EP4237082A1 - Anticorps vectorisés anti-cgrp et anti-récepteur de cgrp et leur administration - Google Patents

Anticorps vectorisés anti-cgrp et anti-récepteur de cgrp et leur administration

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Publication number
EP4237082A1
EP4237082A1 EP21811651.5A EP21811651A EP4237082A1 EP 4237082 A1 EP4237082 A1 EP 4237082A1 EP 21811651 A EP21811651 A EP 21811651A EP 4237082 A1 EP4237082 A1 EP 4237082A1
Authority
EP
European Patent Office
Prior art keywords
seq
serotype
aav
transgene
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21811651.5A
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German (de)
English (en)
Inventor
Jared Smith
Bradley HOLLIDGE
Joseph Bruder
Ye Liu
Randolph QIAN
Chunping Qiao
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Regenxbio Inc
Original Assignee
Regenxbio Inc
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Publication date
Application filed by Regenxbio Inc filed Critical Regenxbio Inc
Priority claimed from PCT/US2021/057155 external-priority patent/WO2022094157A1/fr
Publication of EP4237082A1 publication Critical patent/EP4237082A1/fr
Pending legal-status Critical Current

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/06—Antimigraine agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14145—Special targeting system for viral vectors

Definitions

  • compositions and methods are described for the delivery of a fully human post- translationally modified (HuPTM) therapeutic monoclonal antibody (“mAb”) that binds to CGRP or CGRPR or the HuPTM antigen-binding fragment of a therapeutic mAb that binds to CGRP or CGRPR — e.g., a fully human-glycosylated (HuGly) Fab of the therapeutic mAb — to a human subject diagnosed with a disease or condition indicated for treatment with the therapeutic mAb.
  • diseases include migraine and cluster headaches.
  • Co-delivery of two HuPTM therapeutic mAbs targeting anti- CGRP and anti-CGRPR HuPTM mAb is also disclosed.
  • Therapeutic mAbs have been shown to be effective in treating a number of diseases and conditions. However, because these agents are effective for only a short period of time, repeated injections for long durations are often required, thereby creating considerable treatment burden for patients. Treatments that interfere with the functioning of CGRP in the peripheral trigeminal system are effective against migraine. Blocking sensitization of the trigeminal nerve by attenuating CGRP activity in the periphery may be sufficient to block a migraine attack.
  • Therapeutic antibodies that bind to calcitonin gene-related peptide (CGRP) or its receptor (CGRPR) may be used for preventive treatment of migraine and cluster headaches.
  • CGRP calcitonin gene-related peptide
  • CGRPR its receptor
  • erenumab is approved for the treatment of migraine and three anti-CGRP antibodies, fremanezumab (AJOVY®), eptinezumab (VYETPI®), and galcanezumab (EMGALITY®), are also approved for the preventive treatment for migraine.
  • Galcanezumab is also approved for the treatment of episodic cluster headaches.
  • the recommended dosage of erenumab (AIMOVIG®) is 70 mg injected subcutaneously once monthly. Some patients can benefit from a dosage of 140 mg injected subcutaneously once monthly, which is administered as two consecutive subcutaneous injections of 70 mg each.
  • Therapeutic antibodies delivered by gene therapy have several advantages over inj ected or infused therapeutic antibodies that dissipate over time resulting in peak and trough levels. Sustained expression of the transgene product antibody, as opposed to injecting an antibody repeatedly, allows for a more consistent level of antibody to be present at the site of action, and is less risky and more convenient for patients, since fewer injections need to be made. Furthermore, antibodies expressed from transgenes are post-translationally modified in a different manner than those that are directly inj ected because of the different microenvironment present during and after translation.
  • compositions and methods for anti-CGRP and anti-CGRPR gene therapy designed to target the CNS, particularly arterial smooth muscle cells and/or the trigeminal ganglion (TG), and may also generate a depot in the liver, muscle, or liver and muscle, of transgenes for expression of anti-CGRP or anti-CGRPR antibodies that cross the blood-brain barrier, particularly erenumab, eptinezumab, fremanezumab, and galcanezumab, or an antigen binding fragment thereof, that result in a therapeutic or prophylactic serum levels of the antibody within 20 days, 30 days, 40 days, 50 days, 60 days, or 90 days of administration of the rAAV composition.
  • Serum levels include 2 to 20 pg/ml antibody for an anti-CGRPR antibody, particularly, erenumab, or an antigen binding fragment thereof.
  • the levels of antibody achieved are sufficient to lead to an at least 10%, 20%, 50%, 70% or 90% reduction in headache days per month from a baseline, or a reduction in at least 1, 2, 3, or 4 headache days per month from baseline.
  • compositions and methods are described for the systemic delivery of an anti-CGRP or anti-CGRPR HuPTM mAb or an anti-CGRP or anti-CGRPR HuPTM antigen-binding fragment of a therapeutic mAb (for example, a fully human-glycosylated Fab (HuGlyFab) of a therapeutic mAb, to a patient (human subject) diagnosed with migraine, including episodic and chronic migraine, or cluster headaches or other conditions indicated for treatment with the therapeutic anti-CGRP or anti-CGRPR mAb.
  • a therapeutic mAb for example, a fully human-glycosylated Fab (HuGlyFab) of a therapeutic mAb
  • Such antigen-binding fragments of therapeutic mAbs include a Fab, F(ab')2, or scFv (singlechain variable fragment) (collectively referred to herein as “antigen-binding fragment”).
  • “HuPTM Fab” as used herein may include other antigen binding fragments of a mAb.
  • full-length mAbs can be used.
  • compositions comprising and methods of administering a rAAV that encodes both an anti-CGRP antibody, particularly a Fab, and an anti- CGRPR antibody, particularly a Fab, particularly in which the anti-CGRP antibody and the anti- CGRPR antibody are under the control of different regulatory sequences that direct expression in different tissue types, or a combination of an rAAV encoding an anti-CGRP antibody and an rAAV encoding an anti-CGRP receptor antibody.
  • Delivery may be advantageously accomplished via gene therapy — e.g., by administering a viral vector or other DNA expression construct encoding a therapeutic anti-CGRP and/or anti-CGRPR mAb or its antigen-binding fragment (or a hyperglycosylated derivative of either) diagnosed with a condition indicated for treatment with the therapeutic anti-CGRP or anti-CGRPR mAb — to create a permanent depot in CNS, PNS, arterial smooth muscle, muscle and/or liver cells, of the patient that continuously supplies the HuPTM mAb or antigen-binding fragment of the therapeutic mAb, e.g., a human-glycosylated transgene product, or peptide to dural vessels and/or the trigeminal ganglion, or generally to the circulation or CNS, of the subject where the mAb or antigen-binding fragment thereof or peptide exerts its therapeutic or prophylactic effect.
  • a viral vector or other DNA expression construct encoding a therapeutic anti-CGRP and/or anti-
  • gene therapy vectors particularly rAAV gene therapy vectors, which when administered to a human subject result in expression of an anti-CGRP or anti-CGRPR antibody to achieve a maximum or steady state serum concentration (for example, 20, 30, 40, 50, 60 or 90 days after administration) of 2 pg/ml to 20 pg/ml (or, 2 pg/ml to 10 pg/ml, or 5 pg/ml to 15 pg/ml, or 10 pg/ml to 20 pg/ml) anti-CGRP or anti-CGRPR antibody (including erenumab).
  • a maximum or steady state serum concentration for example, 20, 30, 40, 50, 60 or 90 days after administration
  • 2 pg/ml to 20 pg/ml or, 2 pg/ml to 10 pg/ml, or 5 pg/ml to 15 pg/ml, or 10 pg/ml to 20 pg/ml
  • gene therapy vectors particularly rAAV gene therapy vectors, which when administered to a human subject result in expression of an anti-CGRP or anti-CGRPR antibody to achieve a maximum or steady state serum concentration (for example, 20, 30, 40, 50, 60 or 90 days after administration) of 2 pg/ml to 20 pg/ml (or, 2 pg/ml to 10 pg/ml, or 5 pg/ml to 15 pg/ml, or 10 pg/ml to 20 pg/ml).
  • a maximum or steady state serum concentration for example, 20, 30, 40, 50, 60 or 90 days after administration
  • Methods include a method of treating migraine and/or cluster headaches in a human subject in need thereof, comprising intravenously or intramuscularly administering to the subject a dose of a composition comprising a recombinant AAV comprising a transgene encoding an antibody selected from fremanezumab, eptinezumab, galcanezumab or erenumab or an antigen binding protein comprising a heavy chain variable region, a light chain variable region and optionally an Fc domain of the antibody or an antigen binding fragment thereof, operably linked to one or more regulatory sequences that control expression of the transgene in liver and/or muscle cells, in an amount sufficient to result in expression from the transgene and secretion of the antibody, or the antigen binding protein or the antigen binding fragment thereof into the bloodstream of the human subj ect to produce antibody or the antigen binding protein or antigen binding fragment thereof, plasma levels of at least 1.5 pg/ml to 35 pg/ml antibody or the antigen binding protein
  • administration of a therapeutically effective amount of the anti-CGRPR or anti-CGRP mAb, or antigen-binding fragment thereof is determined to be sufficient to reduce nausea, light sensitivity, sound sensitivity, red eye, eyelid edema, forehead and facial sweating, tearing (lacrimation), abnormal small size of the pupil (miosis), nasal congestion, runny nose (rhinorrhea), and drooping eyelid (ptosis).
  • administration of a therapeutically effective amount of the anti-CGRPR or anti-CGRP mAb, or antigen-binding fragment thereof is determined to be sufficient to reduce the intensity or frequency of migraines or cluster headaches, or a reduction in the amount of acute migraine-specific medication used over a defined period of time.
  • the recombinant vector used for delivering the transgene includes non -replicating recombinant adeno-associated virus vectors (“rAAV”).
  • the AAV type has a tropism for CNS, PNS, arterial smooth muscle, muscle and/or liver cells, for example an AAV8, AAV9, AAV.PHP.eB, AAVrhlO, AAVhu.32, AAV3B, AAVrh46, AAVrh73, AAVS3, AAV-LK03, AAVhu.51, AAVhu.21, AAVhu.12 or AAVhu.26 serotype of AAV.
  • viral vectors including but not limited to lentiviral vectors; vaccinia viral vectors, or non-viral expression vectors referred to as “naked DNA” constructs.
  • Expression of the transgene can be controlled by constitutive or tissue-specific expression control elements, particularly elements that are smooth muscle cellspecific control elements, for example one or more elements of Table 1. Regulatory elements include the C AG promoter, LMTP6 promoter or LMTP24 promoter.
  • the HuPTM mAh or HuPTM antigen-binding fragment encoded by the transgene can include, but is not limited to, a full-length or an antigen-binding fragment of a therapeutic antibody that binds to CGRP or CGRPR, particularly erenumab, eptinezumab, fremanezumab, and galcanezumab, see, for example FIGS. 2A-2D with exemplary transgene construct antibody products for Fab fragments of these antibodies.
  • Gene therapy constructs for the therapeutic antibodies are designed such that both the heavy and light chains are expressed.
  • the coding sequences for the heavy and light chains can be engineered in a single construct in which the heavy and light chains are separated by a cleavable linker or IRES so that separate heavy and light chain polypeptides are expressed.
  • the linker is a Furin T2A linker (SEQ ID NOS: 87 or 88).
  • the coding sequences encode for a Fab or F(ab’)2 or an scFv.
  • the full length heavy and light chains of the antibody are expressed.
  • Gene therapy constructs are also designed such that the construct encodes both an anti -CGRP antibody, particularly a Fab, and an anti-CGRPR antibody, particularly a Fab and, in particular, each operably linked and under the control of different tissue specific promoters such that the anti-CGRP antibody is expressed in a different set of cells from the ant-CGRPR antibody.
  • antibodies expressed from transgenes in vivo are not likely to contain degradation products associated with antibodies produced by recombinant technologies, such as protein aggregation and protein oxidation. Aggregation is an issue associated with protein production and storage due to high protein concentration, surface interaction with manufacturing equipment and containers, and purification with certain buffer systems. These conditions, which promote aggregation, do not exist in transgene expression in gene therapy. Oxidation, such as methionine, tryptophan, and histidine oxidation, is also associated with protein production and storage, and is caused by stressed cell culture conditions, metal and air contact, and impurities in buffers and excipients. The proteins expressed from transgenes in vivo may also oxidize in a stressed condition.
  • HuPTM mAh or HuPTM Fab in the CNS, PNS, arterial smooth muscle cells, and/or liver cells, particularly smooth muscle cells of the dura, of the human subject should result in a “biobetter” molecule for the treatment of disease accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding a full-length HuPTM mAb or HuPTM Fab of a therapeutic mAb to a patient (human subject) diagnosed with a disease indication for that mAb, to create a permanent depot in the subject that continuously supplies the human-glycosylated, sulfated transgene product produced by the subj ect’ s transduced cells.
