide is an engineered signal peptide (e.g., SEQ ID NO: 19). [0097] In some embodiments, the VHH-SGSH fusion protein comprises a signal peptide of SEQ ID NO: 19. In some embodiments, the polynucleotide encoding a fusion protein having a signal peptide comprising SEQ ID NO: 19, an anti-hTfR1 VHH antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7, and a human N- sulfoglucosamine sulfohydrolase (SGSH) having the amino acid sequence of SEQ ID NO: 20 or a functional variant thereof. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding the signal peptide of SEQ ID NO: 19 (e.g., SEQ ID NO: 50 in Table 1), a codon optimized nucleic acid sequence encoding an anti-hTfR1 VHH antibody and a nucleic acid sequence encoding a human N-sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof. As non-limiting examples, the codon optimized nucleic acid sequence encoding the anti-hTfR1 VHH antibody includes any one of SEQ ID NOs.8-14.
Attorney Docket No: JCR-002WO1 [0098] As a non-limiting example, the transgene described herein encodes a fusion protein having the amino acid sequence of SEQ ID NO: 46. 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: 46. [0099] In some embodiments, the transgene encoding a fusion protein described herein having the nucleotide sequence of SEQ ID NO: 47. In some embodiments, the transgene encoding a fusion protein described herein having a nucleotide 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: 47. [0100] In some embodiments, the transgene described herein encodes a fusion protein having the amino acid sequence of SEQ ID NO: 48. 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: 48. [0101] In some embodiments, the transgene encoding a fusion protein described herein having the nucleotide sequence of SEQ ID NO: 49. In some embodiments, the transgene encoding a fusion protein described herein having a nucleotide 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: 49. 2. Recombinant AAV vector elements [0102] In accordance with the present invention, the transgene encoding a VHH-SGSH fusion protein as described herein may be packaged in a viral vector (e.g., retrovirus, lentivirus, adenovirus and adeno-associated virus (AAV)). In some embodiments, the viral vector is a recombinant AAV vector. In some embodiments, the recombinant AAV (rAAV) vector for expressing a VHH-SGSH 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 N-sulfoglucosamine sulfohydrolase (SGSH), 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. [0103] In some embodiments, the rAAV vector comprises a cis-acting regulatory module (CRM). Various kinds of CRM are suitable for use in the vectors described herein and include
Attorney Docket No: JCR-002WO1 tissue-specific enhancers, for example hepatocyte-specific CRM, neuronal-specific CRM. In some embodiments, the vectors described herein include a hepatocyte-specific CRM, for example, CRM8. In some embodiments, the vector includes more than one CRM. In some embodiments, the vector comprises two, three, four, five or six CRMs. In some embodiments, the vector comprises three CRM, for example three CRM8 (e.g., SEQ ID NO: 22). [0104] In some embodiments, the recombinant vector comprises a promoter. Various kinds of promoters can be used in the rAAV vector described herein. These include, for example, ubiquitous, tissue-specific, and regulatable (e.g., inducible or repressible) promoters. [0105] In some embodiments, the rAAV vector comprises a ubiquitous promoter. Use of 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). In some examples, 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: 24). In one embodiment, the CBh promoter comprises the sequence of SEQ ID NO: 25. 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 1α promoter. [0106] In other embodiments, the rAAV vector comprise a tissue-specific promoter, such as a liver-specific promoter. Examples of liver-specific promoters are known in the art and include, for example, human transthyrethin promoter (TTR), α1-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. In some embodiments, the liver specific promoter is TTR promoter comprising SEQ ID NO: 23. [0107] 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 β globin Splice Donor/immunoglobulin heavy chain Splice Acceptor intron, SV40 and/or an alpha globin 1st intron. In some embodiments, the rAAV vector comprises an SV40 intron. In some embodiments, the rAAV vector comprises an MVM intron (e.g., SEQ ID NO: 24). In some embodiments, the MVM intron is grafted to a novel chimeric promoter, such as a CBh promoter (SEQ ID NO: 25).
Attorney Docket No: JCR-002WO1 [0108] In addition to a promoter, an expression cassette and/or a vector may contain other appropriate transcription initiation, termination, enhancer sequences, and efficient RNA processing signals. Such sequences include splicing and polyadenylation (polyA) signals; regulatory elements that enhance expression (e.g., WPRE); sequences that 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. In one embodiment, a KOZAK sequence is included. [0109] In one embodiment, a polyadenylation (polyA) signal is included to mediate efficient termination of the fusion protein mRNA transcripts. A polyA signal may be an artificial polyA. Examples of other suitable polyA sequences include, e.g., bovine growth hormone, SV40, rabbit beta globin, and TK polyA, amongst others. [0110] In some embodiments, the polyA signal is an in silico designed synthetic polyA sequence. As a non-limiting example, the polyA sequence comprises SEQ ID NO: 29. In other embodiments the polyA signal comprises the sequence of SEQ ID NO: 30. [0111] In some embodiments, the rAAV vector described herein comprises a posttranscriptional response element. As used herein, the term “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. Examples of 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 (Hsp705′UTR). In some embodiments, the rAAV vector comprises a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Various optimized or variant forms of WPRE are known in the art, and include WPRE3, WPREmut6delATG, and WPRE3mut5delATG among others. Other variant WPRE forms include, for example, WPRE2, WPRE_wt (GenBank accession no. J04514); WPRE_wt (GenBank accession no. J02442) and WPREmut6. In some embodiments, the WPRE is WPRE3 or a variant thereof, e.g., WPRE3mut5delATG. In some examples, the rAAV vector comprises a WPRE having a sequence of SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28. [0112] In one preferred embodiment, the rAAV vector comprises a WPRE3mut5delATG having SEQ ID NO: 28.
Attorney Docket No: JCR-002WO1 [0113] In some embodiments, the vector comprises a 5’ and a 3’ inverted terminal repeat (ITR). The minimal sequences required to package the expression cassette into an AAV viral particle are the AAV 5′ and 3′ ITRs. The ITRs are the genetic elements responsible for the replication and packaging of the genome during vector production and are the only viral cis elements required to generate rAAV. In accordance, 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). In one embodiment, the ITR sequences from AAV2, or the deleted version thereof (ΔITR), are used. However, 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. [0114] Typically, 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. However, other configurations of these elements may be suitable. A shortened version of the 5′ ITR, termed ΔITR, has been described in which the D- sequence and terminal resolution site (trs) are deleted. In other embodiments, the full- length AAV 5′ and 3′ ITRs are used. [0115] As non-limiting example, the rAAV vector described herein comprises a 5’ITR having SEQ ID NO: 32 and a 3’ITR having SEQ ID NO: 31. [0116] In some embodiments, the rAAV vector further comprises an internal ribosome entry site (IRES), e.g., an IRES having SEQ ID NO: 33. [0117] Exemplary element sequences are shown in Table 2 below. In some embodiments, the rAAV vector comprises a rAAV vector element comprising a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with a vector element sequence shown in Table 2. In some embodiments, the rAAV vector comprises a vector element nucleotide sequence identical to a vector element nucleotide sequence shown in Table 2. Table 2. Exemplary rAAV element sequences 3xCRM8 gggggaggctgctggtgaatattaaccaaggtcaccccagttatcggaggagcaaacaggggctaagtccaccgggggaggct gctggtgaatattaaccaaggtcaccccagttatcggaggagcaaacaggggctaagtccaccgggggaggctgctggtgaat attaaccaaggtcaccccagttatcggaggagcaaacaggggctaagtccac (SEQ ID NO: 22) hTTR promoter
Attorney Docket No: JCR-002WO1 aaatgacctattaagaatatttcatagaacgaatgttccgatgctctaatctctctagacaaggttcatatttgtatgggttacttattctctcttt gttgactaagtcaataatcagaatcagcaggtttgcagtcagattggcagggataagcagcctagctcaggagaagtgagtataaaagccc caggctgggagcagccatcacagaagtccactcattcttggcagg (SEQ ID NO: 23) MVM intron ctaaggtaagttggcgccgtttaagggatggttggttggtggggtattaatgtttaattaccttttttacaggcctg (SEQ ID NO: 24) CBh promoter agatgtactgccaagtaggaaagtcccgtaaggtcatgtactgggcataatgccaggcgggccatttaccgtcattgacgtcaatagggggc gtacttggcatatgatacacttgatgtactgccaagtgggcagtttaccgtaaatactccacccattgacgtcaatggaaagtccctattggcg ttactatgggaacatacgtcattattgacgtcaatgggcgggggtcgttgggcggtcagccaggcgggccatttaccgtaagttatgtaacgc ggaactccatatatgggctatgaactaatgaccccgtaattgattactattaaccacgttctgcttcactctccccatctcccccccctccccacc cccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggcgcgcgccaggcggggcggggcggggcg aggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggc ggcggcggccctataaaaagcgaagcgcgcggcggggagtcgctgcgttgccttcgccccgtgccccgctccgcgccgcctcgcgccgcccg ccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagcgcttaagaggtaagggt ttaagggatggttggttggtggggtattaatgtttaattacctgttttacaggcctgaaatcacttggttttaggttgg (SEQ ID NO: 25) WPREmut6delATG aatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactttgttgctccttttacgctttgtggatacgctgctttattgccttt gtatcttgctattgcttcccgtttggctttcattttctcctccttgtataaatcctggttgctgtctctttttgaggagttgtggcccgttgtcaggca acgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccc cctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttg tcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaat ccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggc cgcctccccgcatc (SEQ ID NO: 26) WPRE3 aatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctt tgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttagttcttgccacggcggaactcatcgccgcctgcctt gcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgtt (SEQ ID NO.27) WPRE3mut5delATG AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTTTGTTGCTCCTTTTACGCTTT GTGGATACGCTGCTTTATTGCCTTTGTATCTTGCTATTGCTTCCCGTTTGGCTTTCATTTTCTCCTCCTTGTATA AATCCTGGTTAGCTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGG CTGTTGGGCACTGACAATTCCGTGGTGTT (SEQ ID NO: 28) Synthetic polyA caataaaagatctttattttcattagatctgtgtgttggttttttgtgtg (SEQ ID NO: 29) BGH polyA cctagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgcc actcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggaca gcaagggggaggattgggaagacaatagcaggcatgctggggaa (SEQ ID NO: 30) 3’ ITR
Attorney Docket No: JCR-002WO1 aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggc tttgcccgggcggcctcagtgagcgagcgagcgcgcagaga (SEQ ID NO: 31) 5’ ITR ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcg cagagagggagtggccaactccatcactaggggttcct (SEQ ID NO: 32) IRES gcccctctccctcccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgc cgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaagg tctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaacccccc acctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttgg atagttgtggaaagagtcaaatggctcacctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctga tctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaa aaacacgatgataat (SEQ ID NO: 33) 3. Exemplary rAAV. VHH-SGSH vectors [0118] In some embodiments, a recombinant AAV vector for expressing a human N- sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof comprises a transgene encoding a fusion protein of an anti-human transferrin receptor (hTfR1) VHH antibody and a human N-sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof, a ubiquitous promoter CBh, a WPRE3mut5ΔATG and a polyA sequence. [0119] In some embodiments, a recombinant AAV vector for expressing a human N- sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof comprises a transgene encoding a fusion protein of an anti-human transferrin receptor (hTfR1) VHH antibody and a human N-sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof, a liver specific promoter TTR, a WPRE3mut5ΔATG and a polyA sequence. [0120] In some embodiments, a recombinant AAV vector for expressing a human N- sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof comprises a transgene encoding a fusion protein of a tandem of anti-human transferrin receptor (hTfR1) VHH antibodies and a human N-sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof, a liver specific promoter TTR, a liver specific enhancer CRM8, a WPRE3mut5ΔATG and a polyA sequence. [0121] In some embodiments, a recombinant AAV vector for expressing a human N- sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof comprises a transgene encoding a fusion protein of a tandem of anti-human transferrin receptor (hTfR1) VHH
Attorney Docket No: JCR-002WO1 antibodies and a human N-sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof, a ubiquitous promoter CBh, a WPRE3mut5ΔATG and a synthetic polyA sequence. [0122] Exemplary rAAV.VHH-SGSH vectors are shown in Table 3. [0123] In some embodiments, the rAAV vector for expressing a human N- sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof comprises the nucleic acid sequence of SEQ ID NO: 34. In other embodiments, the rAAV vector for expressing a human N-sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof 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: 34. [0124] In some embodiments, the rAAV vector for expressing a human N- sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof comprises the nucleic acid sequence of SEQ ID NO: 35. In other embodiments, the rAAV vector for expressing a human N-sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof 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: 35. [0125] In some embodiments, the rAAV vector for expressing a human N- sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof comprises the nucleic acid sequence of SEQ ID NO: 36. In other embodiments, the rAAV vector for expressing a human N-sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof 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:36. [0126] In some embodiments, the rAAV vector for expressing a human N- sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof comprises the nucleic acid sequence of SEQ ID NO: 37. In other embodiments, the rAAV vector for expressing a human N-sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof 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: 37. [0127] In some embodiments, the rAAV vector for expressing a human N- sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof comprises the nucleic acid sequence of SEQ ID NO: 38. In other embodiments, the rAAV vector for expressing a human N-sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof 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: 38.