  • the cDNA construct for the HuPTMmAb or HuPTM Fab should include a signal peptide that ensures proper co- and post-translational processing (glycosylation and protein sulfation) by the transduced human cells.
  • the full-length HuPTM mAb or HuPTM Fab can be produced in human cell lines by recombinant DNA technology, and the glycoprotein can be administered to patients.
  • Dual vector therapy involving systemic delivery of two viral vectors, wherein the first vector expresses as anti-CGRPR antibody or antigen-binding fragment thereof, and the second vector expresses an anti-CGRP antibody or antigen-binding fragment, to a patient in need thereof are encompassed by the methods provided herein.
  • the viral vectors may be the same or different serotypes, for example, an AAV9 serotype and an AAV8 serotype.
  • Combination therapy involving systemic (including IV or IM) or intranasal delivery of the full-length HuPTM anti-CGRPR or anti-CGRP antibody, or an binding-fragment thereof, to the patient accompanied by administration of other available treatments are also encompassed by the methods provided herein.
  • the additional treatments may be administered before, concurrently or subsequent to the gene therapy treatment.
  • Such additional treatments can include but are not limited to co-therapy with the therapeutic mAb.
  • kits for manufacturing the viral vectors particularly the AAV based viral vectors.
  • methods of producing recombinant AAVs comprising culturing a host cell containing an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a transgene encoding a therapeutic antibody operably linked to expression control elements that will control expression of the transgene in human cells; a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and capsid protein operably linked to expression control elements that drive expression of the AAV rep and capsid proteins in the host cell in culture and supply the rep and cap proteins in trans; sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid proteins; and recovering recombinant AAV encapsidating the artificial genome from the cell culture.
  • a pharmaceutical composition for treating migraine or cluster headaches in a human subject in need thereof comprising an adeno-associated virus (AAV) vector having:
  • a viral capsid that has a tropism for CNS, PNS, arterial smooth muscle, skeletal muscle and/or liver cells;
  • an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding a heavy chain and a light chain of a substantially full-length or full-length anti-CGRP or anti-CGRPR antibody, or an antigen-binding fragment thereof, operably linked to one or more regulatory sequences that promote expression of the transgene in human CNS, PNS, arterial smooth muscle, skeletal muscle and/or liver cells; wherein said AAV vector is formulated for intranasal or systemic administration to said human subject.
  • ITRs AAV inverted terminal repeats
  • a pharmaceutical composition for treating migraine or cluster headaches in a human subject in need thereof comprising a first adeno-associated virus (AAV) vector and a second AAV vector, wherein each said first and second AAV vector comprises:
  • a viral capsid that has a tropism for CNS, PNS, arterial smooth muscle, skeletal muscle and/or liver cells; and (b) an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette in said first AAV vector comprises a transgene encoding a heavy chain and a light chain of a substantially full-length or full- length anti-CGRP mAb, or an antigen-binding fragment thereof, operably linked to one or more regulatory sequences that promote expression of the transgene in human CNS, PNS, arterial smooth muscle, skeletal muscle and/or liver cells and the expression cassette in said second AAV vector comprises a transgene encoding a heavy chain and a light chain of a substantially full-length or full-length anti-CGRP receptor mAb, or an antigen-binding fragment thereof, operably linked to one or more regulatory sequences that promote expression of the transgene in human CNS, PNS, arterial smooth muscle, skeletal muscle and/or liver cells
  • a viral capsid that has a tropism for CNS, PNS, arterial smooth muscle, skeletal muscle and/or liver cells;
  • an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a first and a second transgene, wherein the first transgene encodes a heavy chain and a light chain of an antigen-binding fragment of an anti-CGRP operably linked to a first regulatory sequence, and the second transgene encodes a heavy and light chain of an antigen binding fragment of an anti-CGRPR antibody, operably linked to a second regulatory sequence, wherein said first and second regulatory sequences promote expression of the transgene in human CNS, PNS, arterial smooth muscle, skeletal muscle and/or liver cells, and the first and second regulatory sequences promote expression of the first and second transgenes in different human cell types; wherein said AAV vector is formulated for intranasal or systemic administration to said human subject.
  • ITRs AAV inverted terminal repeats
  • the viral capsid is at least 95% identical to the amino acid sequence of AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 3B (AAV3B), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype rh34 (AAVrh34), serotype hu.32 (AAVhu.32), serotype hu.37 (AAVhu.37), serotype hu.60 (AAVrh73), serotype
  • the pharmaceutical composition of paragraph 8 wherein the regulatory sequence is a human smooth muscle protein 22 alpha (sma22a) promoter (SEQ ID NOS: 184 or 185-190), a CAG promoter (SEQ ID NO:25), a human synapsin 1 gene (hSyn) promoter (SEQ ID NO: 191-195) a LMTP6 promoter (SEQ ID NO: 159) or a LMTP24 promoter (SEQ ID NO: 263).
  • the transgene comprises a Furin/2A linker between the nucleotide sequences coding for the heavy and light chains of said mAh or antigen-binding fragment thereof.
  • the pharmaceutical composition of paragraph 10 wherein said Furin 2A linker is a Furin/T2A linker having the amino acid sequence RKRR(GSG)APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NOS: 87 or 88).
  • said signal sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 28) or a signal sequence from Tables 2, 3, or 4.
  • transgene has the structure: signal sequence- Heavy chain - Furin site - 2A site - signal sequence- Light chain - PolyA.
  • the full-length mAb or the antigen-binding fragment comprises a heavy chain with an amino acid sequence of SEQ ID NO: 1 and optionally an Fc polypeptide with an amino acid sequence of SEQ ID NO: 17 and a light chain with an amino acid sequence of SEQ ID NO: 2; a heavy chain with an amino acid sequence of SEQ ID NO: 3 and optionally an Fc polypeptide with an amino acid sequence of SEQ ID NO:22 and a light chain with an amino acid sequence of SEQ ID NO: 4; a heavy chain with an amino acid sequence of SEQ ID NO: 5 and optionally an Fc polypeptide with an amino acid sequence of SEQ ID NO:23 and a light chain with an amino acid sequence of SEQ ID NO: 6; or a heavy chain with an amino acid sequence of SEQ ID NO: 7 and optionally an Fc polypeptide with an amino acid sequence of SEQ ID NO: 20 and a light chain with an amino acid sequence of SEQ ID NO: 8.
  • the transgene comprises a nucleotide sequence of SEQ ID NO: 9 encoding the heavy chain and a nucleotide sequence of SEQ ID NO: 10 encoding the light chain; a nucleotide sequence of SEQ ID NO: 11 encoding the heavy chain and a nucleotide sequence of SEQ ID NO: 12 encoding the light chain; a nucleotide sequence of SEQ ID NO: 13 encoding the heavy chain and a nucleotide sequence of SEQ ID NO: 14 encoding the light chain, or a nucleotide sequence of SEQ ID NO: 15 encoding the heavy chain and a nucleotide sequence of SEQ ID NO: 16 encoding the light chain.
  • transgene comprises a nucleotide sequence that is codon optimized and/or deleted for CpG sequences.
  • the pharmaceutical composition of paragraphs 18, 19 or 20 wherein the artificial genome comprises the nucleotide sequence of pAAV.CAG.erenumab (SEQ ID NO: 268 or 268), pAAV.LMTP6.VH4i.erenumab.T2A (SEQ ID NO: 270 or 271), pAAVLMTP24.VH4i.erenumab.T2A (SEQ ID NO: 272 or 273), pAAV.CAG.fremanezumab (SEQ ID NO: 275 or 276), pAAV.LMTP6.VH4.fremanezumab.T2A (SEQ ID NO: 277 or 278), pAAVLMTP24.VH4i.felumab.T2A (SEQ ID NO: 279 or 280), pAAV.CAG.
  • galcanezumab (SEQ ID NO: 282 or 283), pAAV.LMTP6.VH4i.galcanezumab.T2A (SEQ ID NO: 284 or 285), pAAVLMTP24.VH4i. galcanezumab. T2 A (SEQ ID NO: 286 or 287), pAAV.CAG. eptinezumab (SEQ ID NO: 289 or 290), pAAV.LMTP6.VH4i.eptinezumab.T2A (SEQ ID NO: 291 or 292), or pAAVLMTP24.VH4i. eptinezumab. T2A (SEQ ID NO: 293 or 294).
  • compositions 1 to 20 wherein the antigen-binding fragment is a Fab, a F(ab’)2, or an scFv.
  • AAV adeno-associated virus
  • AAV capsid that is optionally at least 95% identical to the amino acid sequence of AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 3B (AAV3B), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype rh34 (AAVrh34), serotype hu.37 (AAVhu.37), serotype hu.60 (AAVrh60), serotype rh21 (AAVrh21), serotype rhl5 (AAVr
  • an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding a heavy and a light chain of a substantially full-length or full-length anti-CGRP or anti-CGRPR mAb, or an antigen-binding fragment thereof, operably linked to one or more regulatory sequences that promote expression of the transgene in human CNS, PNS, arterial smooth muscle and/or liver tissue cells; c. wherein the transgene encodes a signal sequence at the N-terminus of the heavy chain and the light chain of said mAb that directs secretion and post translational modification of said mAb in CNS, PNS, liver, and/or arterial smooth muscle tissue cells.
  • ITRs AAV inverted terminal repeats
  • a composition comprising a first AAV vector and a second AAV vector, wherein each AAV vector comprises: a) a viral AAV capsid, that is optionally at least 95% identical to the amino acid sequence of AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 3B (AAV3B), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype rh34 (AAVrh34), serotype hu.32 (AAVhu.32), serotype hu.37
  • a composition comprising an adeno-associated virus (AAV) vector having: a.
  • AAV serotype 1 AAV1
  • serotype 2 AAV2
  • serotype 3 AAV3
  • serotype 3B AAV3B
  • serotype 4 AAV4
  • serotype 5 AAV5
  • serotype 6 AAV6
  • serotype 7 AAV7
  • serotype 8 AAV8
  • serotype rh8 AAVrh8
  • serotype 9 AAV9
  • serotype rhlO AAVrhlO
  • serotype rh20 AAVrh20
  • serotype rh39 AAVrh39
  • serotype rh34 AAVrh34
  • serotype hu.32 AAVhu.32
  • serotype hu.37 AAVhu.37
  • serotype hu.60 AAVrh60
  • an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a first and a second transgene, wherein the first transgene encodes a heavy chain and a light chain of an antigen-binding fragment of an anti-CGRP operably linked to a first regulatory sequence, and the second transgene encodes a heavy and light chain of an antigen binding fragment of an anti-CGRPR antibody, operably linked to a second regulatory sequence, wherein said first and second regulatory sequences promote expression of the transgene in human CNS, PNS, arterial smooth muscle, skeletal muscle and/or liver cells, and the first and second regulatory sequences promote expression of the first and second transgenes in different human cell types; c.
  • ITRs AAV inverted terminal repeats
  • transgene encodes a signal sequence at the N-terminus of the heavy chain and the light chain of said mAb that directs secretion and post translational modification of said mAb in CNS, PNS, liver, muscle and/or arterial smooth muscle tissue cells.
  • composition of any of paragraphs 24 to 30, wherein the full-length mAb or the antigenbinding fragment comprises a heavy chain with an amino acid sequence of SEQ ID NO: 1 and optionally an Fc polypeptide with an amino acid sequence of SEQ ID NO:21 and a light chain with an amino acid sequence of SEQ ID NO: 2; a heavy chain with an amino acid sequence of SEQ ID NO: 3 and optionally an Fc polypeptide with an amino acid sequence of SEQ ID NO: 18 and a light chain with an amino acid sequence of SEQ ID NO: 4; a heavy chain with an amino acid sequence of SEQ ID NO: 5 and optionally an Fc polypeptide with an amino acid sequence of SEQ ID NO: 19 and a light chain with an amino acid sequence of SEQ ID NO: 6; or a heavy chain with an amino acid sequence of SEQ ID NO: 7 and optionally an Fc polypeptide with an amino acid sequence of SEQ ID NO: 17 and a light chain with an amino acid sequence of SEQ ID NO: 8
  • composition of any of paragraphs 24 to 32 wherein the transgene comprises a Furin/2A linker between the nucleotide sequences coding for the heavy and light chains of said mAh.
  • composition of paragraphs 37 or 38 wherein the artificial genome comprises the nucleotide sequence of pAAV.CAG.erenumab (SEQ ID NO: 268 or 268), pAAV.LMTP6.VH4i.erenumab.T2A (SEQ ID NO: 270 or 271), pAAVLMTP24.VH4i.erenumab.T2A (SEQ ID NO: 272 or 273), pAAV.CAG.fremanezumab (SEQ ID NO: 275 or 276), pAAV.LMTP6.VH4.fremanezumab.T2A (SEQ ID NO: 277 or 278), pAAVLMTP24.VH4i.felumab.T2A (SEQ ID NO: 279 or 280), pAAV.CAG.
  • galcanezumab (SEQ ID NO: 282 or 283), pAAV.LMTP6.VH4i.galcanezumab.T2A (SEQ ID NO: 284 or 285), pAAVLMTP24.VH4i. galcanezumab. T2 A (SEQ ID NO: 286 or 287), pAAV.CAG. eptinezumab (SEQ ID NO: 289 or 290), pAAV.LMTP6.VH4i.eptinezumab.T2A (SEQ ID NO: 291 or 292), or pAAVLMTP24.VH4i. eptinezumab. T2A (SEQ ID NO: 293 or 294).
  • a method of treating migraine or cluster headaches in a human subject in need thereof comprising intranasally or systemically administering to the subject a therapeutically effective amount of a composition comprising a recombinant AAV comprising a transgene encoding an anti-CGRP or anti-CGRPR mAb, or antigen-binding fragment thereof, operably linked to one or more regulatory sequences that control expression of the transgene in CNS, PNS, liver, skeletal muscle and/or arterial smooth muscle cells.
  • a method of treating migraine or cluster headaches in a human subject in need thereof comprising intranasally or systemically administering to said subject a therapeutically effective amount of a recombinant nucleotide expression vector comprising a transgene encoding an anti- CGRP or anti-CGRPR mAb, or antigen-binding fragment thereof, operably linked to one or more regulatory sequences that control expression of the transgene in human CNS, PNS, liver and/or muscle cells, so that a depot is formed that releases a human post-translationally modified (HuPTM) form of anti-CGRP or anti-CGRPR mAb or antigen-binding fragment thereof.
  • Human post-translationally modified (HuPTM) form of anti-CGRP or anti-CGRPR mAb or antigen-binding fragment thereof comprising intranasally or systemically administering to said subject a therapeutically effective amount of a recombinant nucleotide expression vector comprising a transgene encoding an anti-
  • a method of treating migraine or cluster headaches in a human subject in need thereof comprising intranasally or systemically administering to the subject a therapeutically effective amount of a composition
  • a composition comprising: i) a first recombinant AAV comprising a transgene encoding an anti-CGRP mAb, or antigenbinding fragment thereof, operably linked to one or more regulatory sequences that control expression of the transgene in CNS, PNS, liver, skeletal muscle and/or arterial smooth muscle cells; and a ii) a second recombinant AAV comprising a transgene encoding an anti-CGRPR mAb, or antigen-binding fragment thereof, operably linked to one or more regulatory sequences that control expression of the transgene in CNS, PNS, liver, skeletal muscle and/or arterial smooth muscle cells.
  • the full-length mAb or the antigen-binding fragment comprises a heavy chain with an amino acid sequence of SEQ ID NO: 1 and optionally an Fc polypeptide with an amino acid sequence of SEQ ID NO:21 and a light chain with an amino acid sequence of SEQ ID NO: 2; a heavy chain with an amino acid sequence of SEQ ID NO: 3 and optionally an Fc polypeptide with an amino acid sequence of SEQ ID NO:22 and a light chain with an amino acid sequence of SEQ ID NO: 4; a heavy chain with an amino acid sequence of SEQ ID NO: 5 and optionally an Fc polypeptide with an amino acid sequence of SEQ ID NO:23 and a light chain with an amino acid sequence of SEQ ID NO: 6; or a heavy chain with an amino acid sequence of SEQ ID NO: 7 and optionally an Fc polypeptide with an amino acid sequence of SEQ ID NO:24 and a light chain with an amino acid sequence of SEQ ID NO: 8.
  • the transgene comprises a nucleotide sequence of SEQ ID NO: 9 encoding the heavy chain and a nucleotide sequence of SEQ ID NO: 10 encoding the light chain; a nucleotide sequence of SEQ ID NO: 11 encoding the heavy chain and a nucleotide sequence of SEQ ID NO: 12 encoding the light chain; a nucleotide sequence of SEQ ID NO: 13 encoding the heavy chain and a nucleotide sequence of SEQ ID NO: 14 encoding the light chain, or a nucleotide sequence of SEQ ID NO: 15 encoding the heavy chain and a nucleotide sequence of SEQ ID NO: 16 encoding the light chain.
  • the viral capsid is at least 95% identical to the amino acid sequence of an AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 3B (AAV3B), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype rh34 (AAVrh34), serotype hu.32 (AAVhu.32), serotype hu.37 (AAVhu.37), serotype hu.60 (AAVrh60), ser
  • AAV capsid is AAV8, AAV9, AAV.PHP.eB, or AAVrhlO.
  • the regulator sequence is a human smooth muscle protein 22 alpha (sm22a) promoter (SEQ ID NOS: 184 or 185-191), a CAG promoter (SEQ ID NO: 25), a LMTP6 promoter (SEQ ID NO: 159), a LMTP24 promoter (SEQ ID NO: 263), or a human synapsin 1 gene (hSyn) promoter (SEQ ID NO: 192-195).
  • the transgene comprises a Furin/2A linker between the nucleotide sequences coding for the heavy and light chains of said mAb.
  • said Furin 2A linker is a Furin/T2A linker having the amino acid sequence RKRR(GSG)APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NOS: 87 or 88).
  • the transgene encodes a signal sequence at the N-terminus of the heavy chain and the light chain of said antigen-binding fragment that directs secretion and post translational modification in said human ocular tissue cells.
  • said signal sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 28) or a signal sequence from Tables 2, 3, or 4.
  • transgene has the structure: Signal sequence- Heavy chain - Furin site - 2A site - Signal sequence- Light chain - PolyA.
  • transgene comprises a nucleotide sequence of SEQ ID NO: 267, 274, 281, or 288.
  • the transgene comprises a VH4 intron between the regulatory element and the coding sequence encoding the mAb or antigen binding fragment thereof.
  • the pharmaceutical composition of paragraphs 55 or 56 wherein the artificial genome comprises the nucleotide sequence of pAAV.CAG.erenumab (SEQ ID NO: 268 or 268), pAAV.LMTP6.VH4i.erenumab.T2A (SEQ ID NO: 270 or 271), pAAVLMTP24.VH4i.erenumab.T2A (SEQ ID NO: 272 or 273), pAAV.CAG.fremanezumab (SEQ ID NO: 275 or 276), pAAV.LMTP6.VH4.fremanezumab.T2A (SEQ ID NO: 277 or 278), pAAVLMTP24.VH4i.felumab.T2A (SEQ ID NO: 279 or 280), pAAV.CAG.
  • galcanezumab (SEQ ID NO: 282 or 283), pAAV.LMTP6.VH4i.galcanezumab.T2A (SEQ ID NO: 284 or 285), pAAVLMTP24.VH4i. galcanezumab. T2 A (SEQ ID NO: 286 or 287), pAAV.CAG. eptinezumab (SEQ ID NO: 289 or 290), pAAV.LMTP6.VH4i.eptinezumab.T2A (SEQ ID NO: 291 or 292), or pAAVLMTP24.VH4i. eptinezumab. T2A (SEQ ID NO: 293 or 294).
  • any of paragraphs 40 to 57 wherein the mAb is a hyperglycosylated mutant or wherein the Fc polypeptide of the mAb is glycosylated or aglycosylated.
  • the method of any of paragraphs 40 to 60 wherein the mAb contains a tyrosine sulfation.
  • a method of treating migraine or cluster headaches in a human subject in need thereof comprising intranasally or systemically administering to the subject a therapeutically effective amount of a composition comprising a bicistronic recombinant AAV comprising a first transgene encoding an anti-CGRPR antigen-binding fragment, operably linked to a first regulatory sequence and a second transgene encoding an anti-CGRP antigen-binding fragment, operably linked to a second regulatory sequence wherein said first and second regulatory sequences promote expression of the transgene in CNS, PNS, liver, skeletal muscle and/or arterial smooth muscle cells and promote expression of the first and second transgenes in different human cell types.
  • a method of treating migraine or cluster headaches in a human subject in need thereof comprising: intranasally or systemically administering to said subject first and second recombinant nucleotide expression vectors, wherein said first recombinant nucleotide expression vector comprises a first transgene encoding an anti-CGRPR mAb, or antigen-binding fragment thereof, and said second recombinant nucleotide expression vector comprises a second transgene encoding an anti-CGRP mAb, or antigen-binding fragment thereof, wherein each first and second transgene is operably linked to one or more regulatory sequences that control expression of the transgene in human CNS, PNS, liver, skeletal muscle and/or arterial smooth muscle cells, so that depots are formed that releases a human post-translationally modified (HuPTM) form of the anti-CGRP and anti-CGRPR mAbs or antigen-binding fragments thereof.
  • Human post-translationally modified Human post-translationally modified
  • the first transgene encoding a full-length anti- CGRPR mAb or the antigen-binding fragment comprises a heavy chain with an amino acid sequence of SEQ ID NO: 1 and optionally an Fc polypeptide with an amino acid sequence of SEQ ID NO:21 and a light chain with an amino acid sequence of SEQ ID NO: 2; and wherein the second transgene encoding a full-length anti-CGRP mAh or the antigen-binding fragment comprises a heavy chain with an amino acid sequence of SEQ ID NO: 3 and optionally an Fc polypeptide with an amino acid sequence of SEQ ID NO:22 and a light chain with an amino acid sequence of SEQ ID NO: 4; a heavy chain with an amino acid sequence of SEQ ID NO: 5 and optionally an Fc polypeptide with an amino acid sequence of SEQ ID NO:23 and a light chain with an amino acid sequence of SEQ ID NO: 6; or a heavy chain with an amino acid sequence of SEQ ID NO: 7
  • the first transgene comprises a nucleotide sequence of SEQ ID NO: 9 encoding the heavy chain and a nucleotide sequence of SEQ ID NO: 10 encoding the light chain
  • the second transgene comprises a nucleotide sequence of SEQ ID NO: 11 encoding the heavy chain and a nucleotide sequence of SEQ ID NO: 12 encoding the light chain
  • a nucleotide sequence of SEQ ID NO: 13 encoding the heavy chain and a nucleotide sequence of SEQ ID NO: 14 encoding the light chain
  • a nucleotide sequence of SEQ ID NO: 15 encoding the heavy chain and a nucleotide sequence of SEQ ID NO: 16 encoding the light chain.
  • the viral capsid is at least 95% identical to the amino acid sequence of an AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 3B (AAV3B), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype rh34 (AAVrh34), serotype hu.32 (AAVhu.32), serotype hu.37 (AAVhu.37), serotype hu.60 (AAVrh60
  • any of paragraphs 63 to 68, wherein the AAV capsid is AAV8, AAV9, AAV.PHP.eB, or AAVrhlO.
  • the first regulatory sequence is a human smooth muscle protein 22 alpha (sm22a) promoter (SEQ ID NOS: 184 or 185-190) and the second regulatory sequence is a CAG promoter (SEQ ID NO: 25), a LMTP6 promoter (SEQ ID NO: 159), a LMTP24 promoter (SEQ ID NO: 263), or a human synapsin promoter (SEQ ID NO: 191-195).
  • the first and second transgene comprise a Furin/2A linker between the nucleotide sequences coding for the heavy and light chains of said mAbs.
  • said Furin 2A linker is a Furin/T2A linker having the amino acid sequence RKRR(GSG)APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NOS: 87 or 88).
  • the first and second transgene encode a signal sequence at the N-terminus of the heavy chain and the light chain of said antigen-binding fragment that directs secretion and post translational modification in said human CNS, PNS, liver, skeletal muscle and/or arterial smooth muscle cells.
  • said signal sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 28) or a signal sequence from Tables 2, 3, or 4.
  • transgene comprises a nucleotide sequence of SEQ ID NO: 267, 274, 281, or 288.
  • the artificial genome comprises the nucleotide sequence of pAAV.CAG.erenumab (SEQ ID NO: 268 or 268), pAAV.LMTP6.VH4i.erenumab.T2A (SEQ ID NO: 270 or 271), pAAVLMTP24.VH4i.erenumab.T2A (SEQ ID NO: 272 or 273), pAAV.CAG.fremanezumab (SEQ ID NO: 275 or 276), pAAV.LMTP6.VH4.fremanezumab.T2A (SEQ ID NO: 277 or 278), pAAVLMTP24.VH4i.felumab.T2A (SEQ ID NO: 279 or 280), pAAV.CAG.
  • galcanezumab (SEQ ID NO: 282 or 283), pAAV.LMTP6.VH4i.galcanezumab.T2A (SEQ ID NO: 284 or 285), pAAVLMTP24.VH4i. galcanezumab. T2 A (SEQ ID NO: 286 or 287), pAAV.CAG. eptinezumab (SEQ ID NO: 289 or 290), pAAV.LMTP6.VH4i.eptinezumab.T2A (SEQ ID NO: 291 or 292), or pAAVLMTP24.VH4i. eptinezumab. T2A (SEQ ID NO: 293 or 294).