Attorney Docket No: JCR-002WO1 [0128] In some embodiments, the rAAV vector for expressing a human N- sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof comprises the nucleic acid sequence of SEQ ID NO: 39. In other embodiments, the rAAV vector for expressing a human N-sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof 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: 39. [0129] In some embodiments, the rAAV vector for expressing a human N- sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof comprises the nucleic acid sequence of SEQ ID NO: 40. In other embodiments, the rAAV vector for expressing a human N-sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof 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. [0130] In some embodiments, the rAAV vector for expressing a human N- sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof comprises the nucleic acid sequence of SEQ ID NO: 41. In other embodiments, the rAAV vector for expressing a human N-sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof 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. [0131] In some embodiments, the rAAV vector for expressing a human N- sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof comprises the nucleic acid sequence of SEQ ID NO: 42. In other embodiments, the rAAV vector for expressing a human N-sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof 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. [0132] In some embodiments, the rAAV vector for expressing a human N- sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof comprises the nucleic acid sequence of SEQ ID NO: 43. In other embodiments, the rAAV vector for expressing a human N-sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof 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: 43. [0133] In some embodiments, the rAAV vector for expressing a human N- sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof comprises the nucleic acid sequence of SEQ ID NO: 44. In other embodiments, the rAAV vector for expressing a
Attorney Docket No: JCR-002WO1 human N-sulfoglucosamine sulfohydrolase (SGSH) or a functional variant thereof 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. Table 3: Exemplary rAAV.VHH-SGSH vector sequences (from 5’ ITR to 3’ ITR) TJ013 CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTTAATTAAACGCGTGGGGG AGGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCACCGGGGGA GGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCACCGGGGGAG GCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCACACTAGTAAAT GACCTATTAAGAATATTTCATAGAACGAATGTTCCGATGCTCTAATCTCTCTAGACAAGGTTCATATTTGTATGGGT TACTTATTCTCTCTTTGTTGACTAAGTCAATAATCAGAATCAGCAGGTTTGCAGTCAGATTGGCAGGGATAAGCAG CCTAGCTCAGGAGAAGTGAGTATAAAAGCCCCAGGCTGGGAGCAGCCATCACAGAAGTCCACTCATTCTTGGCAG GCCGCGGCCTGGGCGCGCCGCCACCATGAGCTGCCCCGTGCCCGCCTGCTGCGCGCTGCTGCTAGTCCTGGGGCT CTGCCGGGCGCGTCCCCGGAACGCACTGCTGCTCCTCGCGGATGACGGAGGCTTTGAGAGTGGCGCGTACAACA ACAGCGCCATCGCCACCCCGCACCTGGACGCCTTGGCCCGCCGCAGCCTCCTCTTTCGCAATGCCTTCACCTCGGTC AGCAGCTGCTCTCCCAGCCGCGCCAGCCTCCTCACTGGCCTGCCCCAGCATCAGAATGGGATGTACGGGCTGCAC CAGGACGTGCACCACTTCAACTCCTTCGACAAGGTGCGGAGCCTGCCGCTGCTGCTCAGCCAAGCTGGTGTGCGC ACAGGCATCATCGGGAAGAAGCACGTGGGGCCGGAGACCGTGTACCCGTTTGACTTTGCGTACACGGAGGAGAA TGGCTCCGTCCTCCAGGTGGGGCGGAACATCACTAGAATTAAGCTGCTCGTCCGGAAATTCCTGCAGACTCAGGA TGACCGGCCTTTCTTCCTCTACGTCGCCTTCCACGACCCCCACCGCTGTGGGCACTCCCAGCCCCAGTACGGAACCT TCTGTGAGAAGTTTGGCAACGGAGAGAGCGGCATGGGTCGTATCCCAGACTGGACCCCCCAGGCCTACGACCCA CTGGACGTGCTGGTGCCTTACTTCGTCCCCAACACCCCGGCAGCCCGAGCCGACCTGGCCGCTCAGTACACCACCG TCGGCCGCATGGACCAAGGAGTTGGACTGGTGCTCCAGGAGCTGCGTGACGCCGGTGTCCTGAACGACACACTG GTGATCTTCACGTCCGACAACGGGATCCCCTTCCCCAGCGGCAGGACCAACCTGTACTGGCCGGGCACTGCTGAA CCCTTACTGGTGTCATCCCCGGAGCACCCAAAACGCTGGGGCCAAGTCAGCGAGGCCTACGTGAGCCTCCTAGAC CTCACGCCCACCATCTTGGATTGGTTCTCGATCCCGTACCCCAGCTACGCCATCTTTGGCTCGAAGACCATCCACCT CACTGGCCGGTCCCTCCTGCCGGCGCTGGAGGCCGAGCCCCTCTGGGCCACCGTCTTTGGCAGCCAGAGCCACCA CGAGGTCACCATGTCCTACCCCATGCGCTCCGTGCAGCACCGGCACTTCCGCCTCGTGCACAACCTCAACTTCAAG ATGCCCTTTCCCATCGACCAGGACTTCTACGTCTCACCCACCTTCCAGGACCTCCTGAACCGCACCACAGCTGGTCA GCCCACGGGCTGGTACAAGGACCTCCGTCATTACTACTACCGGGCGCGCTGGGAGCTCTACGACCGGAGCCGGG ACCCCCACGAGACCCAGAACCTGGCCACCGACCCGCGCTTTGCTCAGCTTCTGGAGATGCTTCGGGACCAGCTGG CCAAGTGGCAGTGGGAGACCCACGACCCCTGGGTGTGCGCCCCCGACGGCGTCCTGGAGGAGAAGCTCTCTCCC CAGTGCCAGCCCCTCCACAATGAGCTGTGAGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTG GTATTCTTAACTTTGTTGCTCCTTTTACGCTTTGTGGATACGCTGCTTTATTGCCTTTGTATCTTGCTATTGCTTCCCG TTTGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGCTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGC CCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGGTACCCAATAAAAGATCTTTATTT TCATTAGATCTGTGTGTTGGTTTTTTGTGTGTCTAGACCCCGTGTGAACGATTGGTAAACCCGGTGTCCTGTGAGC GGCGAAAGCCTAAACGGGAAATACGGTTTAAACATTTAAATAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTC TCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTC AGTGAGCGAGCGAGCGCGCAGAGA (SEQ ID NO: 34)
Attorney Docket No: JCR-002WO1 TJ018 CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTTAATTAAACGCGTGGGGG AGGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCACCGGGGGA GGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCACCGGGGGAG GCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCACACTAGTAAAT GACCTATTAAGAATATTTCATAGAACGAATGTTCCGATGCTCTAATCTCTCTAGACAAGGTTCATATTTGTATGGGT TACTTATTCTCTCTTTGTTGACTAAGTCAATAATCAGAATCAGCAGGTTTGCAGTCAGATTGGCAGGGATAAGCAG CCTAGCTCAGGAGAAGTGAGTATAAAAGCCCCAGGCTGGGAGCAGCCATCACAGAAGTCCACTCATTCTTGGCAG GCCGCGGCCTGGGCGCGCCGCCACCATGGGATGGTCCTGCATCATCCTGTTTCTGGTGGCCACAGCCACAGGCGC TCACTCCCAAGTGCAGCTGGTGGAGAGTGGAGGTGGCCTGGTCCAGCCTGGAGGCTCCCTGCGGCTGAGCTGTG CTGCCTCTGGCAGCATCTTTGACATCTACGTGATGAGATGGTACAGACAAGCTCCTGGGAAGGGGCTGGAGTGG GTGGCCTCCATCTATGATGGGGGCAGAAATGACTATGACAATGTGGTGAAGGGCCGCTTCACCATCAGCCGGGA CAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTCAGAGCTGAGGACACCGCTGTGTACTTCTGCAATGTC CTGACCTATGCTGGAAGACCCTACTGGGGCCAAGGCACCCAAGTCACTGTGTCCAGCGGCGGTGGAGGCAGTGG TGGCGGAGGCAGTGGAGGTGGAGGCAGTCGTCCCCGGAACGCACTGCTGCTCCTCGCGGATGACGGAGGCTTTG AGAGTGGCGCGTACAACAACAGCGCCATCGCCACCCCGCACCTGGACGCCTTGGCCCGCCGCAGCCTCCTCTTTC GCAATGCCTTCACCTCGGTCAGCAGCTGCTCTCCCAGCCGCGCCAGCCTCCTCACTGGCCTGCCCCAGCATCAGAA TGGGATGTACGGGCTGCACCAGGACGTGCACCACTTCAACTCCTTCGACAAGGTGCGGAGCCTGCCGCTGCTGCT CAGCCAAGCTGGTGTGCGCACAGGCATCATCGGGAAGAAGCACGTGGGGCCGGAGACCGTGTACCCGTTTGACT TTGCGTACACGGAGGAGAATGGCTCCGTCCTCCAGGTGGGGCGGAACATCACTAGAATTAAGCTGCTCGTCCGGA AATTCCTGCAGACTCAGGATGACCGGCCTTTCTTCCTCTACGTCGCCTTCCACGACCCCCACCGCTGTGGGCACTCC CAGCCCCAGTACGGAACCTTCTGTGAGAAGTTTGGCAACGGAGAGAGCGGCATGGGTCGTATCCCAGACTGGAC CCCCCAGGCCTACGACCCACTGGACGTGCTGGTGCCTTACTTCGTCCCCAACACCCCGGCAGCCCGAGCCGACCTG GCCGCTCAGTACACCACCGTCGGCCGCATGGACCAAGGAGTTGGACTGGTGCTCCAGGAGCTGCGTGACGCCGG TGTCCTGAACGACACACTGGTGATCTTCACGTCCGACAACGGGATCCCCTTCCCCAGCGGCAGGACCAACCTGTAC TGGCCGGGCACTGCTGAACCCTTACTGGTGTCATCCCCGGAGCACCCAAAACGCTGGGGCCAAGTCAGCGAGGCC TACGTGAGCCTCCTAGACCTCACGCCCACCATCTTGGATTGGTTCTCGATCCCGTACCCCAGCTACGCCATCTTTGG CTCGAAGACCATCCACCTCACTGGCCGGTCCCTCCTGCCGGCGCTGGAGGCCGAGCCCCTCTGGGCCACCGTCTTT GGCAGCCAGAGCCACCACGAGGTCACCATGTCCTACCCCATGCGCTCCGTGCAGCACCGGCACTTCCGCCTCGTG CACAACCTCAACTTCAAGATGCCCTTTCCCATCGACCAGGACTTCTACGTCTCACCCACCTTCCAGGACCTCCTGAA CCGCACCACAGCTGGTCAGCCCACGGGCTGGTACAAGGACCTCCGTCATTACTACTACCGGGCGCGCTGGGAGCT CTACGACCGGAGCCGGGACCCCCACGAGACCCAGAACCTGGCCACCGACCCGCGCTTTGCTCAGCTTCTGGAGAT GCTTCGGGACCAGCTGGCCAAGTGGCAGTGGGAGACCCACGACCCCTGGGTGTGCGCCCCCGACGGCGTCCTGG AGGAGAAGCTCTCTCCCCAGTGCCAGCCCCTCCACAATGAGCTGTGAGTCGACAATCAACCTCTGGATTACAAAAT TTGTGAAAGATTGACTGGTATTCTTAACTTTGTTGCTCCTTTTACGCTTTGTGGATACGCTGCTTTATTGCCTTTGTA TCTTGCTATTGCTTCCCGTTTGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGCTCTTGCCACGGCGGAACT CATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGGTACC CAATAAAAGATCTTTATTTTCATTAGATCTGTGTGTTGGTTTTTTGTGTGTCTAGACCCCGTGTGAACGATTGGTAA ACCCGGTGTCCTGTGAGCGGCGAAAGCCTAAACGGGAAATACGGTTTAAACATTTAAATAGGAACCCCTAGTGAT GGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGG CTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGA (SEQ ID NO: 35) TJ020