  • any of paragraphs 63 to 80 wherein the first and/or second mAb is a hyperglycosylated mutant or wherein the Fc polypeptides of the first and/or second mAb is glycosylated or aglycosylated.
  • the method of any of paragraphs 63 to 83 wherein the first and/or mAb contains a tyrosine sulfation.
  • the therapeutically effective amount is determined to be sufficient to reduce nausea, light sensitivity, sound sensitivity, red eye, eyelid edema, forehead and facial sweating, tearing (lacrimation), abnormal small size of the pupil (miosis), nasal congestion, runny nose (rhinorrhea), and drooping eyelid (ptosis).
  • the therapeutically effective amount is determined to be sufficient to reduce the intensity or frequency of migraines, such as change from baseline in the number of headache and/or migraine days per month, number of or cluster headaches, or a reduction in the amount of acute migraine-specific medication used over a defined period of time.
  • a method of treating migraine and/or cluster headaches in a human subject in need thereof comprising intravenously or intramuscularly administering to the subject a dose of a composition comprising a recombinant AAV comprising a transgene encoding an antibody selected from fremanezumab, eptinezumab, galcanezumab or erenumab or an antigen binding protein comprising a heavy chain variable region, a light chain variable region and optionally an Fc domain of the antibody or an antigen binding fragment thereof, operably linked to one or more regulatory sequences that control expression of the transgene in liver and/or muscle cells, in an amount sufficient to result in expression from the transgene and secretion of the antibody, or the antigen binding protein or the antigen binding fragment thereof into the bloodstream of the human subject to produce antibody or the antigen binding protein or antigen binding fragment thereof, plasma levels of at least 1.5 pg/ml to 35 pg/ml antibody or the antigen binding protein or antigen binding fragment thereof,
  • a method of producing recombinant AAVs comprising: (a) culturing a host cell containing:
  • an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises comprising a transgene encoding a substantially full-length or full-length anti-CGRP or anti-CGRPR mAb, or antigenbinding fragment thereof, operably linked to one or more regulatory sequences that promote expression of the transgene in human CNS, PNS, liver, skeletal muscle and/or arterial smooth muscle cells;
  • trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and an AAV capsid protein operably linked to expression control elements that drive expression of the AAV rep and the AAV capsid protein in the host cell in culture and supply the AAV rep and the AAV capsid protein in trans, wherein the capsid has CNS, PNS, liver, skeletal muscle and/or arterial smooth muscle cell tropism;
  • transgene encodes a substantially full-length or full- length mAb or antigen binding fragment that comprises the heavy and light chain variable domains of erenumab, eptinezumab, fremanezumab, or galcanezumab, wherein the AAV capsid protein is an AAV8, AAV9, AAV.PHP.eB, or AAVrhlO capsid protein.
  • a host cell containing one or more polynucleotides comprising: a.
  • an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises comprising a transgene encoding a substantially full-length or full-length anti-CGRP or anti-CGRPR mAb, or antigenbinding fragment thereof, operably linked to one or more regulatory sequences that promote expression of the transgene in human CNS, PNS, skeletal muscle, arterial smooth muscle and/or liver cells; b.
  • trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and an AAV capsid protein operably linked to expression control elements that drive expression of the AAV rep and the AAV capsid protein in the host cell in culture and supply the AAV rep and the AAV capsid protein in trans, wherein the capsid has CNS, PNS, arterial smooth muscle, skeletal muscle and/or liver cell tropism; c. sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid protein.
  • transgene encodes a substantially full-length or full- length mAb or antigen binding fragment that comprises the heavy and light chain variable domains of erenumab, eptinezumab, fremanezumab, or galcanezumab.
  • AAV capsid protein is an AAV8, AAV9, AAV.PHP.eB, or AAVrhlO capsid protein.
  • FIGS 1A-1C A schematic of an rAAV vector genome construct containing an expression cassette encoding the heavy and light chains of a therapeutic mAb separated by a Furin- T2A linker, controlled by expression elements, flanked by the AAV ITRs.
  • the transgene can comprise nucleotide sequences encoding the heavy and light chains of the Fab portion (A) or the full-length heavy (CHI plus hinge) and light chains with Fc regions (B).
  • C human cell types
  • FIGS. 2A-2D The amino acid sequence of a transgene construct for the Fab region of erenumab (A), eptinezumab (B), fremanezumab (C), and galcanezumab (D), therapeutic antibodies to CGRPR and CGRP.
  • Glycosylation sites are boldface.
  • Glutamine glycosylation sites; asparaginal (N) glycosylation sites, non-consensus asparaginal (N) glycosylation sites; and tyrosine-O-sulfation sites (italics) are as indicated in the legend.
  • Complementarity-determining regions (CDR) are underscored. The hinge region is highlighted in italic and grey.
  • FIG. 3. depicts alignment of AAVs l-9e, AAV3B, rhlO, rh20, rh39, rh73, and rh74 version 1 and version 2, hul2, hu21, hu26, hu37, hu51 and hu53 capsid sequences with insertion sites for heterologous peptides after the initiation codon of VP2, and within or near variable region 1 (VR- I), variable region 4 (VR-IV), and variable region 8 (VR-VIII), all highlighted in grey; a particular insertion site within variable region eight (VR-VIII) of each capsid protein is shown by the symbol “#” (after amino acid residue 588 according to the amino acid numbering of AAV9).
  • FIG. 4 Clustal Multiple Sequence Alignment of constant heavy chain regions (CH2 and CH3) of IgGl (SEQ ID NO: 260), IgG2 (SEQ ID NO: 261), and IgG4 (SEQ ID NO: 262).
  • the hinge region from residue 219 to residue 230 of the heavy chain, is shown in italics.
  • the numbering of the amino acids is in EU-format.
  • IV intravenous
  • IM intramuscular
  • AAV9 vectors (2ell gc) were injected either IV or IM and serum antibody levels were determined by ELISA at day 7 (D7), day 21 (D21), day 35 (D35), and day 49 (D49).
  • FIGS. 7A and 7B A Serum expression levels (pg/ml) of therapeutic antibody upon intravenous injection of C/57BL6 mice with 2.5xl0 12 vg/kg of AAV8 vectors encoding a therapeutic antibody regulated by different liver-specific, liver-tandem and liver-muscle regulatory elements (see Table 1).
  • CAG SEQ ID NO: 89
  • TBG SEQ ID NO: 93
  • B. Quantification of viral genomes in liver. C57B1/6 mice were administrated intravenously with AAV8 vectors driven by different liver-specific promoters at equivalent doses (2.5xl0 12 vg/kg). N 5 mice per group. Vector DNA was analyzed by ddPCR in mouse liver samples collected at 49 days post vector administration. Data represent mean + SEM.
  • FIGS. 8 A and 8B A. Route of administration and dose selection in Wistar rats.
  • FIGS. 9A-9D A. Serum anti-kallikrein (pKal) (lanadelumab) antibody concentration following AAV8 delivery. Animals received bilateral injections of 5xl0 10 vg/kg into the GA muscle. Serum was collected biweekly and vectorized antibody concentration was quantified with ELISA. B. Vector genome quantification from relevant tissues with digital droplet PCR (ddPCR). C. Comparison of vector gene expression from liver. Data represent relative fold gene expression as quantified by the AACT method. D. Comparison of AAV transgene expression from tissues using digital droplet PCR (ddPCR). Anti-pKal antibody mRNA copies were normalized to GAPDH mRNA copies across tissues. Data are represented as mean ⁇ SEM. Statistical significance was determined using a one-way ANOVA followed by Tukey’s HSD post-test. *P ⁇ 0.05, **P ⁇ 0.01. DETAILED DESCRIPTION OF THE INVENTION
  • compositions and methods are described for the systemic delivery of a fully human post-translationally modified (HuPTM) therapeutic monoclonal antibody (mAb) or a HuPTM antigenbinding fragment of a therapeutic anti-CGRP or anti-CGRPR mAb (for example, a fully human- glycosylated Fab (HuGlyFab) of a therapeutic mAb) to a patient (human subject) diagnosed with an acute or chronic migraine or cluster headaches, including episodic cluster headaches, or other indications indicated for treatment with the therapeutic mAb.
  • HumanPTM fully human post-translationally modified
  • mAb therapeutic monoclonal antibody
  • HuPTM antigenbinding fragment of a therapeutic anti-CGRP or anti-CGRPR mAb for example, a fully human- glycosylated Fab (HuGlyFab) of a therapeutic mAb
  • compositions comprising and methods of administering a rAAV that encodes both an anti-CGRP antibody, particularly a Fab, and an anti-CGRPR antibody, particularly a Fab, or a combination of an rAAV encoding an anti-CGRP antibody and an rAAV encoding an anti-CGRP receptor antibody, each under the control of different promoter such that the anti-CGRP antibody is expressed in cell types that differ from the cell types where the anti-CGRPR antibody is expressed.
  • Delivery may be advantageously accomplished via gene therapy — e.g., by administering a viral vector or other DNA expression construct encoding a therapeutic anti-CGRP and/or anti-CGRPR mAb or its antigen-binding fragment (or a hyperglycosylated derivative of either) diagnosed with a condition indicated for treatment with the therapeutic anti-CGRP or anti-CGRPR mAb — to create a permanent depot in CNS, PNS, arterial smooth muscles, and/or liver cells, of the patient that continuously supplies the HuPTM mAb or antigen-binding fragment of the therapeutic mAb, e.g., a human-glycosylated transgene product, or to dural vessels and/or the trigeminal ganglion of the subject where the mAb or antigen-binding fragment thereof or peptide exerts its therapeutic or prophylactic effect.
  • a viral vector or other DNA expression construct encoding a therapeutic anti-CGRP and/or anti-CGRPR mAb or its antigen-binding fragment
  • the HuPTM mAb or HuPTM antigen-binding fragment encoded by the transgene is a full-length or an antigen-binding fragment of a HuPTM mAb or HuPTM that binds CGRP, particularly erenumab, or CGRPR, particularly fremanezumab, eptinezumab, and galcanezumab (see FIGS. 2A-2D for the heavy and light chain sequences of the Fab portions of erenumab, fremanezumab, eptinezumab, and galcanezumab).
  • compositions and methods provided herein systemically deliver anti-CGRP or anti-
  • CGRPR antibodies particularly, erenumab, fremanezumab, eptinezumab, and galcanezumab, from a depot of viral genomes, for example, in the subject’s CNS, PNS, arterial smooth muscle, and/or liver cells at a level that is therapeutically or prophylactically effective to treat or ameliorate the symptoms of or to reduce the incidence of (for example, reducing the number of headache days per month) acute or chronic migraine or cluster headaches or other indication that may be treated with an anti-CGRP or anti-CGRPR antibody.
  • viral vectors for delivery of transgenes encoding the therapeutic anti-CGRP or anti-CGRPR antibodies to cells in the human subject including, in embodiments, CNS, PNS, arterial smooth muscle, and/or liver cells, and regulatory elements operably linked to the nucleotide sequence encoding the heavy and light chains of the anti-CGRP or anti- CGRPR antibody that promote the expression of the antibody in the cells, in embodiments, in CNS, PNS, arterial smooth muscle, and/or liver cells.
  • viral vectors for delivery of a first and a second transgene wherein the first transgene encodes a heavy chain and a light chain of an antigen-binding fragment of an anti-CGRP operably linked to a first regulatory sequence (e.g. a CAG promoter), and the second transgene encodes a heavy and light chain of an antigen binding fragment of an anti-CGRPR antibody, operably linked to a second regulatory sequence (e.g.
  • sm22a promoter wherein said first and second regulatory sequences promote expression of the first and second transgenes in different human cell types encoding the therapeutic anti-CGRP or anti- CGRPR antibodies to cells in the human subject, including, in embodiments, CNS, PNS, arterial smooth muscle, and/or liver cells, and regulatory elements operably linked to the nucleotide sequence encoding the heavy and light chains of the anti-CGRP or anti-CGRPR antibody that promote the expression of the antibody in the cells, in embodiments, in CNS, PNS, arterial smooth muscle, and/or liver cells.
  • Such regulatory elements, including smooth muscle cell-specific promoters are provided in Table 1 herein.
  • such viral vectors may be delivered to the human subject at appropriate dosages such that at least 20, 30, 40, 50 or 60 days after administration, the anti-CGRPR antibody or erenumab is present in the serum of said human subject at a level of at least 2 pg/ml to 20 pg/ml anti- CGRPR antibody or erenumab in said subject, or of at least 5 pg/ml to 35 pg/ml anti-CGRPR antibody or erenumab, or of at least 2 pg/ml to 10 pg/ml anti-CGRPR antibody or erenumab or of at least 2 pg/ml to 20 pg/ml anti-CGRPR antibody or erenumab or of at least 5 pg/ml to 20 pg/ml anti-CGRPR antibody or erenumab within at least 20, 30, 40, 50, or 60 days of said administering.