Attorney Docket No: JCR-002WO1 CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTTAATTAAACGCGTGGGGG AGGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCACCGGGGGA GGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCACCGGGGGAG GCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCACACTAGTAAAT GACCTATTAAGAATATTTCATAGAACGAATGTTCCGATGCTCTAATCTCTCTAGACAAGGTTCATATTTGTATGGGT TACTTATTCTCTCTTTGTTGACTAAGTCAATAATCAGAATCAGCAGGTTTGCAGTCAGATTGGCAGGGATAAGCAG CCTAGCTCAGGAGAAGTGAGTATAAAAGCCCCAGGCTGGGAGCAGCCATCACAGAAGTCCACTCATTCTTGGCAG GCCGCGGCCTGGGCGCGCCGCCACCATGAGCTGCCCCGTGCCCGCCTGCTGCGCGCTGCTGCTAGTCCTGGGGCT CTGCCGGGCGCGTCCCCGGAACGCACTGCTGCTCCTCGCGGATGACGGAGGCTTTGAGAGTGGCGCGTACAACA ACAGCGCCATCGCCACCCCGCACCTGGACGCCTTGGCCCGCCGCAGCCTCCTCTTTCGCAATGCCTTCACCTCGGTC AGCAGCTGCTCTCCCAGCCGCGCCAGCCTCCTCACTGGCCTGCCCCAGCATCAGAATGGGATGTACGGGCTGCAC CAGGACGTGCACCACTTCAACTCCTTCGACAAGGTGCGGAGCCTGCCGCTGCTGCTCAGCCAAGCTGGTGTGCGC ACAGGCATCATCGGGAAGAAGCACGTGGGGCCGGAGACCGTGTACCCGTTTGACTTTGCGTACACGGAGGAGAA TGGCTCCGTCCTCCAGGTGGGGCGGAACATCACTAGAATTAAGCTGCTCGTCCGGAAATTCCTGCAGACTCAGGA TGACCGGCCTTTCTTCCTCTACGTCGCCTTCCACGACCCCCACCGCTGTGGGCACTCCCAGCCCCAGTACGGAACCT TCTGTGAGAAGTTTGGCAACGGAGAGAGCGGCATGGGTCGTATCCCAGACTGGACCCCCCAGGCCTACGACCCA CTGGACGTGCTGGTGCCTTACTTCGTCCCCAACACCCCGGCAGCCCGAGCCGACCTGGCCGCTCAGTACACCACCG TCGGCCGCATGGACCAAGGAGTTGGACTGGTGCTCCAGGAGCTGCGTGACGCCGGTGTCCTGAACGACACACTG GTGATCTTCACGTCCGACAACGGGATCCCCTTCCCCAGCGGCAGGACCAACCTGTACTGGCCGGGCACTGCTGAA CCCTTACTGGTGTCATCCCCGGAGCACCCAAAACGCTGGGGCCAAGTCAGCGAGGCCTACGTGAGCCTCCTAGAC CTCACGCCCACCATCTTGGATTGGTTCTCGATCCCGTACCCCAGCTACGCCATCTTTGGCTCGAAGACCATCCACCT CACTGGCCGGTCCCTCCTGCCGGCGCTGGAGGCCGAGCCCCTCTGGGCCACCGTCTTTGGCAGCCAGAGCCACCA CGAGGTCACCATGTCCTACCCCATGCGCTCCGTGCAGCACCGGCACTTCCGCCTCGTGCACAACCTCAACTTCAAG ATGCCCTTTCCCATCGACCAGGACTTCTACGTCTCACCCACCTTCCAGGACCTCCTGAACCGCACCACAGCTGGTCA GCCCACGGGCTGGTACAAGGACCTCCGTCATTACTACTACCGGGCGCGCTGGGAGCTCTACGACCGGAGCCGGG ACCCCCACGAGACCCAGAACCTGGCCACCGACCCGCGCTTTGCTCAGCTTCTGGAGATGCTTCGGGACCAGCTGG CCAAGTGGCAGTGGGAGACCCACGACCCCTGGGTGTGCGCCCCCGACGGCGTCCTGGAGGAGAAGCTCTCTCCC CAGTGCCAGCCCCTCCACAATGAGCTGGGCGGTGGAGGCAGTGGTGGCGGAGGCAGTGGAGGTGGAGGCAGTC AAGTGCAGCTGGTGGAGAGTGGAGGTGGCCTGGTCCAGCCTGGAGGCTCCCTGCGGCTGAGCTGTGCTGCCTCT GGCAGCATCTTTGACATCTACGTGATGAGATGGTACAGACAAGCTCCTGGGAAGGGGCTGGAGTGGGTGGCCTC CATCTATGATGGGGGCAGAAATGACTATGACAATGTGGTGAAGGGCCGCTTCACCATCAGCCGGGACAACAGCA AGAACACCCTGTACCTGCAGATGAACTCCCTCAGAGCTGAGGACACCGCTGTGTACTTCTGCAATGTCCTGACCTA TGCTGGAAGACCCTACTGGGGCCAAGGCACCCAAGTCACTGTGTCCAGCTGAGTCGACAATCAACCTCTGGATTA CAAAATTTGTGAAAGATTGACTGGTATTCTTAACTTTGTTGCTCCTTTTACGCTTTGTGGATACGCTGCTTTATTGCC TTTGTATCTTGCTATTGCTTCCCGTTTGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGCTCTTGCCACGGCG GAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTG GTACCCAATAAAAGATCTTTATTTTCATTAGATCTGTGTGTTGGTTTTTTGTGTGTCTAGACCCCGTGTGAACGATT GGTAAACCCGGTGTCCTGTGAGCGGCGAAAGCCTAAACGGGAAATACGGTTTAAACATTTAAATAGGAACCCCTA GTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGC CCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGA (SEQ ID NO: 36) TJ035
Attorney Docket No: JCR-002WO1 CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTTAATTAAACGCGTGGGGG AGGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCACCGGGGGA GGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCACCGGGGGAG GCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCACACTAGTAAAT GACCTATTAAGAATATTTCATAGAACGAATGTTCCGATGCTCTAATCTCTCTAGACAAGGTTCATATTTGTATGGGT TACTTATTCTCTCTTTGTTGACTAAGTCAATAATCAGAATCAGCAGGTTTGCAGTCAGATTGGCAGGGATAAGCAG CCTAGCTCAGGAGAAGTGAGTATAAAAGCCCCAGGCTGGGAGCAGCCATCACAGAAGTCCACTCATTCTTGGCAG GCCGCGGCTAAGGTAAGTTGGCGCCGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTTTTTTA CAGGCCTGGGCGCGCCGCCACCATGGGATGGTCCTGCATCATCCTGTTTCTGGTGGCCACAGCCACAGGCGCTCA CTCCCAAGTGCAGCTGGTGGAGAGTGGAGGTGGCCTGGTCCAGCCTGGAGGCTCCCTGCGGCTGAGCTGTGCTG CCTCTGGCAGCATCTTTGACATCTACGTGATGAGATGGTACAGACAAGCTCCTGGGAAGGGGCTGGAGTGGGTG GCCTCCATCTATGATGGGGGCAGAAATGACTATGACAATGTGGTGAAGGGCCGCTTCACCATCAGCCGGGACAAC AGCAAGAACACCCTGTACCTGCAGATGAACTCCCTCAGAGCTGAGGACACCGCTGTGTACTTCTGCAATGTCCTGA CCTATGCTGGAAGACCCTACTGGGGCCAAGGCACCCAAGTCACTGTGTCCAGCGGCGGTGGAGGCAGTGGTGGC GGAGGCAGTGGAGGTGGAGGCAGTCGTCCCCGGAACGCACTGCTGCTCCTCGCGGATGACGGAGGCTTTGAGA GTGGCGCGTACAACAACAGCGCCATCGCCACCCCGCACCTGGACGCCTTGGCCCGCCGCAGCCTCCTCTTTCGCAA TGCCTTCACCTCGGTCAGCAGCTGCTCTCCCAGCCGCGCCAGCCTCCTCACTGGCCTGCCCCAGCATCAGAATGGG ATGTACGGGCTGCACCAGGACGTGCACCACTTCAACTCCTTCGACAAGGTGCGGAGCCTGCCGCTGCTGCTCAGC CAAGCTGGTGTGCGCACAGGCATCATCGGGAAGAAGCACGTGGGGCCGGAGACCGTGTACCCGTTTGACTTTGC GTACACGGAGGAGAATGGCTCCGTCCTCCAGGTGGGGCGGAACATCACTAGAATTAAGCTGCTCGTCCGGAAATT CCTGCAGACTCAGGATGACCGGCCTTTCTTCCTCTACGTCGCCTTCCACGACCCCCACCGCTGTGGGCACTCCCAGC CCCAGTACGGAACCTTCTGTGAGAAGTTTGGCAACGGAGAGAGCGGCATGGGTCGTATCCCAGACTGGACCCCCC AGGCCTACGACCCACTGGACGTGCTGGTGCCTTACTTCGTCCCCAACACCCCGGCAGCCCGAGCCGACCTGGCCG CTCAGTACACCACCGTCGGCCGCATGGACCAAGGAGTTGGACTGGTGCTCCAGGAGCTGCGTGACGCCGGTGTCC TGAACGACACACTGGTGATCTTCACGTCCGACAACGGGATCCCCTTCCCCAGCGGCAGGACCAACCTGTACTGGC CGGGCACTGCTGAACCCTTACTGGTGTCATCCCCGGAGCACCCAAAACGCTGGGGCCAAGTCAGCGAGGCCTACG TGAGCCTCCTAGACCTCACGCCCACCATCTTGGATTGGTTCTCGATCCCGTACCCCAGCTACGCCATCTTTGGCTCG AAGACCATCCACCTCACTGGCCGGTCCCTCCTGCCGGCGCTGGAGGCCGAGCCCCTCTGGGCCACCGTCTTTGGCA GCCAGAGCCACCACGAGGTCACCATGTCCTACCCCATGCGCTCCGTGCAGCACCGGCACTTCCGCCTCGTGCACAA CCTCAACTTCAAGATGCCCTTTCCCATCGACCAGGACTTCTACGTCTCACCCACCTTCCAGGACCTCCTGAACCGCA CCACAGCTGGTCAGCCCACGGGCTGGTACAAGGACCTCCGTCATTACTACTACCGGGCGCGCTGGGAGCTCTACG ACCGGAGCCGGGACCCCCACGAGACCCAGAACCTGGCCACCGACCCGCGCTTTGCTCAGCTTCTGGAGATGCTTC GGGACCAGCTGGCCAAGTGGCAGTGGGAGACCCACGACCCCTGGGTGTGCGCCCCCGACGGCGTCCTGGAGGA GAAGCTCTCTCCCCAGTGCCAGCCCCTCCACAATGAGCTGTGAGTCGACAATCAACCTCTGGATTACAAAATTTGT GAAAGATTGACTGGTATTCTTAACTTTGTTGCTCCTTTTACGCTTTGTGGATACGCTGCTTTATTGCCTTTGTATCTT GCTATTGCTTCCCGTTTGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGCTCTTGCCACGGCGGAACTCATC GCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGGTACCCAAT AAAAGATCTTTATTTTCATTAGATCTGTGTGTTGGTTTTTTGTGTGTCTAGACCCCGTGTGAACGATTGGTAAACCC GGTGTCCTGTGAGCGGCGAAAGCCTAAACGGGAAATACGGTTTAAACATTTAAATAGGAACCCCTAGTGATGGA GTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTT TGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGA (SEQ ID NO: 37) TJ036
Attorney Docket No: JCR-002WO1 CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTTAATTAAACGCGTGGGGG AGGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCACCGGGGGA GGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCACCGGGGGAG GCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCACACTAGTAAAT GACCTATTAAGAATATTTCATAGAACGAATGTTCCGATGCTCTAATCTCTCTAGACAAGGTTCATATTTGTATGGGT TACTTATTCTCTCTTTGTTGACTAAGTCAATAATCAGAATCAGCAGGTTTGCAGTCAGATTGGCAGGGATAAGCAG CCTAGCTCAGGAGAAGTGAGTATAAAAGCCCCAGGCTGGGAGCAGCCATCACAGAAGTCCACTCATTCTTGGCAG GCCGCGGCTAAGGTAAGTTGGCGCCGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTTTTTTA CAGGCCTGGGCGCGCCGCCACCATGAGCTGCCCCGTGCCCGCCTGCTGCGCGCTGCTGCTAGTCCTGGGGCTCTG CCGGGCGCGTCCCCGGAACGCACTGCTGCTCCTCGCGGATGACGGAGGCTTTGAGAGTGGCGCGTACAACAACA GCGCCATCGCCACCCCGCACCTGGACGCCTTGGCCCGCCGCAGCCTCCTCTTTCGCAATGCCTTCACCTCGGTCAG CAGCTGCTCTCCCAGCCGCGCCAGCCTCCTCACTGGCCTGCCCCAGCATCAGAATGGGATGTACGGGCTGCACCA GGACGTGCACCACTTCAACTCCTTCGACAAGGTGCGGAGCCTGCCGCTGCTGCTCAGCCAAGCTGGTGTGCGCAC AGGCATCATCGGGAAGAAGCACGTGGGGCCGGAGACCGTGTACCCGTTTGACTTTGCGTACACGGAGGAGAATG GCTCCGTCCTCCAGGTGGGGCGGAACATCACTAGAATTAAGCTGCTCGTCCGGAAATTCCTGCAGACTCAGGATG ACCGGCCTTTCTTCCTCTACGTCGCCTTCCACGACCCCCACCGCTGTGGGCACTCCCAGCCCCAGTACGGAACCTTC TGTGAGAAGTTTGGCAACGGAGAGAGCGGCATGGGTCGTATCCCAGACTGGACCCCCCAGGCCTACGACCCACT GGACGTGCTGGTGCCTTACTTCGTCCCCAACACCCCGGCAGCCCGAGCCGACCTGGCCGCTCAGTACACCACCGTC GGCCGCATGGACCAAGGAGTTGGACTGGTGCTCCAGGAGCTGCGTGACGCCGGTGTCCTGAACGACACACTGGT GATCTTCACGTCCGACAACGGGATCCCCTTCCCCAGCGGCAGGACCAACCTGTACTGGCCGGGCACTGCTGAACC CTTACTGGTGTCATCCCCGGAGCACCCAAAACGCTGGGGCCAAGTCAGCGAGGCCTACGTGAGCCTCCTAGACCT CACGCCCACCATCTTGGATTGGTTCTCGATCCCGTACCCCAGCTACGCCATCTTTGGCTCGAAGACCATCCACCTCA CTGGCCGGTCCCTCCTGCCGGCGCTGGAGGCCGAGCCCCTCTGGGCCACCGTCTTTGGCAGCCAGAGCCACCACG AGGTCACCATGTCCTACCCCATGCGCTCCGTGCAGCACCGGCACTTCCGCCTCGTGCACAACCTCAACTTCAAGAT GCCCTTTCCCATCGACCAGGACTTCTACGTCTCACCCACCTTCCAGGACCTCCTGAACCGCACCACAGCTGGTCAGC CCACGGGCTGGTACAAGGACCTCCGTCATTACTACTACCGGGCGCGCTGGGAGCTCTACGACCGGAGCCGGGAC CCCCACGAGACCCAGAACCTGGCCACCGACCCGCGCTTTGCTCAGCTTCTGGAGATGCTTCGGGACCAGCTGGCC AAGTGGCAGTGGGAGACCCACGACCCCTGGGTGTGCGCCCCCGACGGCGTCCTGGAGGAGAAGCTCTCTCCCCA GTGCCAGCCCCTCCACAATGAGCTGGGCGGTGGAGGCAGTGGTGGCGGAGGCAGTGGAGGTGGAGGCAGTCAA GTGCAGCTGGTGGAGAGTGGAGGTGGCCTGGTCCAGCCTGGAGGCTCCCTGCGGCTGAGCTGTGCTGCCTCTGG CAGCATCTTTGACATCTACGTGATGAGATGGTACAGACAAGCTCCTGGGAAGGGGCTGGAGTGGGTGGCCTCCAT CTATGATGGGGGCAGAAATGACTATGACAATGTGGTGAAGGGCCGCTTCACCATCAGCCGGGACAACAGCAAGA ACACCCTGTACCTGCAGATGAACTCCCTCAGAGCTGAGGACACCGCTGTGTACTTCTGCAATGTCCTGACCTATGC TGGAAGACCCTACTGGGGCCAAGGCACCCAAGTCACTGTGTCCAGCTGAGTCGACAATCAACCTCTGGATTACAA AATTTGTGAAAGATTGACTGGTATTCTTAACTTTGTTGCTCCTTTTACGCTTTGTGGATACGCTGCTTTATTGCCTTT GTATCTTGCTATTGCTTCCCGTTTGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGCTCTTGCCACGGCGGA ACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGGT ACCCAATAAAAGATCTTTATTTTCATTAGATCTGTGTGTTGGTTTTTTGTGTGTCTAGACCCCGTGTGAACGATTGG TAAACCCGGTGTCCTGTGAGCGGCGAAAGCCTAAACGGGAAATACGGTTTAAACATTTAAATAGGAACCCCTAGT GATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCC GGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGA (SEQ ID NO: 38) TJ039
Attorney Docket No: JCR-002WO1 CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTTAATTAAAGATGTACTGC CAAGTAGGAAAGTCCCGTAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATA GGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGAC GTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGG GCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCGGAACTCCATATATGGGCTATGAACTAATGACC CCGTAATTGATTACTATTAACCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTA TTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGG GGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTT CCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGGAGTCGCTGCGTTGC CTTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGT GAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTAAGAGGTAAGGGTTTAAGGGATGGTTGGT TGGTGGGGTATTAATGTTTAATTACCTGTTTTACAGGCCTGAAATCACTTGGTTTTAGGTTGGGAATTCGGCGCGC CGCCACCATGAGCTGCCCCGTGCCCGCCTGCTGCGCGCTGCTGCTAGTCCTGGGGCTCTGCCGGGCGCGTCCCCG GAACGCACTGCTGCTCCTCGCGGATGACGGAGGCTTTGAGAGTGGCGCGTACAACAACAGCGCCATCGCCACCCC GCACCTGGACGCCTTGGCCCGCCGCAGCCTCCTCTTTCGCAATGCCTTCACCTCGGTCAGCAGCTGCTCTCCCAGCC GCGCCAGCCTCCTCACTGGCCTGCCCCAGCATCAGAATGGGATGTACGGGCTGCACCAGGACGTGCACCACTTCA ACTCCTTCGACAAGGTGCGGAGCCTGCCGCTGCTGCTCAGCCAAGCTGGTGTGCGCACAGGCATCATCGGGAAGA AGCACGTGGGGCCGGAGACCGTGTACCCGTTTGACTTTGCGTACACGGAGGAGAATGGCTCCGTCCTCCAGGTG GGGCGGAACATCACTAGAATTAAGCTGCTCGTCCGGAAATTCCTGCAGACTCAGGATGACCGGCCTTTCTTCCTCT ACGTCGCCTTCCACGACCCCCACCGCTGTGGGCACTCCCAGCCCCAGTACGGAACCTTCTGTGAGAAGTTTGGCAA CGGAGAGAGCGGCATGGGTCGTATCCCAGACTGGACCCCCCAGGCCTACGACCCACTGGACGTGCTGGTGCCTTA CTTCGTCCCCAACACCCCGGCAGCCCGAGCCGACCTGGCCGCTCAGTACACCACCGTCGGCCGCATGGACCAAGG AGTTGGACTGGTGCTCCAGGAGCTGCGTGACGCCGGTGTCCTGAACGACACACTGGTGATCTTCACGTCCGACAA CGGGATCCCCTTCCCCAGCGGCAGGACCAACCTGTACTGGCCGGGCACTGCTGAACCCTTACTGGTGTCATCCCCG GAGCACCCAAAACGCTGGGGCCAAGTCAGCGAGGCCTACGTGAGCCTCCTAGACCTCACGCCCACCATCTTGGAT TGGTTCTCGATCCCGTACCCCAGCTACGCCATCTTTGGCTCGAAGACCATCCACCTCACTGGCCGGTCCCTCCTGCC GGCGCTGGAGGCCGAGCCCCTCTGGGCCACCGTCTTTGGCAGCCAGAGCCACCACGAGGTCACCATGTCCTACCC CATGCGCTCCGTGCAGCACCGGCACTTCCGCCTCGTGCACAACCTCAACTTCAAGATGCCCTTTCCCATCGACCAG GACTTCTACGTCTCACCCACCTTCCAGGACCTCCTGAACCGCACCACAGCTGGTCAGCCCACGGGCTGGTACAAGG ACCTCCGTCATTACTACTACCGGGCGCGCTGGGAGCTCTACGACCGGAGCCGGGACCCCCACGAGACCCAGAACC TGGCCACCGACCCGCGCTTTGCTCAGCTTCTGGAGATGCTTCGGGACCAGCTGGCCAAGTGGCAGTGGGAGACCC ACGACCCCTGGGTGTGCGCCCCCGACGGCGTCCTGGAGGAGAAGCTCTCTCCCCAGTGCCAGCCCCTCCACAATG AGCTGTGAGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTTTGTTGCTCCT TTTACGCTTTGTGGATACGCTGCTTTATTGCCTTTGTATCTTGCTATTGCTTCCCGTTTGGCTTTCATTTTCTCCTCCTT GTATAAATCCTGGTTAGCTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCG GCTGTTGGGCACTGACAATTCCGTGGTGTTGGTACCCAATAAAAGATCTTTATTTTCATTAGATCTGTGTGTTGGTT TTTTGTGTGTCTAGACCCCGTGTGAACGATTGGTAAACCCGGTGTCCTGTGAGCGGCGAAAGCCTAAACGGGAAA TACGGTTTAAACATTTAAATAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACT GAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCA GAGA (SEQ ID NO: 39) TJ040
Attorney Docket No: JCR-002WO1 CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTTAATTAAAGATGTACTGC CAAGTAGGAAAGTCCCGTAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATA GGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGAC GTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGG GCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCGGAACTCCATATATGGGCTATGAACTAATGACC CCGTAATTGATTACTATTAACCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTA TTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGG GGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTT CCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGGAGTCGCTGCGTTGC CTTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGT GAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTAAGAGGTAAGGGTTTAAGGGATGGTTGGT TGGTGGGGTATTAATGTTTAATTACCTGTTTTACAGGCCTGAAATCACTTGGTTTTAGGTTGGGAATTCGGCGCGC CGCCACCATGGGATGGTCCTGCATCATCCTGTTTCTGGTGGCCACAGCCACAGGCGCTCACTCCCAAGTGCAGCTG GTGGAGAGTGGAGGTGGCCTGGTCCAGCCTGGAGGCTCCCTGCGGCTGAGCTGTGCTGCCTCTGGCAGCATCTTT GACATCTACGTGATGAGATGGTACAGACAAGCTCCTGGGAAGGGGCTGGAGTGGGTGGCCTCCATCTATGATGG GGGCAGAAATGACTATGACAATGTGGTGAAGGGCCGCTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGT ACCTGCAGATGAACTCCCTCAGAGCTGAGGACACCGCTGTGTACTTCTGCAATGTCCTGACCTATGCTGGAAGACC CTACTGGGGCCAAGGCACCCAAGTCACTGTGTCCAGCGGCGGTGGAGGCAGTGGTGGCGGAGGCAGTGGAGGT GGAGGCAGTCGTCCCCGGAACGCACTGCTGCTCCTCGCGGATGACGGAGGCTTTGAGAGTGGCGCGTACAACAA CAGCGCCATCGCCACCCCGCACCTGGACGCCTTGGCCCGCCGCAGCCTCCTCTTTCGCAATGCCTTCACCTCGGTCA GCAGCTGCTCTCCCAGCCGCGCCAGCCTCCTCACTGGCCTGCCCCAGCATCAGAATGGGATGTACGGGCTGCACC AGGACGTGCACCACTTCAACTCCTTCGACAAGGTGCGGAGCCTGCCGCTGCTGCTCAGCCAAGCTGGTGTGCGCA CAGGCATCATCGGGAAGAAGCACGTGGGGCCGGAGACCGTGTACCCGTTTGACTTTGCGTACACGGAGGAGAAT GGCTCCGTCCTCCAGGTGGGGCGGAACATCACTAGAATTAAGCTGCTCGTCCGGAAATTCCTGCAGACTCAGGAT GACCGGCCTTTCTTCCTCTACGTCGCCTTCCACGACCCCCACCGCTGTGGGCACTCCCAGCCCCAGTACGGAACCTT CTGTGAGAAGTTTGGCAACGGAGAGAGCGGCATGGGTCGTATCCCAGACTGGACCCCCCAGGCCTACGACCCAC TGGACGTGCTGGTGCCTTACTTCGTCCCCAACACCCCGGCAGCCCGAGCCGACCTGGCCGCTCAGTACACCACCGT CGGCCGCATGGACCAAGGAGTTGGACTGGTGCTCCAGGAGCTGCGTGACGCCGGTGTCCTGAACGACACACTGG TGATCTTCACGTCCGACAACGGGATCCCCTTCCCCAGCGGCAGGACCAACCTGTACTGGCCGGGCACTGCTGAAC CCTTACTGGTGTCATCCCCGGAGCACCCAAAACGCTGGGGCCAAGTCAGCGAGGCCTACGTGAGCCTCCTAGACC TCACGCCCACCATCTTGGATTGGTTCTCGATCCCGTACCCCAGCTACGCCATCTTTGGCTCGAAGACCATCCACCTC ACTGGCCGGTCCCTCCTGCCGGCGCTGGAGGCCGAGCCCCTCTGGGCCACCGTCTTTGGCAGCCAGAGCCACCAC GAGGTCACCATGTCCTACCCCATGCGCTCCGTGCAGCACCGGCACTTCCGCCTCGTGCACAACCTCAACTTCAAGA TGCCCTTTCCCATCGACCAGGACTTCTACGTCTCACCCACCTTCCAGGACCTCCTGAACCGCACCACAGCTGGTCAG CCCACGGGCTGGTACAAGGACCTCCGTCATTACTACTACCGGGCGCGCTGGGAGCTCTACGACCGGAGCCGGGA CCCCCACGAGACCCAGAACCTGGCCACCGACCCGCGCTTTGCTCAGCTTCTGGAGATGCTTCGGGACCAGCTGGC CAAGTGGCAGTGGGAGACCCACGACCCCTGGGTGTGCGCCCCCGACGGCGTCCTGGAGGAGAAGCTCTCTCCCC AGTGCCAGCCCCTCCACAATGAGCTGTGAGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGG TATTCTTAACTTTGTTGCTCCTTTTACGCTTTGTGGATACGCTGCTTTATTGCCTTTGTATCTTGCTATTGCTTCCCGT TTGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGCTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCC CGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGGTACCCAATAAAAGATCTTTATTTT CATTAGATCTGTGTGTTGGTTTTTTGTGTGTCTAGACCCCGTGTGAACGATTGGTAAACCCGGTGTCCTGTGAGCG GCGAAAGCCTAAACGGGAAATACGGTTTAAACATTTAAATAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCT
Attorney Docket No: JCR-002WO1 CTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGA (SEQ ID NO: 40) TJ050 CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTTAATTAAAGATGTACTGC CAAGTAGGAAAGTCCCGTAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATA GGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGAC GTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGG GCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCGGAACTCCATATATGGGCTATGAACTAATGACC CCGTAATTGATTACTATTAACCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTA TTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGG GGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTT CCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGGAGTCGCTGCGTTGC CTTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGT GAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTAAGAGGTAAGGGTTTAAGGGATGGTTGGT TGGTGGGGTATTAATGTTTAATTACCTGTTTTACAGGCCTGAAATCACTTGGTTTTAGGTTGGGAATTCGGCGCGC CGCCACCATGGGATGGTCCTGCATCATCCTGTTTCTGGTGGCCACAGCCACAGGCGCTCACTCCCAAGTGCAGCTG GTGGAGAGTGGAGGTGGCCTGGTCCAGCCTGGAGGCTCCCTGCGGCTGAGCTGTGCTGCCTCTGGCAGCATCTTT GACATCTACGTGATGAGATGGTACAGACAAGCTCCTGGGAAGGGGCTGGAGTGGGTGGCCTCCATCTATGATGG GGGCAGAAATGACTATGACAATGTGGTGAAGGGCCGCTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGT ACCTGCAGATGAACTCCCTCAGAGCTGAGGACACCGCTGTGTACTTCTGCAATGTCCTGACCTATGCTGGAAGACC CTACTGGGGCCAAGGCACCCAAGTCACTGTGTCCAGCGGCGGTGGAGGCAGTGGTGGCGGAGGCAGTGGAGGT GGAGGCAGTAGGCCTAGGAATGCCCTCCTGCTGCTGGCAGATGATGGAGGCTTTGAGAGTGGGGCCTACAACAA CTCAGCCATTGCCACCCCCCATTTGGATGCCTTGGCCAGAAGGAGTCTCCTCTTCAGAAATGCTTTCACAAGTGTCA GCTCCTGCTCCCCATCAAGGGCCTCCCTGCTCACTGGGCTGCCCCAGCACCAGAATGGGATGTATGGCCTGCACCA AGATGTCCACCACTTCAACAGCTTTGACAAGGTGAGGAGCCTGCCTCTGTTGCTGTCACAAGCTGGAGTGCGCACT GGAATCATTGGGAAGAAGCATGTGGGCCCTGAGACAGTCTACCCTTTTGATTTTGCCTACACAGAGGAGAATGGT TCTGTCCTGCAAGTTGGGAGAAACATCACCAGAATCAAGCTGCTGGTAAGAAAATTCCTGCAGACCCAAGATGAC AGACCATTCTTCCTCTATGTGGCCTTCCATGATCCCCACAGATGTGGCCACAGCCAGCCTCAGTATGGGACTTTCTG TGAGAAGTTTGGAAATGGAGAATCTGGCATGGGCAGAATTCCTGACTGGACTCCACAAGCATATGACCCACTGGA TGTGTTGGTTCCTTACTTTGTGCCCAACACCCCTGCTGCAAGAGCTGACCTGGCTGCCCAGTACACCACAGTGGGA AGAATGGACCAAGGAGTTGGACTTGTCCTCCAAGAGCTCAGAGATGCTGGGGTGCTGAATGACACCCTGGTCATC TTCACCTCTGACAATGGCATCCCCTTTCCATCTGGACGCACCAACCTCTACTGGCCCGGCACAGCAGAACCCCTTCT GGTGTCCAGCCCAGAGCACCCCAAGAGATGGGGCCAAGTGTCTGAAGCCTATGTTTCTCTGCTTGACCTCACCCCA ACAATCCTGGACTGGTTCTCCATTCCCTACCCATCCTATGCCATCTTTGGGAGCAAGACCATCCACCTGACTGGTAG GTCCCTCCTTCCTGCCCTGGAGGCTGAGCCCCTGTGGGCCACTGTGTTTGGCTCCCAGAGCCACCATGAAGTGACC ATGTCCTACCCCATGAGATCTGTGCAGCATAGGCATTTCCGCCTGGTGCACAACCTGAACTTCAAGATGCCCTTCCC TATTGATCAAGACTTCTATGTCAGTCCCACCTTCCAAGATCTTCTCAACAGAACCACTGCTGGCCAGCCCACTGGCT GGTACAAGGACTTGAGACACTACTACTATAGAGCCCGCTGGGAGCTGTATGATAGAAGCAGAGACCCTCATGAG ACCCAGAACCTTGCCACAGATCCAAGATTTGCTCAGCTCTTGGAGATGCTGAGGGACCAGCTGGCCAAGTGGCAG TGGGAAACACATGACCCCTGGGTGTGTGCACCTGATGGTGTGCTGGAAGAAAAACTGTCTCCACAGTGCCAGCCA CTCCACAATGAACTGTGAGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTT TGTTGCTCCTTTTACGCTTTGTGGATACGCTGCTTTATTGCCTTTGTATCTTGCTATTGCTTCCCGTTTGGCTTTCATT