  • Viral vectors may be delivered to the human subject at appropriate dosages such that at least 20, 30, 40, 50 or 60 days after administration, the anti-CGRP antibody or fremanezumab, eptinezumab, or galcanezumab is present in the serum of said human subject at a level of at least 5 pg/ml to 40 pg/ml anti-CGRP antibody or fremanezumab, eptinezumab, or galcanezumab in said subject, or of at least 5 pg/ml to 35 pg/ml anti-CGRP antibody or fremanezumab, eptinezumab, or galcanezumab, or of at least 5 pg/ml to 20 pg/ml anti-CGRPR antibody or erenumab or of at least 2 pg/ml to 20 pg/ml anti-CGRP antibody or fremanezumab, eptinezumab, or galcanezumab within at least 20, 30,
  • the HuPTM mAb or HuPTM antigen-binding fragment encoded by the transgene can include, but is not limited to, a full-length or an antigen-binding fragment of a therapeutic antibody that binds to CGRP or CGRPR, including but not limited to, erenumab, fremanezumab, eptinezumab, and galcanezumab.
  • the amino acid sequences of the heavy and light chain of antigen binding fragments of the foregoing are provided in Table 8, infra.
  • Heavy chain variable domain having an amino acid sequence of SEQ ID NO: 1, 3, 5, or 7 and light chain variable domain having an amino acid sequence of SEQ ID NO: 2, 4, 6, or 8 (encoded by nucleotide sequence SEQ ID NO: 9, 11, 13, or 15 and 10, 12, 14, or 16, respectively).
  • the HuPTM mAb or HuPTM anti gen -binding fragment encoded by the transgene can include, but is not limited to, a full-length or an antigen-binding fragment of a therapeutic antibody or antigen-binding fragments engineered to contain additional glycosylation sites on the Fab domain (e.g., see Courtois et al., 2016, mAbs 8: 99-112 which is incorporated by reference herein in its entirety for its description of derivatives of antibodies that are hyperglycosylated on the Fab domain of the full-length antibody).
  • the recombinant vector used for delivering the transgene includes non-replicating recombinant adeno-associated virus vectors (“rAAV”).
  • rAAVs are particularly attractive vectors for a number of reasons -they can be modified to preferentially target a specific organ of choice; and there are hundreds of capsid serotypes to choose from to obtain the desired tissue specificity, and/or to avoid neutralization by pre-existing patient antibodies to some AAVs.
  • AAV “serotype” refers to an AAV having an immunologically distinct capsid, a naturally-occurring capsid, or an engineered capsid.”
  • Such rAAVs include but are not limited to AAV based vectors comprising capsid components from one or more of AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 3B (AAV3B), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype rh34 (AAVrh34), serotype hu.32 (AAVhu.32), serotype hu.37 (AAVhu.37), serotype hu.60 (AAVrh73), ser
  • viral vectors including but not limited to lentiviral vectors; vaccinia viral vectors, or non-viral expression vectors referred to as “naked DNA” constructs. Expression of the transgene can be controlled by constitutive or tissue-specific expression control elements.
  • Gene therapy constructs are designed such that both the heavy and light chains are expressed.
  • the full length heavy and light chains of the antibody are expressed.
  • the coding sequences encode for a Fab or F(ab’)2 or an scFv.
  • the heavy and light chains should be expressed at about equal amounts, in other words, the heavy and light chains are expressed at approximately a 1 : 1 ratio of heavy chains to light chains.
  • the coding sequences for the heavy and light chains can be engineered in a single construct in which the heavy and light chains are separated by a cleavable linker or IRES so that separate heavy and light chain polypeptides are expressed.
  • the linker separating the heavy and light chains is a Furin-2A linker, for example a Furin-F2A linker RKRR(GSG)APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NOS: 85 or 86) or a Furin-T2A linker RKRR(GSG)EGRGSLLTCGDVEENPGP (SEQ ID NOS: 87 or 88).
  • the construct expresses, from the N-terminus to C-terminus, NH2-VL- linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • the construct expresses, from the N-terminus to C-terminus, NH2-signal or localization sequence- VL-linker-VH-COOH or NH2- signal or localization sequence- VH-linker-VL-COOH.
  • the constructs express an scFv in which the heavy and light chain variable domains are connected via a flexible, non- cleavable linker.
  • Gene therapy constructs are designed such that both the heavy and light chains of an anti-CGRP antibody (first antibody), particularly a Fab, and the heavy and light chains of an anti- CGRPR antibody (second antibody), particularly a Fab, are expressed.
  • the heavy and light chains of the first and second antibody should be expressed at about equal amounts, in other words, the heavy and light chains of the first and second antibody are expressed at approximately a 1 : 1 ratio of heavy chains to light chains.
  • the coding sequences for both the heavy and light chains of the first and second antibody can be engineered in a single construct in which the heavy and light chains of the first and second antibody are separated by a cleavable linker or IRES so that separate heavy and light chain polypeptides are expressed.
  • the linker separating the heavy and light chains is a Furin-2A linker, for example a Furin-F2A linker RKRR(GSG)APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NOS: 85 or 86) or a Furin-T2A linker RKRR(GSG)EGRGSLLTCGDVEENPGP (SEQ ID NOS: 87 or 88).
  • a Furin-2A linker for example a Furin-F2A linker RKRR(GSG)APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NOS: 85 or 86) or a Furin-T2A linker RKRR(GSG)EGRGSLLTCGDVEENPGP (SEQ ID NOS: 87 or 88).
  • nucleic acids e.g., polynucleotides
  • nucleic acid sequences disclosed herein may be codon-optimized, for example, via any codon-optimization technique known to one of skill in the art (see, e.g., review by Quax et al., 2015, Mol Cell 59: 149-161) and may also be optimized to reduce CpG dimers.
  • Each heavy and light chain requires a signal sequence to ensure proper post-translation processing and secretion (unless expressed as an scFv, in which only the N-terminal chain requires a signal sequence sequence).
  • Useful signal sequences for the expression of the heavy and light chains of the therapeutic antibodies in human cells are disclosed herein.
  • Table 9 discloses optimized nucleotide sequences encoding the vectorized antibodies erenumab, fremanezumab, galcanezumab or eptinezumab (SEQ ID Nos 267, 274, 281, and 288, respectively, with leader sequence coding sequences underlined). Exemplary recombinant expression constructs are shown in FIGS. 1A-1C. Table 9 provides the nucleotide sequences for the constructs pAAV.CAG.erenumab (SEQ ID NO: 268 or 268), pAAV.LMTP6.VH4i. erenumab. T2A (SEQ ID NO: 270 or 271), pAAVLMTP24.VH4i.
  • T2 A (SEQ ID NO: 272 or 273), pAAVCAG.felanezumab (SEQ ID NO: 275 or 276), pAAV.LMTP6.VH4. fremanezumab. T2A(SEQ ID NO: 277 or 278), pAAVLMTP24.VH4i.fremanezumab.T2A (SEQ ID NO: 279 or 280), pAAVCAG. galcanezumab (SEQ ID NO: 282 or 283), pAAV.LMTP6.VH4i. galcanezumab. T2A(SEQ ID NO: 284 or 285), pAAVLMTP24.VH4i.
  • galcanezumab.T2A (SEQ ID NO: 286 or 287), pAAVCAG. eptinezumab (SEQ ID NO: 289 or 290), pAAV.LMTP6.VH4i.eptinezumab.T2A (SEQ ID NO: 291 or 292), and pAAVLMTP24.VH4i.eptinezumab.T2A (SEQ ID NO: 293 or 294).
  • HuPTM mAb or HuPTM Fab should result in a “biobetter” molecule for the treatment of disease accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding a full-length HuPTM mAb or HuPTM Fab or other antigen binding fragment, such as an scFv, of a therapeutic mAb to a patient (human subject) diagnosed with a disease indication for that mAb, to create a permanent depot in the subject that continuously supplies the human-glycosylated, sulfated transgene product produced by the subject’s transduced cells.
  • a viral vector or other DNA expression construct encoding a full-length HuPTM mAb or HuPTM Fab or other antigen binding fragment, such as an scFv
  • the cDNA construct for the HuPTM mAb or HuPTM Fab or HuPTM scFv should include a signal peptide that ensures proper co- and post-translational processing (glycosylation and protein sulfation) by the transduced human cells.
  • compositions suitable for administration to human subjects comprise a suspension of the recombinant vector in a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant and optional excipients.
  • a formulation buffer can comprise one or more of a polysaccharide, a surfactant, polymer, or oil.
  • formulations adapted for intranasal administration are provided.
  • the full-length HuPTM mAb or HuPTM Fab or other antigen binding fragment thereof can be produced in human cell lines by recombinant DNA technology, and the glycoprotein can be administered to patients.
  • Human cell lines that can be used for such recombinant glycoprotein production include but are not limited to human embryonic kidney 293 cells (HEK293), fibrosarcoma HT-1080, HKB-11, CAP, HuH-7, and retinal cell lines, PER.C6, or RPE to name a few (e.g., see Dumont et al., 2015, Crit. Rev. Biotechnol.
  • HuPTM Fab glycoprotein e.g., HuPTM Fab glycoprotein
  • the cell line used for production can be enhanced by engineering the host cells to co-express a-2,6-sialyltransferase (or both a-2,3- and a-2,6-sialyltransferases) and/or TPST-1 and TPST-2 enzymes responsible for tyrosine-O-sulfation in human cells.
  • Combination therapies involving delivery of the full-length HuPTM mAb or HuPTM Fab or antigen binding fragment thereof to the patient accompanied by administration of other available treatments are encompassed by the methods of the invention.
  • the additional treatments may be administered before, concurrently or subsequent to the gene therapy treatment.
  • Such additional treatments can include but are not limited to co-therapy with the therapeutic mAb.
  • kits for manufacturing the viral vectors particularly the AAV based viral vectors.
  • methods of producing recombinant AAVs comprising culturing a host cell containing an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a transgene encoding a therapeutic antibody operably linked to expression control elements that will control expression of the transgene in human cells; a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and capsid protein operably linked to expression control elements that drive expression of the AAV rep and capsid proteins in the host cell in culture and supply the rep and cap proteins in trans; sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid proteins; and recovering recombinant AAV encapsidating the artificial genome from the cell culture.
  • Viral vectors or other DNA expression constructs encoding an anti-CGRP or anti- CGRPR HuPTM mAb or antigen-binding fragment thereof, particularly a HuGlyFab, or a hyperglycosylated derivative of a HuPTM mAb antigen-binding fragment are provided herein.
  • the viral vectors and other DNA expression constructs provided herein include any suitable method for delivery of a transgene to a target cell.
  • the means of delivery of a transgene include viral vectors, liposomes, other lipid-containing complexes, other macromolecular complexes, synthetic modified mRNA, unmodified mRNA, small molecules, non-biologically active molecules (e.g., gold particles), polymerized molecules (e.g., dendrimers), naked DNA, plasmids, phages, transposons, cosmids, or episomes.
  • the vector is a targeted vector, e.g., CNS, PNS, arterial smooth muscle, skeletal muscle, and/or liver cells, or a vector that has a tropism for CNS, PNS, arterial smooth muscle, skeletal muscle, and/or liver cells, particularly arterial smooth muscle cells.
  • the disclosure provides for a nucleic acid for use, wherein the nucleic acid comprises a nucleotide sequence that encodes a HuPTM mAb or HuGlyFab or other antigenbinding fragment thereof, as a transgene described herein, operatively linked to an ubiquitous promoter, a CNS-specific, skeletal muscle-specific, liver-specific and/or smooth muscle cell-specific promoter, or an inducible promoter, wherein the promoter is selected for expression in tissue targeted for expression of the transgene.
  • a nucleic acid comprises a nucleotide sequence that encodes a HuPTM mAb or HuGlyFab or other antigenbinding fragment thereof, as a transgene described herein, operatively linked to an ubiquitous promoter, a CNS-specific, skeletal muscle-specific, liver-specific and/or smooth muscle cell-specific promoter, or an inducible promoter, wherein the promoter is selected for expression in tissue targeted for expression of the transgene.
  • Promoters may, for example, be a CAG promoter (SEQ ID NO: 25) and associated upstream regulatory sequences, cytomegalovirus (CMV) promoter, EF-1 alpha promoter (SEQ ID NO:27), mUla (SEQ ID NO:26), UB6 promoter, chicken beta-actin (CBA) promoter, and liver-specific promoters, such as TBG (Thyroxine-binding Globulin) promoter (SEQ ID NO: 183), APOA2 promoter, SERPINA1 (hAAT) promoter, ApoE.hAAT (SEQ ID NO: 166), or muscle-specific promoters, such as a human desmin promoter, CK8 promoter (SEQ ID NO: 182), LMTP6 promoter (SEQ ID NO: 169), LMTP24 promoter (SEQ ID NO: 263), or Pitx3 promoter, inducible promoters, such as a hypoxia-inducible promoter or a rapamycin-
  • the promoter is a smooth muscle cell-specific promoter or a CNS-specific promoter.