Attorney Docket No: JCR-002WO1 TTCTCCTCCTTGTATAAATCCTGGTTAGCTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGAC AGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGGTACCCAATAAAAGATCTTTATTTTCATTAGATCTG TGTGTTGGTTTTTTGTGTGTCTAGACCCCGTGTGAACGATTGGTAAACCCGGTGTCCTGTGAGCGGCGAAAGCCTA AACGGGAAATACGGTTTAAACATTTAAATAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGC TCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCG AGCGCGCAGAGA (SEQ ID NO: 41) TJ051 CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTTAATTAAAGATGTACTGC CAAGTAGGAAAGTCCCGTAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATA GGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGAC GTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGG GCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCGGAACTCCATATATGGGCTATGAACTAATGACC CCGTAATTGATTACTATTAACCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTA TTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGG GGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTT CCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGGAGTCGCTGCGTTGC CTTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGT GAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTAAGAGGTAAGGGTTTAAGGGATGGTTGGT TGGTGGGGTATTAATGTTTAATTACCTGTTTTACAGGCCTGAAATCACTTGGTTTTAGGTTGGGAATTCGGCGCGC CGCCACCATGGGATGGTCCTGCATCATCCTGTTTCTGGTGGCCACAGCCACAGGCGCTCACTCCCAAGTGCAGCTG GTGGAGAGTGGAGGTGGCCTGGTCCAGCCTGGAGGCTCCCTGCGGCTGAGCTGTGCTGCCTCTGGCAGCATCTTT GACATCTACGTGATGAGATGGTACAGACAAGCTCCTGGGAAGGGGCTGGAGTGGGTGGCCTCCATCTATGATGG GGGCAGAAATGACTATGACAATGTGGTGAAGGGCCGCTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGT ACCTGCAGATGAACTCCCTCAGAGCTGAGGACACCGCTGTGTACTTCTGCAATGTCCTGACCTATGCTGGAAGACC CTACTGGGGCCAAGGCACCCAAGTCACTGTGTCCAGCGGCGGTGGAGGCAGTGGTGGCGGAGGCAGTGGAGGT GGAGGCAGTAGGCCCAGAAATGCCCTCCTTCTCCTGGCTGATGATGGAGGATTTGAATCTGGGGCCTACAACAAC TCAGCCATTGCCACCCCTCACCTGGATGCCCTGGCAAGAAGGTCTCTGCTTTTTAGGAATGCTTTCACCAGTGTCAG TTCCTGCTCCCCATCAAGGGCCTCCTTGCTGACTGGGCTGCCCCAGCACCAGAATGGCATGTATGGCCTGCACCAA GATGTGCACCATTTCAACAGCTTTGACAAGGTGAGGAGCCTTCCTCTGCTCCTCTCACAAGCTGGAGTAAGAACTG GCATCATTGGCAAGAAACATGTGGGCCCTGAGACAGTCTATCCCTTTGATTTTGCCTACACAGAAGAAAATGGTTC TGTTCTCCAAGTTGGGAGAAACATCACCAGAATCAAGCTGCTGGTTAGAAAATTTCTGCAGACCCAAGATGACAG ACCTTTCTTCCTGTATGTGGCCTTCCATGATCCCCACAGATGTGGCCACAGCCAGCCTCAGTATGGAACATTCTGTG AGAAGTTTGGAAATGGAGAGAGTGGGATGGGCAGAATTCCTGACTGGACTCCACAAGCATATGACCCATTGGAT GTCCTTGTTCCTTACTTTGTGCCCAACACCCCAGCTGCCAGAGCTGACCTTGCAGCCCAGTACACCACAGTGGGGA GGATGGACCAAGGTGTGGGACTGGTGCTGCAAGAGCTGAGAGATGCTGGGGTCCTGAATGACACCCTGGTCATC TTCACATCTGACAATGGAATCCCATTCCCAAGTGGCCGCACCAACCTCTACTGGCCCGGCACAGCTGAGCCCCTGC TGGTGTCCAGCCCAGAGCACCCCAAGAGATGGGGCCAAGTGTCAGAAGCCTATGTCTCCCTCTTGGACCTCACCC CCACCATTCTGGACTGGTTCTCCATCCCCTACCCTTCCTATGCCATCTTTGGGAGCAAGACCATCCACCTCACTGGA AGAAGCCTGCTGCCTGCTCTGGAAGCAGAGCCACTGTGGGCCACTGTGTTTGGCTCCCAGAGCCATCATGAAGTG ACCATGTCCTACCCCATGAGATCTGTGCAGCATAGACACTTCAGACTTGTCCACAACCTGAACTTCAAGATGCCCTT CCCTATTGATCAAGACTTCTACGTGTCTCCTACCTTCCAAGACTTGCTCAACAGAACCACTGCTGGCCAGCCCACTG GCTGGTACAAGGACCTGAGGCACTACTACTATAGAGCCCGCTGGGAGCTCTATGATAGGTCCAGAGACCCTCATG
Attorney Docket No: JCR-002WO1 AGACCCAGAACTTGGCCACAGATCCAAGATTTGCTCAGCTCCTGGAGATGCTGCGGGACCAGCTGGCCAAGTGGC AGTGGGAAACACATGACCCCTGGGTGTGTGCTCCTGATGGGGTGTTGGAGGAGAAACTGTCTCCACAGTGCCAG CCACTTCATAATGAACTGTGAGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAA CTTTGTTGCTCCTTTTACGCTTTGTGGATACGCTGCTTTATTGCCTTTGTATCTTGCTATTGCTTCCCGTTTGGCTTTC ATTTTCTCCTCCTTGTATAAATCCTGGTTAGCTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTG GACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGGTACCCAATAAAAGATCTTTATTTTCATTAGAT CTGTGTGTTGGTTTTTTGTGTGTCTAGACCCCGTGTGAACGATTGGTAAACCCGGTGTCCTGTGAGCGGCGAAAGC CTAAACGGGAAATACGGTTTAAACATTTAAATAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCT CGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGA GCGAGCGCGCAGAGA (SEQ ID NO: 42) TJ052 CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTTAATTAAAGATGTACTGC CAAGTAGGAAAGTCCCGTAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATA GGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGAC GTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGG GCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCGGAACTCCATATATGGGCTATGAACTAATGACC CCGTAATTGATTACTATTAACCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTA TTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGG GGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTT CCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGGAGTCGCTGCGTTGC CTTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGT GAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTAAGAGGTAAGGGTTTAAGGGATGGTTGGT TGGTGGGGTATTAATGTTTAATTACCTGTTTTACAGGCCTGAAATCACTTGGTTTTAGGTTGGGAATTCGGCGCGC CGCCACCATGGGATGGTCCTGCATCATCCTGTTTCTGGTGGCCACAGCCACAGGCGCTCACTCCCAAGTGCAGCTG GTGGAGAGTGGAGGTGGCCTGGTCCAGCCTGGAGGCTCCCTGCGGCTGAGCTGTGCTGCCTCTGGCAGCATCTTT GACATCTACGTGATGAGATGGTACAGACAAGCTCCTGGGAAGGGGCTGGAGTGGGTGGCCTCCATCTATGATGG GGGCAGAAATGACTATGACAATGTGGTGAAGGGCCGCTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGT ACCTGCAGATGAACTCCCTCAGAGCTGAGGACACCGCTGTGTACTTCTGCAATGTCCTGACCTATGCTGGAAGACC CTACTGGGGCCAAGGCACCCAAGTCACTGTGTCCAGCGGCGGTGGAGGCAGTGGTGGCGGAGGCAGTGGAGGT GGAGGCAGTAGACCAAGAAATGCTCTGCTGCTGCTGGCAGATGATGGGGGCTTTGAATCTGGGGCCTACAACAA CTCTGCCATTGCCACCCCCCATCTGGATGCCCTGGCCAGAAGGTCCTTGCTGTTTAGGAATGCCTTTACCTCAGTGT CCTCCTGCTCCCCATCTAGGGCCTCCCTGCTCACTGGCCTGCCCCAGCACCAGAATGGGATGTATGGACTGCACCA AGATGTCCACCACTTCAACAGCTTTGACAAGGTGAGAAGCCTGCCTCTGCTTCTGTCCCAAGCTGGAGTGAGAACT GGAATCATTGGCAAGAAGCATGTGGGCCCTGAGACAGTCTATCCCTTTGATTTTGCCTACACAGAGGAGAATGGA AGTGTGCTGCAAGTTGGGAGAAACATCACCAGAATCAAGCTGTTGGTGAGGAAGTTCCTGCAGACCCAAGATGA CAGACCTTTCTTCCTTTATGTGGCCTTCCATGACCCCCACAGATGTGGCCACAGCCAGCCACAGTATGGCACTTTCT GTGAGAAGTTTGGAAATGGAGAGAGTGGCATGGGCAGAATTCCTGACTGGACTCCACAAGCATATGATCCATTG GATGTGTTGGTCCCCTACTTTGTGCCCAACACCCCTGCTGCTAGAGCTGACCTGGCTGCCCAGTACACCACAGTGG GAAGAATGGACCAAGGTGTGGGGCTGGTGCTCCAAGAGCTGAGAGATGCTGGGGTGCTGAATGACACCCTGGTC ATCTTCACCTCTGACAATGGCATCCCATTCCCTTCTGGAAGGACCAACCTGTACTGGCCTGGCACAGCAGAGCCAC TGCTTGTGAGCAGCCCAGAGCACCCCAAGAGGTGGGGCCAAGTGTCGGAGGCCTATGTTTCTCTGTTGGACCTGA CCCCAACAATCCTGGACTGGTTCTCCATCCCTTACCCTTCCTATGCCATCTTTGGGAGCAAGACCATCCACCTGACT
Attorney Docket No: JCR-002WO1 GGGAGGAGCCTCCTCCCAGCCTTGGAGGCTGAGCCCCTGTGGGCCACTGTGTTTGGCTCCCAGAGCCACCATGAA GTGACCATGTCCTACCCCATGAGATCTGTGCAGCATAGGCATTTCAGACTGGTGCACAACCTCAACTTCAAGATGC CCTTCCCCATTGATCAAGACTTCTATGTCAGCCCCACCTTCCAAGATCTGCTGAACAGAACCACTGCTGGCCAGCCC ACTGGCTGGTACAAGGACCTCAGACACTACTACTATAGAGCAAGATGGGAGCTCTATGACAGATCCAGAGACCCT CATGAGACCCAGAACTTGGCCACAGACCCCAGATTTGCTCAGCTCCTGGAGATGCTGAGGGACCAGCTGGCCAAG TGGCAGTGGGAAACACATGATCCCTGGGTGTGTGCTCCTGATGGAGTCCTGGAAGAAAAACTGTCTCCTCAGTGC CAGCCTCTCCACAATGAACTGTGAGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTC TTAACTTTGTTGCTCCTTTTACGCTTTGTGGATACGCTGCTTTATTGCCTTTGTATCTTGCTATTGCTTCCCGTTTGGC TTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGCTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTG CTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGGTACCCAATAAAAGATCTTTATTTTCATTA GATCTGTGTGTTGGTTTTTTGTGTGTCTAGACCCCGTGTGAACGATTGGTAAACCCGGTGTCCTGTGAGCGGCGAA AGCCTAAACGGGAAATACGGTTTAAACATTTAAATAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGC GCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG CGAGCGAGCGCGCAGAGA (SEQ ID NO: 43) TJ054 CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCA GTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTTAATTAAAGATGTACTGC CAAGTAGGAAAGTCCCGTAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATA GGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGAC GTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGG GCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCGGAACTCCATATATGGGCTATGAACTAATGACC CCGTAATTGATTACTATTAACCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTA TTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGG GGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTT CCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGGAGTCGCTGCGTTGC CTTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGT GAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTAAGAGGTAAGGGTTTAAGGGATGGTTGGT TGGTGGGGTATTAATGTTTAATTACCTGTTTTACAGGCCTGAAATCACTTGGTTTTAGGTTGGGAATTCGGCGCGC CGCCACCATGGGATGGTCCTGCATCATCCTGTTTCTGGTGGCCACAGCCACAGGCGCTCACTCCCAAGTGCAGCTG GTGGAGAGTGGAGGTGGCCTGGTCCAGCCTGGAGGCTCCCTGCGGCTGAGCTGTGCTGCCTCTGGCAGCATCTTT GACATCTACGTGATGAGATGGTACAGACAAGCTCCTGGGAAGGGGCTGGAGTGGGTGGCCTCCATCTATGATGG GGGCAGAAATGACTATGACAATGTGGTGAAGGGCCGCTTCACCATCAGCCGGGACAACAGCAAGAACACCCTGT ACCTGCAGATGAACTCCCTCAGAGCTGAGGACACCGCTGTGTACTTCTGCAATGTCCTGACCTATGCTGGAAGACC CTACTGGGGCCAAGGCACCCAAGTCACTGTGTCCAGCGGCGGTGGAGGCAGTGGTGGCGGAGGCAGTGGAGGT GGAGGCAGTAGACCAAGGAATGCTCTGCTGCTCCTGGCAGATGATGGAGGCTTTGAGAGTGGGGCCTACAACAA CTCTGCCATTGCCACCCCCCACCTGGATGCCCTGGCTAGGAGGTCCCTCCTCTTCAGAAATGCCTTTACCTCAGTGT CTTCCTGCAGCCCTTCAAGGGCCTCCTTGCTGACTGGGCTGCCTCAGCACCAGAATGGGATGTATGGCCTGCACCA AGATGTGCACCACTTCAACAGCTTTGACAAGGTGAGGAGCCTGCCCCTGTTGCTCTCACAAGCTGGAGTCAGAAC TGGAATCATTGGCAAGAAGCATGTGGGCCCTGAGACTGTGTATCCCTTTGATTTTGCTTACACAGAGGAGAATGG TTCTGTCCTCCAAGTTGGAAGAAACATCACCAGAATCAAGCTGCTGGTGAGAAAATTCCTGCAGACCCAAGATGA CAGACCTTTCTTCCTCTATGTGGCCTTCCATGACCCCCATAGATGTGGCCATTCCCAGCCCCAGTATGGGACCTTCT GTGAGAAGTTTGGAAATGGAGAATCTGGCATGGGCAGAATTCCTGACTGGACTCCACAAGCATATGACCCTCTTG ATGTCTTGGTTCCTTACTTTGTGCCCAACACCCCAGCTGCCAGAGCTGACTTGGCTGCTCAGTACACCACAGTGGG