  • the promoter is the sm22a (SEQ ID NO: 184, 185, 186, 187, 188, 189, or 190) promoter.
  • the promoter is a hSyn promoter (SEQ ID NO: 191-195).
  • transgene expression is controlled by engineered nucleic acid regulatory elements that have more than one regulatory element (promoter or enhancer), including regulatory elements that are arranged in tandem (two or three copies) that promote liver-specific expression, or both liver-specific expression and muscle-specific expression.
  • LSPX1 SEQ ID NO: 154
  • LSPX2 SEQ ID NO: 155
  • LTP1 SEQ ID NO: 156
  • LTP2 SEQ ID NO: 157
  • LTP3 SEQ ID NO: 158
  • LMTP6 SEQ ID NO: 159
  • LMTP13 SEQ ID NO: 160
  • LMTP14 SEQ ID NO: 161
  • LMTP15 SEQ ID NO: 162
  • LMTP18 SEQ ID NO: 163
  • LMTP19 SEQ ID NO: 164
  • LMTP20 SEQ ID NO: 165
  • LMTP24 SEQ ID NO: 263
  • the disclosure provides for a nucleic acid for use, wherein the nucleic acid encodes a first and a second transgene encoding an CGRP antibody (particularly a Fab fragment thereof) and CGRPR antibody (particularly a Fab fragment thereof), respectively, including vice versa, wherein each transgene is operatively linked to an ubiquitous promoter, a CNS-specific and/or smooth muscle cell-specific promoter, or an inducible promoter, wherein the promoter is selected such that the promoters promote expression of the first and second transgenes in different human cell types.
  • the first transgene is operably linked to a smooth muscle cell-specific promoter and the second transgene is operably linked to a CNS specific promoter.
  • the first transgene is operably linked to the sm22a promoter (SEQ ID NOS: 184, 185-190) and the second transgene to the hSyn promoter (SEQ ID NOS: 191-195) or alternatively the first transgene is operably linked to the hSyn promoter (SEQ ID NOS: 191-195) and the second transgene is operably linked to the sm22a promoter (SEQ ID NOS: 184, 185-190).
  • nucleic acids e.g., polynucleotides
  • the nucleic acids may comprise DNA, RNA, or a combination of DNA and RNA.
  • the DNA comprises one or more of the sequences selected from the group consisting of promoter sequences, the sequence of the gene of interest (the transgene, e.g., the nucleotide sequences encoding the heavy and light chains of the HuPTMmAb or HuGlyFab or other antigen-binding fragment), untranslated regions, and termination sequences.
  • viral vectors provided herein comprise a promoter operably linked to the gene of interest.
  • nucleic acids e.g., polynucleotides
  • nucleic acid sequences disclosed herein may be codon-optimized, for example, via any codon-optimization technique known to one of skill in the art (see, e.g., review by Quax et al., 2015, Mol Cell 59: 149- 161).
  • the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank the expression cassette; (2) one or more control elements, b) optionally, a chicken P-actin or other intron (such as a VH4 intron), c) optionally, a Kozak sequence, and d) a rabbit P-globin poly A signal; and (3) nucleic acid sequences coding for the heavy and light chains of a mAb or Fab, separated by a self-cleaving furin (F)/(F/T)2 A linker (SEQ ID NOS:85, 86, 87, or 88), ensuring expression of equal amounts of the heavy and the light chain polypeptides.
  • An exemplary construct is shown in FIG. 1 A.
  • the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank the expression cassette; (2) sm22a promoter, b) optionally, a chicken P-actin or other intron (such as a VH4 intron), c) optionally a Kozak sequence, d) a rabbit P-globin polyA signal; and (3) nucleic acid sequences coding for a full-length antibody comprising the heavy and light chain sequences using sequences that encode the Fab portion of the heavy chain, including the hinge region sequence, plus the Fc polypeptide of the heavy chain for the appropriate isotype and the light chain, wherein heavy and light chain nucleotide sequences are separated by a self-cleaving furin (F)/(F/T)2A linker (SEQ ID NOS: 85, 86, 87, or 88), ensuring expression of equal amounts of the heavy and the light chain polypeptides.
  • An exemplary construct is shown in FIG. IB.
  • the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank the expression cassette; (2) sm22a promoter, (3) a first nucleic acid sequences coding for the heavy and light chains of a first Fab (e.g. an anti-CGRP Fab), separated by a self-cleaving furin (F)/(F/T)2 A linker (SEQ ID NOS:85, 86, 87, or 88), (4) a rabbit P-globin polyA signal; (5) a hSyn promoter, (6) a second nucleic acid sequences coding for the heavy and light chains of a second Fab (e.g.
  • an anti-CGRPR Fab separated by a selfcleaving furin (F)/(F/T)2A linker (SEQ ID NOS: 85, 86, 87, or 88), and (7) a rabbit P-globin polyA signal.
  • F furin
  • F/T furin2A linker
  • the vectors provided herein are modified mRNA encoding for the gene of interest (e.g., the transgene, for example, HuPTMmAb or HuGlyFab or other antigen binding fragment thereof).
  • the transgene for example, HuPTMmAb or HuGlyFab or other antigen binding fragment thereof.
  • the synthesis of modified and unmodified mRNA for delivery of a transgene to retinal pigment epithelial cells is taught, for example, in Hansson et al., J. Biol. Chem., 2015, 290(9):5661-5672, which is incorporated by reference herein in its entirety.
  • provided herein is a modified mRNA encoding for a HuPTMmAb, HuPTM Fab, or HuPTM scFv.
  • Viral vectors include adenovirus, adeno-associated virus (AAV, e.g., AAV8, AAV9, AAVrhlO, AAV.PHP.B), lentivirus, helper-dependent adenovirus, herpes simplex virus, poxvirus, hemagglutinin virus of Japan (HVJ), alphavirus, vaccinia virus, and retrovirus vectors.
  • Retroviral vectors include murine leukemia virus (MLV) and human immunodeficiency virus (HlV)-based vectors.
  • Alphavirus vectors include semliki forest virus (SFV) and Sindbis virus (SIN).
  • the viral vectors provided herein are recombinant viral vectors.
  • the viral vectors provided herein are altered such that they are replication-deficient in humans.
  • the viral vectors are hybrid vectors, e.g., an AAV vector placed into a “helpless” adenoviral vector.
  • viral vectors comprising a viral capsid from a first virus and viral envelope proteins from a second virus.
  • the second virus is vesicular stomatitus virus (VSV).
  • VSV vesicular stomatitus virus
  • the envelope protein is VSV- G protein.
  • the viral vectors provided herein are HIV based viral vectors.
  • HIV-based vectors provided herein comprise at least two polynucleotides, wherein the gag and pol genes are from an HIV genome and the env gene is from another virus.
  • the viral vectors provided herein are herpes simplex virusbased viral vectors.
  • herpes simplex virus-based vectors provided herein are modified such that they do not comprise one or more immediately early (IE) genes, rendering them non-cytotoxic.
  • IE immediately early
  • the viral vectors provided herein are MLV based viral vectors.
  • MLV-based vectors provided herein comprise up to 8 kb of heterologous DNAin place of the viral genes.
  • the viral vectors provided herein are lentivirus-based viral vectors.
  • lentiviral vectors provided herein are derived from human lentiviruses.
  • lentiviral vectors provided herein are derived from non-human lentiviruses.
  • lentiviral vectors provided herein are packaged into a lentiviral capsid.
  • lentiviral vectors provided herein comprise one or more of the following elements: long terminal repeats, a primer binding site, a polypurine tract, att sites, and an encapsidation site.
  • the viral vectors provided herein are alphavirus-based viral vectors.
  • alphavirus vectors provided herein are recombinant, replicationdefective alphaviruses.
  • alphavirus replicons in the alphavirus vectors provided herein are targeted to specific cell types by displaying a functional heterologous ligand on their virion surface.
  • the viral vectors provided herein are AAV based viral vectors.
  • the AAV-based vectors provided herein do not encode the AAV rep gene (required for replication) and/or the AAV cap gene (required for synthesis of the capsid proteins) (the rep and cap proteins may be provided by the packaging cells in trans). Multiple AAV serotypes have been identified.
  • AAV-based vectors provided herein comprise components from one or more serotypes of AAV.
  • AAV-based vectors provided herein comprise components from one or more serotypes of AAV with tropism to CNS, liver and/or muscle.
  • AAV based vectors provided herein comprise capsid components from one or more of AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 3B (AAV3B), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype rh34 (AAVrh34), serotype hu.37 (AAVhu.37), serotype hu.60 (AAVrh73), serotype rh21 (AAVrh21), serotype rhl5 (AAVrh
  • AAV based vectors provided herein are or comprise components from one or more of AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 3B (AAV3B), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype rh34 (AAVrh34), serotype hu.37 (AAVhu.37), serotype hu.60 (AAVhu.60), serotype rh21 (AAVrh21), serotype rhl5 (AAVhu
  • the encoded AAV capsid has the sequence of SEQ ID NO: 139 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions and retaining the biological function of the AAV9 capsid.
  • FIG. 4 provides a comparative alignment of the amino acid sequences of the capsid proteins of different AAV serotypes with potential amino acids that may be substituted at certain positions in the aligned sequences based upon the comparison in the row labeled SUBS.
  • the AAV vector comprises an AAV8, AAV9, AAVrhlO, or AAV.PHP.eB, capsid variant that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions that are not present at that position in the native AAV capsid sequence as identified in the SUBS row of FIG. 4.
  • AAV9 vectors comprising a viral genome comprising an expression cassette for expression of the transgene, under the control of regulatory elements, and flanked by ITRs and an engineered viral capsid as described herein or is at least 95%, 96%, 97%, 98%, 99% or 99.9% identical to the amino acid sequence of the AAV9 capsid protein, while retaining the biological function of the engineered AAV9 capsid.
  • the encoded AAV9 capsid has the sequence of wild type AAV9, with the peptide insertion as described herein, with, in addition, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions with respect to the wild type AAV sequence and retains biological function of the AAV9 capsid.
  • engineered AAV vectors other than AAV9 vectors such as engineered AAV1, AAV2 AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAVe9, AAVrhlO, AAVrh20, AAVrh39, AAVrh34, AAVhu.37, AAV.hu60, AAVrh21, AAVrhl5, AAVrh24, AAVhu.5, AAVhu.10, AAVrh73, AAVrh74, or AAV.PHP.eB vectors with the amino acid substitutions and/or peptide insert as described herein and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions relative to the wild type or unengineered sequence for that AAV type and that retains its biological function.
  • the amino acid sequence of hu37 capsid can be found in international application PCT WO 2005/033321 (SEQ ID NO: 88 thereof) and the amino acid sequence for the rh8 capsid can be found in international application PCT WO 03/042397 (SEQ ID NO:97).
  • the amino acid sequence for the rh64Rl sequence is found in W02006/110689 (a R697W substitution of the Rh.64 sequence, which is SEQ ID NO: 43 of WO 2006/110689).
  • the rh64Rl sequence is:
  • AAV-based vectors comprise components from one or more serotypes of AAV.
  • AAV based vectors provided herein comprise capsid components from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVS3, AAVrh8, AAV.rhlO, AAVrh20, AAVrh39, AAVrh46, AAVrh73, AAVRh74, AAV.RHM4-1, AAVhu37, AAVAnc80, AAVAnc80L65, AAV7m8, AAVPHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAVLK03, AAVHSC1, AAVHSC2, AAVHSC3, AAVHSC4, AAV.HSC5,
  • AAV based vectors provided herein comprise components from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVS3, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.rh46, AAV.rh73, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAVPHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HS
  • rAAV particles comprise a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e.
  • AAV capsid serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.rh46, AAV.rh73, AAVRh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, rAAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAVHSC10, AAV.HSC11, AAV.HSC
  • the recombinant AAV for us in compositions and methods herein is AAVS3 (including variants thereof) (see e.g., US Patent Application No. 20200079821, which is incorporated herein by reference in its entirety).
  • rAAV particles comprise the capsids of AAV-LK03 or AAV3B, as described in Puzzo et al., 2017, Sci. Transl. Med. 29(9): 418, which is incorporated by reference in its entirety.
  • the AAV for use in compositions and methods herein is any AAV disclosed in US 10,301,648, such as AAV.rh46 or AAV.rh73.
  • the recombinant AAV for use in compositions and methods herein is Anc80 or Anc80L65 (see, e.g., Zinn et al., 2015, Cell Rep. 12(6): 1056-1068, which is incorporated by reference in its entirety).
  • the AAV for use in compositions and methods herein is any AAV disclosed in US 9,585,971, such as AAV-PHP.B.
  • the AAV for use in compositions and methods herein is an AAV2/Rec2 or AAV2/Rec3 vector, which has hybrid capsid sequences derived from AAV8 and serotypes cy5, rh20 or rh39 (see, e.g., Issa et al., 2013, PLoS One 8(4): e60361, which is incorporated by reference herein for these vectors).
  • the AAV for use in compositions and methods herein is an AAV disclosed in any of the following, each of which is incorporated herein by reference in its entirety: US 7,282,199; US 7,906,111; US 8,524,446; US 8,999,678; US 8,628,966; US 8,927,514; US 8,734,809; US9,284,357; US 9,409,953; US 9,169,299; US 9,193,956; US 9,458,517; US 9,587,282; US 2015/0374803; US 2015/0126588; US 2017/0067908; US 2013/0224836; US 2016/0215024; US 2017/0051257; PCT/US2015/034799; and PCT/EP2015/053335.
  • rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos.
  • rAAV particles comprise any AAV capsid disclosed in United States Patent No. 9,840,719 and WO 2015/013313, such as AAV.Rh74 and RHM4-1, each of which is incorporated herein by reference in its entirety.
  • rAAV particles comprise any AAV capsid disclosed in WO 2014/172669, such as AAV rh.74, which is incorporated herein by reference in its entirety.
  • rAAV particles comprise the capsid of AAV2/5, as described in Georgiadis et al., 2016, Gene Therapy 23: 857-862 and Georgiadis et al., 2018, Gene Therapy 25: 450, each of which is incorporated by reference in its entirety.
  • rAAV particles comprise any AAV capsid disclosed in WO 2017/070491, such as AAV2tYF, which is incorporated herein by reference in its entirety.
  • rAAV particles comprise any AAV capsid disclosed in US Pat Nos. 8,628,966; US 8,927,514; US 9,923,120 and WO 2016/049230, such as HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16, each of which is incorporated by reference in its entirety.
  • rAAV particles have a capsid protein disclosed in Inti. Appl. Publ. No. WO 2003/052051 (see, e.g., SEQ ID NO: 2 of '051 publication), WO 2005/033321 (see, e.g., SEQ ID NOs: 123 and 88 of '321 publication), WO 03/042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of '397 publication), WO 2006/068888 (see, e.g., SEQ ID NOs: 1 and 3-6 of '888 publication), WO 2006/110689, (see, e.g., SEQ ID NOs: 5-38 of '689 publication) W02009/104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31 of '964 publication), WO 2010/127097 (see, e.g., SEQ ID NOs:
  • rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of an AAV capsid disclosed in Inti. Appl. Publ. No.
  • WO 2003/052051 see, e.g., SEQ ID NO: 2 of '051 publication
  • WO 2005/033321 see, e.g., SEQ ID NOs: 123 and 88 of '321 publication
  • WO 03/042397 see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of '397 publication
  • WO 2006/068888 see, e.g., SEQ ID NOs: 1 and 3-6 of '888 publication
  • WO 2006/110689 see, e.g., SEQ ID NOs: 5-38 of '689 publication
  • W02009/104964 see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31 of 964 publication
  • WO 2010/127097 see, e.g., SEQ ID NOs: 5-38 of '097 publication
  • WO 2015/191508 see, e.g., SEQ ID NOs: 80-294 of
  • rAAV particles comprise a pseudotyped AAV capsid.
  • the pseudotyped AAV capsids are rAAV2/8 or rAAV2/9 pseudotyped AAV capsids.
  • Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74: 1524-1532 (2000); Zolotukhin et al., Methods 28: 158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001).
  • AAV8-based, AAV9-based, and AAVrh 10-based viral vectors are used in certain of the methods described herein.
  • Nucleotide sequences of AAV based viral vectors and methods of making recombinant AAV and AAV capsids are taught, for example, in United States Patent No. 7,282,199 B2, United States Patent No. 7,790,449 B2, United States Patent No. 8,318,480 B2, United States Patent No. 8,962,332 B2 and International Patent Application No. PCT/EP2014/076466, each of which is incorporated herein by reference in its entirety.
  • AAV e.g.
  • AAV8 AAV9 or AAVrh 10 -based viral vectors encoding a transgene (e.g., an HuPTM Fab).
  • a transgene e.g., an HuPTM Fab.
  • the amino acid sequences of AAV capsids, including AAV8, AAV9 and AAVrhlO are provided in Figure 21.
  • a single-stranded AAV may be used supra.
  • a self-complementary vector e.g., scAAV
  • scAAV single-stranded AAV
  • the viral vectors used in the methods described herein are adenovirus based viral vectors.
  • a recombinant adenovirus vector may be used to transfer in the transgene encoding the HuPTMmAb or HuGlyFab or antigen-binding fragment.
  • the recombinant adenovirus can be a first-generation vector, with an El deletion, with or without an E3 deletion, and with the expression cassette inserted into either deleted region.
  • the recombinant adenovirus can be a second-generation vector, which contains full or partial deletions of the E2 and E4 regions.
  • a helper- dependent adenovirus retains only the adenovirus inverted terminal repeats and the packaging signal (phi).
  • the transgene is inserted between the packaging signal and the 3’ITR, with or without stuffer sequences to keep the genome close to wild-type size of approximately 36 kb.
  • An exemplary protocol for production of adenoviral vectors may be found in Alba et al., 2005, “Gutless adenovirus: last generation adenovirus for gene therapy,” Gene Therapy 12:S18-S27, which is incorporated by reference herein in its entirety.
  • the viral vectors used in the methods described herein are lentivirus based viral vectors.
  • a recombinant lenti virus vector may be used to transfer in the transgene encoding the HuPTM mAb antigen binding fragment.
  • Four plasmids are used to make the construct: Gag/pol sequence containing plasmid, Rev sequence containing plasmids, Envelope protein containing plasmid (e.g., VSV-G), and Cis plasmid with the packaging elements and the anti-CGRP or anti-CGRPR antigen-binding fragment gene.
  • the four plasmids are co-transfected into cells (e.g., HEK293 based cells), whereby polyethylenimine or calcium phosphate can be used as transfection agents, among others.
  • the lentivirus is then harvested in the supernatant (lentiviruses need to bud from the cells to be active, so no cell harvest needs/should be done).
  • the supernatant is filtered (0.45 pm) and then magnesium chloride and benzonase added.
  • Further downstream processes can vary widely, with using TFF and column chromatography being the most GMP compatible ones. Others use ultracentrifugation with/without column chromatography.
  • Exemplary protocols for production of lentiviral vectors may be found in Lesch et al., 2011, “Production and purification of lentiviral vector generated in 293T suspension cells with baculoviral vectors,” Gene Therapy 18:531-538, andAusubel et al., 2012, “Production of CGMP-Grade Lentiviral Vectors,” Bioprocess Int. 10(2):32-43, both of which are incorporated by reference herein in their entireties.
  • a vector for use in the methods described herein is one that encodes an HuPTM mAb, such that, upon introduction of the vector into a relevant cell, a glycosylated and/or tyrosine sulfated variant of the HuPTM mAb is expressed by the cell.
  • the vectors provided herein comprise components that modulate gene delivery or gene expression (e.g., “expression control elements”). In certain embodiments, the vectors provided herein comprise components that modulate gene expression. In certain embodiments, the vectors provided herein comprise components that influence binding or targeting to cells. In certain embodiments, the vectors provided herein comprise components that influence the localization of the polynucleotide (e.g., the transgene) within the cell after uptake. In certain embodiments, the vectors provided herein comprise components that can be used as detectable or selectable markers, e.g., to detect or select for cells that have taken up the polynucleotide.
  • the viral vectors provided herein comprise one or more promoters that control expression of the transgene.
  • These promoters may be constitutive (promote ubiquitous expression) or may specifically or selectively express in the liver (including promoting expression in the liver only or expressing in the liver at least at 1 to 100 fold greater levels than in a non-liver tissue).
  • the promoter is a constitutive promoter.
  • the promoter is a CB7 (also referred to as a CAG promoter) (see Dinculescu et al., 2005, Hum Gene Ther 16: 649-663, incorporated by reference herein in its entirety).
  • the CAG (SEQ ID NO: 25) includes other expression control elements that enhance expression of the transgene driven by the vector.
  • the other expression control elements include chicken P-actin intron and/or rabbit P-globin polyA signal.
  • the promoter comprises a TATA box.
  • the promoter comprises one or more elements. In certain embodiments, the one or more promoter elements may be inverted or moved relative to one another.
  • the elements of the promoter are positioned to function cooperatively. In certain embodiments, the elements of the promoter are positioned to function independently.
  • the viral vectors provided herein comprise one or more promoters selected from the group consisting of the human CMV immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus (RS) long terminal repeat, and rat insulin promoter. In certain embodiments, the vectors provided herein comprise one or more long terminal repeat (LTR) promoters selected from the group consisting of AAV, MLV, MMTV, SV40, RSV, HIV-1, and HIV-2 LTRs.
  • LTR long terminal repeat
  • the promoter is the smooth muscle cell-specific promoter, particularly the sm22a promoter (SEQ ID NOS: 184, 185-190) (see Li,L., et al, J Cell Biol 132 (5), 849-859 (1996) and Li,L., et al, J Cell Biol 132 (5), 849-859 (1996); incorporated by reference herein in its entirety).
  • the promoter is a CNS-specific promoter (see Table 1; SEQ ID NOs: 191-195).
  • the vectors provided herein comprise one or more tissue specific promoters (e.g., a liver-specific promoter or a dual liver-muscle specific promoter).
  • the viral vectors provided herein comprises a liver cell specific promoter, such as, a TBG (Thyroxine-binding Globulin) promoter (SEQ ID NO: 183), an APOA2 promoter, a SERPINA1 (hAAT) promoter, or an ApoE.hAAT promoter (SEQ ID NO: 166).
  • the viral vector provided herein comprises a muscle specific promoter, such as a human desmin promoter (Jonuschies et al., 2014, Curr. Gene Ther.
  • the viral vector comprises a VMD2 promoter.
  • nucleic acid regulatory elements that are chimeric with respect to arrangements of elements in tandem in the expression cassette. Regulatory elements, in general, have multiple functions as recognition sites for transcription initiation or regulation, coordination with cellspecific machinery to drive expression upon signaling, and to enhance expression of the downstream gene.
  • nucleic acid regulatory elements that promote transgene expression in liver tissue, or liver and muscle (skeletal and/or cardiac) tissue.
  • certain elements are arranged with two or more copies of the individual enhancer and promoter elements arranged in tandem and operably linked to a transgene to promote expression, particularly tissue specific expression.
  • Exemplary nucleotide sequences of the individual promoter and enhancer elements are provided in Table 1.
  • Table 1 Also provided in Table 1 are exemplary composite nucleic acid regulatory elements comprising the individual tandem promoter and enhancer elements.
  • the downstream promoter is an hAAT promoter (in certain embodiments the hAAT promoter is an hAAT(AATG) promoter) and the other promoter is another hAAT promoter or is a TBG promoter).
  • hAAT promoter in certain embodiments the hAAT promoter is an hAAT(AATG) promoter
  • the other promoter is another hAAT promoter or is a TBG promoter.
  • tissue-specific promoter cassettes such as those targeting the liver
  • promoter cassettes to achieve dual expression in two separate tissue populations (such as liver and skeletal muscle, and in certain embodiments cardiac muscle, and liver and bone).
  • these designs aim to improve the therapeutic efficacy of gene transfer by providing more robust levels of transgene expression, improved stability/persistence, and induction of immune tolerance to the transgene product.
  • the hAAT promoter with the start codon deleted (AATG) is used in an expression cassette provided herein.
  • nucleic acid regulatory elements that comprise or consist of promoters and/or other nucleic acid elements, such as enhancers, that promote liver expression, such as ApoE enhancers, Mic/BiKE elements or hAAT promoters. These may be present as single copies or with two or more copies in tandem.
  • the nucleic acid regulatory element may also comprise, in addition to the one or more elements that promote liver specific expression, one or more elements that promote muscle specific expression (including skeletal and/or cardiac muscle), for example, one or more copies, for example two copies, of the MckE element, which may be arranged as two or more copies in tandem or an MckE and MhcE elements arranged in tandem.
  • a promoter element is deleted for the initiation codon to prevent translation initiation at that site, and preferably, the element with the modified start codon is the promoter that is the element at the 3’ end or the downstream end of the nucleic acid regulatory element, for example, closest within the nucleic acid sequence of the expression cassette to the transgene.
  • the composite nucleic acid regulatory element comprises an hAAT promoter, in embodiments an hAAT which is start-codon modified (AATG) as the downstream promoter, and a second promoter in tandem with the hAAT promoter, which is an hAAT promoter, a CK8 promoter, an Spc5.12 promoter or an minSpc5.12 promoter.