Attorney Docket No: JCR-002WO1 GAGGATGGACCAAGGAGTGGGTCTGGTTCTGCAAGAGCTCAGAGATGCTGGGGTCCTGAATGACACCCTGGTCA TCTTCACCTCTGACAATGGCATCCCATTCCCATCTGGAAGGACCAACCTCTACTGGCCTGGCACAGCTGAGCCCCTT CTTGTGTCCAGCCCAGAGCACCCCAAGAGGTGGGGCCAAGTGTCAGAAGCCTATGTTTCTCTGCTGGACCTGACA CCCACCATTTTGGACTGGTTCTCCATCCCCTACCCTTCCTATGCCATCTTTGGGAGCAAGACCATCCACCTCACTGGT AGATCCCTGCTTCCAGCCTTGGAAGCAGAGCCTCTGTGGGCCACAGTCTTTGGCAGCCAGAGCCACCATGAAGTG ACCATGTCCTACCCCATGAGATCTGTGCAGCATAGGCATTTTAGGTTGGTCCACAACCTGAACTTCAAGATGCCCTT CCCTATTGATCAAGACTTCTATGTCAGCCCCACTTTCCAAGATCTTCTCAACAGAACCACTGCTGGCCAGCCAACTG GCTGGTACAAGGACCTCAGACACTACTACTATAGAGCAAGATGGGAGCTGTATGATAGGAGTAGGGACCCACAT GAGACCCAGAACTTGGCCACTGACCCCAGATTTGCCCAGCTCCTGGAGATGCTGAGAGACCAGCTGGCCAAGTGG CAGTGGGAAACACATGATCCCTGGGTGTGTGCTCCTGATGGGGTGCTGGAAGAAAAACTGTCCCCACAGTGCCA GCCTCTCCACAATGAACTGTGAGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTT AACTTTGTTGCTCCTTTTACGCTTTGTGGATACGCTGCTTTATTGCCTTTGTATCTTGCTATTGCTTCCCGTTTGGCTT TCATTTTCTCCTCCTTGTATAAATCCTGGTTAGCTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCT GGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGGTACCCAATAAAAGATCTTTATTTTCATTAGA TCTGTGTGTTGGTTTTTTGTGTGTCTAGACCCCGTGTGAACGATTGGTAAACCCGGTGTCCTGTGAGCGGCGAAAG CCTAAACGGGAAATACGGTTTAAACATTTAAATAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGC TCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCG AGCGAGCGCGCAGAGA (SEQ ID NO: 44) 4. Recombinant AAV.VHH-SGSH viral particles [0134] In some embodiments, the present disclosure provides isolated adeno-associated viruses (AAVs) comprising an AAV capsid and a transgene encoding the fusion protein described herein. As used herein with respect to AAVs, 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 (rAAVs) 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. [0135] Methods for obtaining recombinant AAVs having a desired capsid protein are well known in the art. (See, for example, US 2003/0138772), the contents of which are incorporated herein by reference in their entirety). Typically the methods involve culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; a recombinant AAV vector composed of, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the recombinant AAV vector into the AAV capsid proteins. In some embodiments, capsid proteins are structural proteins encoded by the cap gene of an AAV. AAVs comprise three capsid proteins, virion proteins 1 to 3 (named
Attorney Docket No: JCR-002WO1 VP1, VP2 and VP3), all of which are transcribed from a single cap gene via alternative splicing. In some embodiments, the molecular weights of VP1, VP2 and VP3 are respectively about 87 kDa, about 72 kDa and about 62 kDa. In some embodiments, upon translation, capsid proteins form a spherical 60-mer protein shell around the viral genome. In some embodiments, the functions of the capsid proteins are to protect the viral genome, deliver the genome and interact with the host. In some aspects, capsid proteins deliver the viral genome to a host in a tissue specific manner. [0136] In some embodiments, 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. In one preferred embodiment, the capsid is an AAV9 capsid protein or a variant thereof. [0137] As a non-limiting example, 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 N-sulfoglucosamine sulfohydrolase (SGSH). [0138] In a specific embodiment described herein, 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-SGSH-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. [0139] In one example, a rAAV viral particle comprises an AAV9 capsid and the transgene polynucleotide of SEQ ID NO: 34. In one example, a rAAV viral particle comprises an AAV9 capsid and the transgene polynucleotide of SEQ ID NO: 35. In one example, a rAAV viral particle comprises an AAV9 capsid and the transgene polynucleotide of SEQ ID NO: 36. In one example, a rAAV viral particle comprises an AAV9 capsid and the transgene polynucleotide of SEQ ID NO: 37. In one example, a rAAV viral particle comprises an AAV9 capsid and the transgene polynucleotide of SEQ ID NO: 38. In one example, a rAAV viral particle comprises an AAV9 capsid and the transgene polynucleotide of SEQ ID NO: 39. In one example, a rAAV viral particle comprises an AAV9 capsid and the transgene polynucleotide of SEQ ID NO: 40. In one example, a rAAV viral particle comprises an AAV9 capsid and the transgene polynucleotide of SEQ ID NO: 41. In one example, a rAAV viral particle comprises an AAV9 capsid and the transgene polynucleotide of SEQ ID NO: 42. In one example, a rAAV viral particle comprises an AAV9 capsid and the transgene polynucleotide of SEQ ID NO: 43. In one example, a rAAV viral particle comprises an AAV9 capsid and the transgene polynucleotide of
Attorney Docket No: JCR-002WO1 SEQ ID NO: 44. In some embodiments, a rAAV viral particle comprises an AAV9 capsid and a transgene polynucleotide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to a nucleotide sequence of any one of SEQ ID NOs: 34-44 (Table 3). [0140] 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. Alternatively, any one or more of the required components (e.g., recombinant AAV vector, rep sequences, cap sequences, and/or helper functions) may be provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art. In some embodiments, the host cell is a mammalian cell (e.g., HEK293 cell, or MPNST cells) or an insect cell (e.g., SF9 cell). In some embodiments, the disclosure relates to a composition comprising the host cell described above. In some embodiments, the composition comprising the host cell above further comprises a cryopreservative. [0141] 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. The methods used to construct 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. [0142] In some embodiments, recombinant AAV viral particles may be produced using the triple transfection method (described in detail in U.S. Pat. No.6,001,650). Typically, 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. Preferably, 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). Non-limiting examples of 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
Attorney Docket No: JCR-002WO1 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 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. [0143] 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. Thus, a “host cell” as used herein may also refer to a cell which has been transfected with an exogenous DNA sequence. [0144] 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 Pat. No.7,588,772. In a one system, 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. In a second system, a packaging cell line that stably supplies rep and cap is transiently transfected with a construct encoding the transgene flanked by ITRs. In each of these systems, AAV virions are produced in response to infection with helper adenovirus or herpesvirus, requiring the separation of the rAAVs from contaminating virus. More recently, 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. In these newer systems, 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. [0145] In some embodiments, 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.
Attorney Docket No: JCR-002WO1 [0146] In some embodiments, the expression cassette flanked by ITRs and rep/cap genes are introduced into insect cells by infection with baculovirus-based vectors. For reviews on these production systems, see generally, e.g., Zhang et al, 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, the contents of which is incorporated herein by reference in its entirety. Methods of making and using these and other AAV production systems are also described in the following U.S. patents, the contents of each of which is incorporated herein by reference in its entirety: 5, 139,941 ; 5,741,683; 6,057, 152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7, 172,893; 7,201,898; 7,229,823; and 7,439,065. See generally, e.g., Grieger & Samulski, 2005, "Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications," Adv. Biochem. Engin/Biotechnol.99: 119- 145; Buning et al, 2008, "Recent developments in adeno-associated virus vector technology," J. Gene Med 10:717-733; and the references cited below, each of which is incorporated herein by reference in its entirety. [0147] The methods used to construct any embodiment of this invention are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Similarly, methods of generating rAAV virions are well known and the selection of a suitable method is not a limitation on the present invention. See, e.g., K. Fisher et al, (1993) J. Virol, 70:520-532 and US Patent No.5,478,745. [0148] Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known and readily available to those of skill in the art. Moreover, those of skill readily may construct any number of other plasmids suitable for use in the invention. The properties, construction and use of such plasmids, as well as other vectors, in the present invention will be readily apparent to those of skill from the present disclosure. [0149] In one embodiment, the production plasmid is that described herein, or as described in WO2012/158757, which is incorporated herein by reference. Various plasmids are known in the art for use in producing rAAV vectors, and are useful herein. 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.
Attorney Docket No: JCR-002WO1 [0150] In certain embodiments, the rAAV expression cassette, the vector (such as rAAV vector), the virus (such as rAAV), the production plasmid comprises AAV inverted terminal repeat sequences, and expression control sequences that direct expression of the encoded proteins (e.g., VHH-SGSH fusion protein) in a host cell. In other embodiments, the rAAV expression cassette, the virus, the vector (such as rAAV vector), the production plasmid further comprises one or more of an intron, a Kozak sequence, a polyA, posttranscriptional regulatory elements and others. In one embodiment, the post-transcriptional regulatory element is Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE). [0151] Various methods are known in the art relating to the production and purification of AAV vectors. See, e.g., Mizukami, Hiroaki, et al. A Protocol for AAV vector production and purification; U.S. Patent Publication Numbers US20070015238 and US20120322861. For example, 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 cell such that the rAAV can be packaged and subsequently purified. [0152] In some embodiments, 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-1573™, ATCC® CRL-1651™, ATCC® CRL-1650™, ATCC® CCL-2, ATCC® CCL- 10™, or ATCC® CCL-61™). Exemplary insect cells include, but are not limited to Sf9 cells (see, e.g., ATCC® CRL-1711™). 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. In some embodiments, the packaging is performed in vitro. [0153] In some embodiments, 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. [0154] In some embodiments, 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. For clarity, helper genes are genes that encode helper proteins Ela, Elb, E4, E2a, and VA. In some embodiments, the cap gene is modified such that one or more of the proteins VP1, VP2 and VP3
Attorney Docket No: JCR-002WO1 do not get expressed. In some embodiments, 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). [0155] 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. (1998), Novel Tools for Production and Purification of Recombinant Adeno associated Virus Vectors, Human Gene Therapy, Vol.9, 2745-2760; Kem, A. et al. (2003), Identification of a Heparin-Binding Motif on Adeno- Associated Virus Type 2 Capsids, Journal of Virology, Vol.77, 11072-11081.; Grimm et al. (2003), Helper Virus-Free, Optically Controllable, and Two-Plasmid-Based Production of Adeno-associated Virus Vectors of Serotypes 1 to 6, Molecular Therapy, Vol.7, 839-850; Kronenberg et al. (2005). 5. Non-viral vectors [0156] In some embodiments, the gene therapy transgene encoding a VHH-SGSH fusion protein described herein may be cloned into a non-viral vector including but not limited to a plasmid, a phagemid, a phage derivative, an bacterial artificial chromosome, or a cosmid. In one embodiment, the gene therapy transgene encoding a VHH-SGSH fusion protein is cloned into a DNA plasmid. Pharmaceutical compositions [0157] In another aspect of the present disclosure, the VHH-SGSH gene therapy construct is provided in a pharmaceutical composition which comprises an aqueous carrier, excipient, diluent or buffer. In some embodiments, the rAAV.VHH-SGSH gene therapy virus formulation is a suspension containing an effective amount of a rAAV.VHH-SGSH vector described herein. Various 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. [0158] For example, a suspension comprising a rAAV.VHH-SGSH 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, or pH 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7., 7.8, 7.9, or 8.0. A suitable surfactant, or combination of surfactants, may be selected from among Poloxamers, i.e.,
Attorney Docket No: JCR-002WO1 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. In one embodiment, the formulation contains a poloxamer. [0159] In some embodiments, a pharmaceutical composition comprising a VHH-SGSH vector of the present disclosure 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. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). [0160] 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. [0161] Optionally, 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. [0162] Formulation of pharmaceutically-acceptable excipients and carrier solutions is well- known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens. Typically, 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. Naturally, 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 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. [0163] 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
Attorney Docket No: JCR-002WO1 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. 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 and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include 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. [0164] For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a suitable sterile aqueous medium may be employed. For example, 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. The person responsible for administration will, in any event, determine the appropriate dose for the individual host. [0165] 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. Generally, 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. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the
Attorney Docket No: JCR-002WO1 active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. [0166] 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. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like. [0167] As used herein, “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. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host. [0168] In addition to recombinant AAV viruses, some recombinant retroviruses may also be used to package the transgene encoding the fusion protein described herein, or the vector for expressing the fusion protein. The recombinant retroviruses may adenoviruses and lentiviruses. [0169] In accordance with the present disclosure, 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. [0170] In particular, 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.
Attorney Docket No: JCR-002WO1 [0171] Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids or the fusion protein constructs disclosed herein. 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). [0172] 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. [0173] 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 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 Å, containing an aqueous solution in the core. [0174] Alternatively, 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 μm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl- cyanoacrylate nanoparticles that meet these requirements are contemplated for use. [0175] In one embodiment, the formulation is suitable for use in human subjects and is administered intravenously. In one embodiment, the formulation is delivered via a peripheral vein by bolus injection. In one embodiment, 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.
Attorney Docket No: JCR-002WO1 [0176] In additional aspects, there are provided pharmaceutical compositions comprising any one of the gene therapy vectors described herein and a pharmaceutically acceptable carrier or excipient for use in treating a genetic disorder. In further aspects, there are provided pharmaceutical composition comprising any one of the nucleic acid constructs provided herein and a pharmaceutically acceptable carrier or excipient for use in preparation of a medicament for treatment of a genetic disorder. In some embodiments, the genetic disorder is a lysosomal storage disorder. In some embodiments, the genetic disorder is Sanfilippo Syndrome type A (MPS IIIA). Applications and Methods of use thereof [0177] In another aspect of the present disclosure, methods of using rAAV.VHH-SGSH transgenes, viral vectors and viral particles described herein for treating a disorder associated with SGSH enzyme deficiency are provided. Accordingly, in some embodiments, the transgenes, rAAV vectors described herein are suitable for treating a subject that has a SGSH deficiency, such as Sanfilippo Syndrome type A (also known as mucopolysaccharidosis type IIIA (MPS IIIA)). MPS IIIA is caused by the genetic defect of N-sulfoglucosamine sulfohydrolase (SGSH) gene, which could cause lysosomal enzyme sulfite enzyme deficiency, leading to accumulation of glycosaminoglycans (GAGs) and heparan sulfate (HS) in various organs of the body, followed by a series of rapid progression of lethal diseases characterized by severe neurological symptoms and skeletal deformities. [0178] The method of treating Sanfilippo Syndrome type A (MPS IIIA) includes administering to the subject in need thereof a VHH-SGSH transgene or a recombinant adeno- associated virus (rAAV) vector expressing the VHH-SGSH transgene as described herein. [0179] A subject having Sanfilippo Syndrome A of any severity is the intended recipient of the compositions and methods described herein. Methods for treating Sanfilippo Syndrome A 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. [0180] In some embodiments, the rAAV vector remains episomal following administration to a subject in need thereof. In some embodiments, the rAAV vector does not remain episomal following administration to a subject in need thereof. For example, in some embodiments, the rAAV vector integrates into the genome of the subject. Such integration can be achieved, for example, by using various gene-editing technologies, such as, zinc finger nucleases (ZFNs),
Attorney Docket No: JCR-002WO1 Transcription activator-like effector nucleases (TALENS), ARCUS genome editing, and/or CRISPR-Cas systems. [0181] In some embodiments, the VHH-SGSH transgenes, rAAV vectors, viral particles and compositions thereof are administered to a subject in need thereof via a suitable route. In some embodiments, the VHH-SGSH transgenes, rAAV vectors, viral particles and compositions thereof are administered by intravenous, intraperitoneal, subcutaneous, intradermal, intrathecal injection, or Intracerebroventricular (ICV) administration. In some embodiments, the VHH- SGSH transgenes, rAAV vectors, viral particles and compositions thereof are administered intravenously (IV). In some embodiments, the VHH-SGSH transgenes, rAAV vectors, viral particles and compositions thereof are administered by intrathecal (IT) injection. In some embodiments, the VHH-SGSH transgenes, rAAV vectors, viral particles and compositions thereof are administered by ICV injection. [0182] In some embodiments, the VHH-SGSH transgenes, rAAV vectors, and compositions thereof are administered via non-viral delivery vehicles, such as lipid nanoparticles. [0183] Upon administration of a gene therapy product that expresses a VHH-SGSH fusion protein described herein, the expression of SGSH or a functional variant thereof is detectable in the serum, liver, brain, lung, kidney, spleen, quadriceps, heart and/or bone marrow of the subject. In some embodiments, the expression of SGSH or a functional variant thereof is detectable in the plasma of the subject. In some embodiments, the expression of SGSH or a functional variant thereof is detectable in the liver of the subject. In some embodiments, the expression of SGSH or a functional variant thereof is detectable in the central nervous system of the subject. Particularly, the expression of SGSH or a functional variant thereof is detectable in the brain of the subject. In some embodiments, the expression of SGSH or a functional variant thereof is detectable in various tissues and organs of the subject at one day, two days, three days, four days, five days, 1 week, 2 weeks, 3 weeks, 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 gene therapy product. In some embodiments, the expression of SGSH or a functional variant thereof is detectable in various tissues and organs of the subject for the remainder of the subject’s life following administration of the gene therapy product. [0184] In some embodiments, the administered gene therapy construct expressing a VHH- SGSH fusion protein results in the production of functional SGSH to the same extent as found following administration of purified SGSH protein delivered intravenously. In some
Attorney Docket No: JCR-002WO1 embodiments, the administered gene therapy construct expressing a VHH- SGSH fusion protein results in production of a greater amount of functional SGSH as compared to administration of purified SGSH protein delivered intravenously. In some embodiments, the administered gene therapy construct expressing a VHH- SGSH fusion protein results in the production of functional SGSH to the same extent as found in a normal subject, i.e., a subject having a normal functional SGSH. [0185] In some embodiments, following administration of the gene therapy construct expressing a VHH- SGSH fusion protein to the subject the levels of functional SGSH detectable in the circulation are between about 2-100 times, or between 2-50 times, or between 2-20 times, or between 10-50 times greater than the amount of functional SGSH detectable in the subject before administration of the construct. [0186] In some embodiments, following the administration of the gene therapy construct expressing a VHH- SGSH fusion protein to the subject, the levels of functional SGSH detectable in the cerebrospinal fluid (CSF) and the brain are between 2-100 times, o between 2-50 times, or between 2-20 times, or between 10-50 times greater than the amount of functional SGSH detectable in the subject before administration of the gene therapy construct. [0187] In some embodiments, following the administration of the gene therapy construct expressing a VHH- SGSH fusion protein to the subject, the levels of functional SGSH detectable in the affected tissues, for example, liver, spleen, kidney, heart, lung, bone marrow, muscle, are between about 2-100 times, or between 2-50 times, or between 2-20 times, or between 10-50 times greater than the amount of functional SGSH detectable in the subject before administration of the gene therapy construct. [0188] Use of a transgene encoding a VHH-SGSH fusion protein described here facilitates penetration of the gene therapy construct across the blood-brain barrier (BBB). The VHH antibody binds to a transferrin receptor present on the BBB that allows entry in the CNS through receptor-mediated transcytosis. [0189] In some embodiments, following the administration of the gene therapy construct expressing a VHH- SGSH fusion protein to the subject, the enzymatic activity of SGSH (sulfamidase) is recovered in various tissues and organs, for example, brain, liver, spleen, kidney, heart, lung, bone marrow, and muscle. In some embodiments, about 5–100% of enzyme activity can be recovered. In some embodiments, about 5%, 10%, 15%.20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% of enzyme activity is recovered. In some embodiments, about 5%, 10%, 15%.20%, 25%, 30%, 35%, 40%, 45%, 60%, 70%, 80%, 90%,
Attorney Docket No: JCR-002WO1 or 100% of enzyme activity is recovered in the brain of the subject. In some embodiments, about 5%, 10%, 15%.20%, 25%, 30%, 35%, 40%, 45%, 60%, 70%, 80%, 90%, or 100% of enzyme activity is recovered in the central nervous system of the subject. [0190] In some embodiments, the enzyme activity is increased to a therapeutic level. For example, the enzyme activity is at least about 5%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, more than 100%, about 2-fold, about 3-fold, or about 5-fold of a healthy control. [0191] The enzymatic activity of sulfamidase includes the breakdown of heparan sulfate (HS). In patients with MPS IIIA, accumulations of HS in the lysosome cause progressive neurodegeneration with subsequent mental decline and a shortened lifespan. [0192] In some embodiments, such therapeutic levels of SGSH may result in alleviation of the MPS III-A related symptoms; improvement of MPS IIIA-related biomarkers of disease; or facilitation of other treatment(s) for MPS IIIA, e.g., HS levels in the cerebrospinal fluid (CSF), serum, urine and/or other biological samples; prevention of neurocognitive decline; reversal of certain MPS IIIA-related symptoms and/or prevention of progression of MPS IIIA-related certain symptoms; or any combination thereof. [0193] In some embodiments, the administration of a therapy construct expressing a VHH- SGSH fusion protein described herein results in the reduction of heparan sulfate (HS) (a glycosaminoglycan (GAG)) in the subject. [0194] In some embodiments, the administered gene therapy construct expressing a VHH- SGSH fusion protein described herein reduces the HS accumulation 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 HS levels prior to the treatment. In some embodiments, the accumulated HS is reduced in the treated subject by about 95%. In some embodiments, the accumulated HS is reduced in the treated subject by about 90%. In some embodiments, the accumulated HS is reduced in the treated subject by about 85%. In some embodiments, the accumulated HS is reduced in the treated subject by about 80%. In some embodiments, the accumulated HS is reduced in the treated subject by about 75%. In some embodiments, the accumulated HS is reduced in the treated subject by about 70%. In some embodiments, the accumulated HS is reduced in the treated subject by about 65%. In some embodiments, the accumulated HS is reduced in the treated subject by about 60%. In some embodiments, the accumulated HS is reduced in the treated subject by about 55%. In some
Attorney Docket No: JCR-002WO1 embodiments, the accumulated HS is reduced in the treated subject by about 50%. In some embodiments, the accumulated HS is reduced in the treated subject by about 45%. In some embodiments, the accumulated HS is reduced in the treated subject by about 40%. In some embodiments, the accumulated HS is reduced in the treated subject by about 35%. In some embodiments, the accumulated HS is reduced in the treated subject by about 30%. In some embodiments, the accumulated HS is reduced in the treated subject by about 25%. In some embodiments, the accumulated HS is reduced in the treated subject by about 20%. In some embodiments, the accumulated HS is reduced in the treated subject by about 15%. In some embodiments, the accumulated HS is reduced in the treated subject by about 10%. [0195] In some embodiments, the accumulated HS is reduced in the serum, liver, brain, lung, kidney, spleen, quadriceps, heart and/or bone marrow of the treated subject. In some embodiments, the accumulated HS is reduced in the serum of the treated subject. In some embodiments, the accumulated HS is reduced in the liver of the treated subject. In some embodiments, the accumulated HS is reduced in the central nervous system of the treated subject. Particularly, the accumulated HS is reduced in the brain of the subject. In some cases, the accumulated HS is reduced in the cerebrospinal fluid (CSF) of the treated subject. [0196] In some embodiments, the administered gene therapy construct expressing a VHH- SGSH fusion protein described herein reduces the HS accumulation in the brain of the treated 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 HS levels in the brain prior to the treatment. In some embodiments, the administered gene therapy construct expressing a VHH- SGSH fusion protein described herein reduces the HS accumulation in the cerebrospinal fluid of the treated 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 HS levels in the CSF prior to the treatment. [0197] In some embodiments, the administered gene therapy construct expressing a VHH- SGSH fusion protein described herein results in reduction of lysosomal storage stress in the treated subject. [0198] In some embodiments, the administered gene therapy construct expressing a VHH- SGSH fusion protein described herein results in reduction of neuro-inflammation in the subject. [0199] In Accordance. One or more symptoms of MPS IIIA in a treated patient are improved. For example, improvements in behavior, attention, and sleep of the treated patients are observed.