  • the composite promoter comprises a transcriptionally active portion of a muscle enhancer, such as a cis regulatory element or transcription factor binding site.
  • the muscle enhancer is active in muscle cells.
  • the muscle enhancer is active in skeletal muscle cells, and not active in cardiac cells.
  • the muscle enhancer is upstream of a composite nucleic acid regulatory element which comprises a muscle promoter and an hAAT promoter which is start-codon modified (hAATAATG) and downstream of the muscle promoter.
  • the muscle enhancer is Mus022.
  • an ApoE enhancer or a portion thereof may be placed upstream of the muscle enhancer or downstream of the muscle enhancer.
  • the composite nucleic acid regulatory element comprises LMTP24 of Table 1.
  • the nucleotide sequence encoding the CGRP or anti-CGRPR antibody heavy and light chains is operably linked to a composite nucleic acid regulatory element comprising a) two copies of Mic/BiKE arranged in tandem or two copies of ApoE arranged in tandem or two copies of Mic/BiKE arranged in tandem with one copy of ApoE, b) one promoter or, in tandem promoter embodiments, two promoters arranged in tandem comprising at least one copy of hAAT which is start-codon modified (AATG) (where in certain embodiments the hAAT promoter is the downstream or 3’ promoter).
  • the composite nucleic acid regulatory element comprises LSPX1, LSPX2, LTP1, LTP2, or LTP3 of Table 1.
  • the promoter is a LMTP24 (SEQ ID NO: 263), which is a tandem liver/muscle specific enhancer promoter which, in embodiments, has lower expression in cardiac muscle cells.
  • the LMTP24 promoter is comprised of (i) synthetic ApoE enhancer region (SEQ ID NO: 264). (ii) a muscle enhancer region (for example, Mus022, SEQ ID NO: 265)), (iii) a CK promoter (SEQ ID NO: 266), and (IV) a hAAT promoter (AATG) (SEQ ID NO: 172).
  • the anti-CGRP or CGRPR therapeutic antibody coding sequence is operably linked to composite nucleic acid regulatory elements for enhancing gene expression in the liver LSPX1 (SEQ ID NO: 154, LSPX2 (SEQ ID NO: 155), LTP1 (SEQ ID NO: 156), LTP2 (SEQ ID NO: 157), or LTP3 (SEQ ID NO: 158), liver and muscle expression, LMTP6 (SEQ ID NO: 159), LMTP13 (SEQ ID NO: 160), LMTP14 (SEQ ID NO: 161), LMTP15 (SEQ ID NO: 162), LMTP18 (SEQ ID NO:163), LMTP19 (SEQ ID NO: 164), LMTP20 (SEQ ID NO: 165), or LMTP24 (SEQ ID NO: 263) the sequences of which are provided in Table 1 below.
  • composite regulatory elements that enhance gene expression in the liver, and in certain embodiments, also muscle or bone, which have 99%, 95%, 90%, 85% or 80% sequence identity with one of nucleic acid sequences LSPX1 (SEQ ID NO: 154), LSPX2 (SEQ ID NO: 155), LTP1 (SEQ ID NO: 156), LTP2 (SEQ ID NO: 157), or LTP3 (SEQ ID NO: 15869), LMTP6 (SEQ ID NO: 159), LMTP13 (SEQ ID NO: 160), LMTP14 (SEQ ID NO: 161), LMTP15 (SEQ ID NO: 162), LMTP18 (SEQ ID NO: 163), LMTP19 (SEQ ID NO: 164), LMTP20 (SEQ ID NO: 165), or LMTP24 (SEQ ID NO: 263).
  • LSPX1 SEQ ID NO: 154
  • LSPX2 SEQ ID NO: 155
  • LTP1 SEQ ID NO: 156
  • the constructs described herein result in preferred transcription start sites within the promoter region.
  • the constructs described herein have a tandem or composite nucleic acid regulatory sequence that comprises an hAAT promoter (particularly a modified start codon hAAT promoter) and has a transcription start site of TCTCC (corresponding to nt 1541-1545 of LMTP6 (SEQ ID NO: 159), which overlaps with the active TTS found in hAAT (nt 355-359 of SEQ ID NO: 171) or GGTACAATGACTCCTTTCG (SEQ ID NO: 181), which corresponds to nucleotides 139-157 of SEQ ID NO: 171, or GGTACAGTGACTCCTTTCG (SEQ ID NO: 180), which corresponds to nucleotides 139-157 of SEQ ID NO: 172.
  • the constructs described herein have a tandem or composite regulatory sequence that comprises a CK8 promoter and has a transcription start site at TCATTCTACC (SEQ ID NO:249), which corresponds to nucleotides 377-386 of SEQ ID NO: 182, particularly starting at the nucleotide corresponding to nucleotide 377 of SEQ ID NO: 182 or corresponding to nucleotide 1133 of SEQ ID NO: 159.
  • the promoter is an inducible promoter. In certain embodiments the promoter is a hypoxia-inducible promoter. In certain embodiments, the promoter comprises a hypoxia-inducible factor (HIF) binding site. In certain embodiments, the promoter comprises a HIF- la binding site. In certain embodiments, the promoter comprises a HIF -2a binding site. In certain embodiments, the HIF binding site comprises an RCGTG (SEQ ID NO: 153) motif. For details regarding the location and sequence of HIF binding sites, see, e.g., Schodel, et al., Blood, 2011, 117(23):e207-e217, which is incorporated by reference herein in its entirety.
  • the promoter comprises a binding site for a hypoxia induced transcription factor other than a HIF transcription factor.
  • the viral vectors provided herein comprise one or more IRES sites that is preferentially translated in hypoxia.
  • the hypoxiainducible promoter is the human N-WASP promoter, see, e.g., Salvi, 2017, Biochemistry and Biophysics Reports 9: 13-21 (incorporated by reference for the teaching of the N-WASP promoter) or is the hypoxia-induced promoter of human Epo, see, e.g., Tsuchiya et al., 1993, J. Biochem. 113:395- 400 (incorporated by reference for the disclosure of the Epo hypoxia-inducible promoter).
  • the promoter is a drug inducible promoter, for example, a promoter that is induced by administration of rapamycin or analogs thereof.
  • constructs containing certain ubiquitous and tissue-specific promoters include synthetic and tandem promoters. Examples and nucleotide sequences of promoters are provided in Table 1 below. Table 1 also includes the nucleotide sequences of other regulatory elements useful for the expression cassettes provided herein
  • the viral vectors provided herein comprise one or more regulatory elements other than a promoter. In certain embodiments, the viral vectors provided herein comprise an enhancer. In certain embodiments, the viral vectors provided herein comprise a repressor. In certain embodiments, the viral vectors provided herein comprise an intron (e.g. VH4 intron (SEQ ID NO: 54) SV40 Intron (SEQ ID NO: 55) or a chimeric intron (P-globin/Ig Intron) (SEQ ID NO: 53).
  • VH4 intron SEQ ID NO: 54
  • SV40 Intron SEQ ID NO: 55
  • a chimeric intron P-globin/Ig Intron
  • the viral vectors provided herein comprise a polyadenylation sequence downstream of the coding region of the transgene.
  • Any polyA site that signals termination of transcription and directs the synthesis of a polyA tail is suitable for use in AAV vectors of the present disclosure.
  • Exemplary polyA signals are derived from, but not limited to, the following: the SV40 late gene, the rabbit P-globin gene (SEQ ID NO: 57), the bovine growth hormone (BPH) gene, the human growth hormone (hGH) gene, the synthetic polyA (SPA) site, and the bovine growth hormone (bGH) gene. See, e.g., Powell and Rivera-Soto, 2015, Discov. Med., 19(102):49-57. 5.1.4 Signal Peptides
  • the vectors provided herein comprise components that modulate protein delivery.
  • the viral vectors provided herein comprise one or more signal peptides.
  • Signal peptides also referred to as “signal sequences” may also be referred to herein as “leader sequences” or “leader peptides”.
  • the signal peptides allow for the transgene product to achieve the proper packaging (e.g., glycosylation) in the cell.
  • the signal peptides allow for the transgene product to achieve the proper localization in the cell.
  • the signal peptides allow for the transgene product to achieve secretion from the cell.
  • a signal sequence for protein production in a gene therapy context or in cell culture There are two general approaches to select a signal sequence for protein production in a gene therapy context or in cell culture.
  • One approach is to use a signal peptide from proteins homologous to the protein being expressed.
  • a human antibody signal peptide may be used to express IgGs in CHO or other cells.
  • Another approach is to identify signal peptides optimized for the particular host cells used for expression. Signal peptides may be interchanged between different proteins or even between proteins of different organisms, but usually the signal sequences of the most abundant secreted proteins of that cell type are used for protein expression.
  • the signal peptide of human albumin the most abundant protein in plasma, was found to substantially increase protein production yield in CHO cells.
  • the signal peptide may retain function and exert activity after being cleaved from the expressed protein as “post-targeting functions”.
  • the signal peptide is selected from signal peptides of the most abundant proteins secreted by the cells used for expression to avoid the post-targeting functions.
  • the signal sequence is fused to both the heavy and light chain sequences.
  • An exemplary sequence is MYRMQLLLLIALSLALVTNS (SEQ ID NO: 28) which can be encoded by a nucleotide sequence of SEQ ID NO:29 (see Table 2, FIGS. 1A and IB).
  • signal sequences that are appropriate for expression, and may cause selective expression or directed expression of the HuPTM mAb or Fab or scFv in CNS/eye, muscle, or liver are provided in Tables 2, 3 and 4, respectively, below. Table 2.
  • Signal peptides for expression in eye/CNS tissue are provided in Tables 2, 3 and 4, respectively, below. Table 2.
  • a single construct can be engineered to encode both the heavy and light chains separated by a cleavable linker or IRES so that separate heavy and light chain polypeptides are expressed by the transduced cells.
  • the viral vectors provided herein provide polycistronic (e.g., bicistronic) messages.
  • the viral construct can encode the heavy and light chains separated by an internal ribosome entry site (IRES) elements (for examples of the use of IRES elements to create bicistronic vectors see, e.g., Gurtu et al., 1996, Biochem. Biophys. Res. Comm. 229(l):295-8, which is herein incorporated by reference in its entirety).
  • IRES internal ribosome entry site
  • the bicistronic message is contained within a viral vector with a restraint on the size of the polynucleotide(s) therein.
  • the bicistronic message is contained within an AAV virus-based vector e.g., an AAV8- based, AAV9-based or A AVrh 10-based vector).
  • Furin-2A linkers encode the heavy and light chains separated by a cleavable linker such as the self-cleaving 2A and 2A-like peptides, with or without upstream furin cleavage sites, e.g. Furin/2A linkers, such as furin/F2A (F/F2A) or furin/T2A (F/T2A) linkers (Fang et al., 2005, Nature Biotechnology 23: 584-590, Fang, 2007, Mol Ther 15: 1153-9, and Chang, J. et al, MAbs 2015, 7(2):403-412, each of which is incorporated by reference herein in its entirety).
  • a furin/2A linker may be incorporated into an expression cassette to separate the heavy and light chain coding sequences, resulting in a construct with the structure:
  • a 2A site or 2A-like site such as an F2A site comprising the amino acid sequence RKRR(GSG)APVKQTLNFDLLKLAGDVESNPGP(SEQ ID NOS: 87 or 88) or a T2A site comprising the amino acid sequence RKRR(GSG)EGRGSLLTCGDVEENPGP (SEQ ID NOS: 85 or 86), is self-processing, resulting in “cleavage” between the final G and P amino acid residues.
  • linkers, with or without an upstream flexible Gly-Ser-Gly (GSG) linker sequence SEQ ID NO: 72
  • T2A (GSG)EGRGSLLTCGDVEENPGP (SEQ ID NOS:77 or 78);
  • P2A (GSG)ATNFSLLKQAGDVEENPGP (SEQ ID NOS:79 or 80);
  • E2A (GSG)QCTNYALLKLAGDVESNPGP (SEQ ID NOS:81 or 82);
  • F2A (GSG)APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NOS: 83 or 84)
  • an additional proteolytic cleavage site e.g. a furin cleavage site
  • the self-processing cleavage site e.g. 2A or 2A like sequence
  • a peptide bond is skipped when the ribosome encounters the 2A sequence in the open reading frame, resulting in the termination of translation, or continued translation of the downstream sequence (the light chain).
  • This self-processing sequence results in a string of additional amino acids at the end of the C-terminus of the heavy chain.
  • additional amino acids can then be cleaved by host cell Furin at the furin cleavage site(s), e.g. located immediately prior to the 2A site and after the heavy chain sequence, and further cleaved by carboxypeptidases.
  • the resultant heavy chain may have one, two, three, or more additional amino acids included at the C-terminus, or it may not have such additional amino acids, depending on