Attorney Docket No: JCR-002WO1 EXAMPLES [0200] Other features, objects, and advantages of the present invention are apparent in the examples that follow. It should be understood, however, that the examples, while indicating embodiments of the present invention, are given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the examples. Example 1: Design and generation of rAAV therapeutic vectors [0201] This example relates to the design of a recombinant adeno-associated virus (rAAV) vector encoding a human sulfamidase (SGSH) protein described herein. rAAV VHH-SGSH or rAAV SGSH-VHH vector design [0202] A schematic that illustrates exemplary rAAV vectors of the present disclosure is illustrated in Figure 1. As shown in Figure 1, the key components of a rAAV vector of the present disclosure comprises 5‘ inverted terminal repeat (ITR), a promoter, a transgene encoding a fusion protein comprising an anti-human transferrin receptor (anti-hTfR1) VHH antibody fused to a human sulfamidase (SGSH), a WPRE 3’ regulatory sequence, a polyadenylation (polyA) signal, and a 3’ ITR sequences flanking the transgene coding sequence (CDS). The transgene CDS encoding the anti-hTfR1 VHH-SGSH or SGSH-VHH fusion proteins comprise a nucleic acid sequence encoding a signal peptide, the anti-TfR1 VHH antibody, a linker peptide, and the human SGSH. The nucleic acid sequence encoding anti-hTfR1 VHH can be a wild-type or a codon-optimized variant. The nucleic acid sequence encoding the human SGSH can be a wild-type or a codon-optimized variant. [0203] To demonstrate the receptor-mediated transcytosis across the blood-brain barrier, a liver-specific enhancer-promoter combination was used, with or without an MVM intron, to drive the expression of VHH fused to SGSH CDS at the N-terminus (VHH-SGSH) or C- terminus (SGSH-VHH). The fusion is separated by a linker peptide. A 3’ regulatory element, WPRE, for enhancing mRNA transcript stability was appended between the stop codon and a synthetic polyA sequence. [0204] Exemplary methods and designs of generating rAAV expression constructs (rAAV vectors) comprising coding sequences for a fusion protein comprising at least one anti-hTfR1 VHH antibody fused to a human SGSH and variations thereof are provided in this example. In this study, recombinant AAV vector (rAAV9) was used. The basic design of a rAAV vector
Attorney Docket No: JCR-002WO1 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. Typically, an expression cassette contains a promoter, a coding sequence, a polyA tail and/or a tag, as shown in Figure 1. An expression construct encoding human SGSH was designed and prepared using standard molecular biology techniques. The coding sequence for the human SGSH was inserted downstream of a promoter, which can be liver-specific. Additionally, liver-specific enhancers or cis-acting regulatory modules (CRM) were 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. Furthermore, the WPRE sequence was inserted downstream of the coding region, after the stop codon but before the polyA tail. Without wishing to be bound by theory, this element creates a tertiary structure that increases the mRNA stability. Other mechanisms of function have been described for WPRE including, for example, improving transcript termination and facilitating mRNA nuclear export. Figure 1 shows schematic representations of the expression constructs described above. The expression construct 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 Additionally, the coding sequences for anti-hTfR1 VHH were codon-optimized based on multiple parameters, such as codon adaptation index (CAI), individual codon usage and codon context (ICU-CC), in addition to variables that negatively impact the open reading frame in terms of protein product levels: 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. The characteristics of the optimized VHH coding sequences are summarized in Table 4, and the schematics for the representative constructs of VHH-SGSH 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
Attorney Docket No: JCR-002WO1 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 VHH 1 optimized sequences Design GC (%) CpG count SEQ ID NO: 8 ICU-CC 57.06 5 SEQ ID NO: 9 ICU-CC 56.78 5 SEQ ID NO: 10 CAI 56.50 5 Example 2: In vivo evaluation of rAAV.VHH-SGSH and rAAV.SGSH-VHH vectors Materials and methods [0205] Plasmids: Plasmids expressing the CDS for human sulfamidase (SGSH) with and without an anti-hTfR1 receptor binding single domain antibody (VHH) tag under a ubiquitous promoter were tested first in Huh7 cells via transfection and then in a mouse model of MPS IIIA disease. rAAV SGSH-VHH expressed SGSH tagged with VHH at the C terminus while rAAV VHH-SGSH expressed SGSH with a N-terminal VHH tag. pSGSH expressed untagged SGSH. [0206] Animals: Mus musculus, Sgsh (D31N)hTFRC/mTfrc model harbors the hTFRC knock-in to the native mouse Tfrc locus in the disease background, Sgsh (D31N), “SanA mouse model”, was used in the study. Mice were acclimated 6-7 days before injecting with test articles. Wild type C57Bl6 animals were used as controls. In vivo studies [0207] Sgsh (D31N)hTFRC/mTfrc mice at 12-13 weeks old were administered with AAV vector test articles according to Table 5 intravenously through the tail vein. A group of Sgsh (D31N)hTFRC/mTfrc mice and one group of WT mice were injected with AAV formulation buffer only as controls. The animals were sacrificed at 4 weeks post injection. Cerebrospinal fluid (CSF) and serum were collected at termination and tissues such as the brain, liver, kidney, spleen heart and bone marrow were collected after perfusion with PBS. Samples were snap frozen and stored at -80oC. Cerebrospinal fluid (CSF), serum and tissue samples were analyzed for SGSH concentrations and HS-GAGs levels. Table 5: Experimental Design
Attorney Docket No: JCR-002WO1 Number of Group No. Genotype Test article Dose level (vg/kg) animals (Animal number) 1 rAAV9-TJ018 (LSP-VHH-SGSH) 1E+12 8 (01-08) 2 rAAV9-TJ020 (LSP-SGSH-VHH) 1E+12 8 (09-16) 3 rAAV9-TJ035 (LSP-intron-VHH- 1E+12 SGSH) 8 (17-24) 4 rAAV9-TJ036 (LSP-intron-SGSH- 1E+12 VHH) 8 (25-32) 5 rAAV9-TJ013 (LSP-SGSH) 1E+12 8 (33-40) 6 Sgsh rAAV9-TJ018 (LSP-VHH-SGSH) 1E+13 8 (41-48) (D31N)hTFRC/mTfrc 7 rAAV9-TJ020 (LSP-SGSH-VHH) 1E+13 8 (49-56) 8 rAAV9-TJ035 (LSP-intron-VHH- 1E+13 8 (57-64) SGSH) 9 rAAV9-TJ036 (LSP-intron-SGSH- 1E+13 8 (65-72) VHH) 10 rAAV9-TJ013 (LSP-SGSH) 1E+13 8 (73-80) 11 Vehicle - 8 (81-88) 12 WT Vehicle - 8 (89-96) SGSH ELISA [0208] Immuno-quantification of SGSH is performed using electrochemiluminescence method. The assay plate is blocked and coated with mouse anti-human SGSH monoclonal antibody conjugated with biotin at 25°C for 60 ± 5 minutes with shaking (500 rpm). Each plate well is washed three times with PBS-Tween 20 before adding serum, CSF or tissue lysate samples and incubated at 25°C for 60 ± 5 minutes with shaking. The detection antibody solution containing rabbit polyclonal anti-SGSH antibodies conjugated with SULFO tag is added after the plate has been washed three times. Last incubation is carried out at 25°C for 60 ± 5 minutes with shaking, then washed and read with an electrochemiluminescence plate reader after the reading buffer T (Meso Scale diagnostics, Rockville, MD, USA) has been added. [0209] Systemic intravenous delivery of all AAV vector test articles that encode an anti- TfR1 VHH tag showed a dose-dependent brain exposure of the SGSH protein, whereas the vectors that encode the untagged SGSH (rAAV9-TJ013) did not show any CNS detection of protein at either vector doses (Figure 2). Using vectors wherein the SGSH expression was driven by a liver-specific promoter, the observed CNS exposure clearly demonstrated that the anti-TfR1 VHH tag facilitated the brain penetration of SGSH proteins, whether the VHH fusion is VHH-SGSH (N-terminal, TJ018, TJ035) or SGSH-VHH (C-terminal TJ020, TJ036). Serum transgene protein level was also detected in a dose-response manner (Figure 3). Mice that
Attorney Docket No: JCR-002WO1 received vectors encoding the N-terminal fusions (TJ018, TJ035) at showed SGSH levels that were higher than those that received the vectors with C-terminal fusion (TJ020, TJ036). Constructs with intron (TJ035 and TJ036) showed a lower SGSH exposure in serum and brain than the corresponding construct without an intron as shown in Figure 2 and Figure 3. SGSH Activity Assay [0210] SGSH activity is measured by combining 10 µl of sample (tissue lysate) with 20 µl of 1mM 4MUGlcNS in Michaelis' barbital CH3COONa buffer (29 mM sodium barbital, 29mM CH3COONa, and 0.68% (w/v) NaCl; pH 6.5) and incubating the mixture at 37°C for 17 h before addition of 16µl of PiCi buffer (400 mL of milliQ H2O, 10.36 g of Sodium Phosphate Dibasic, 2.3 g Citric Acid, pH to 6.5) with a-glucosidase 500 U/mL in H2O). Samples are incubated for a further 5 h at 37°C before the reaction is stopped with the addition of 0.2 mL of glycine buffer (0.5M Na2CO3/NaHCO30.025% Triton X-100; pH 10.7). The samples are aliquoted into black microtiter plates and fluorescence is determined using a Spectramax M3 reader with an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Enzyme activity is determined by relating the fluorescence of the sample to that of a known concentration of 4MU and expressed in nmol/mg protein. Total protein concentration is quantified by bi-cinchonicic acid (BCA) assay. HS-GAG Quantification by LC-MS-MS [0211] Methods for quantifying HS and DS concentrations were discussed by 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; 2018, 125: 53-58; the contents are incorporated herein by reference). In general, tissues were homogenized in lysis buffer and centrifuged to collect the supernatant as tissue extracts. The extracts were subjected to acidic methanolysis by addition of 2,2- dimethoxypropane and anhydrous 3M HCl-methanol. The reaction was stopped by addition of 10% ammonium carbonate. After mixing with HS- and DS internal standards, the solvent was evaporated under nitrogen gas and each residue was then suspended in water and subjected to LC/MS/MS analysis. [0212] Figure 4 and Figure 5 demonstrate the HS-GAG levels in CSF and brain, respectively. All vectors encoding VHH fusion proteins with SGSH (TJ018, TJ029, TJ035, TJ036) reduced HS-GAG accumulation significantly compared to the disease control. All mice
Attorney Docket No: JCR-002WO1 treated with vectors that encode untagged SGSH (rAAV9-TJ013) did not result in clearance of HS-GAG level in CSF or brain. At 1e12 vg/kg dose, rAAV9-TJ036 which encodes an intron and a liver-specific promoter driving the expression of SGSH-VHH C-terminal fusion protein cleared HS-GAG in CSF and brain less robustly, though still effectively, as the other VHH- tagged test articles (rAAV9-TJ018, rAAV9-TJ029, rAAV9-TJ035). EQUIVALENTS AND SCOPE [0213] 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 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: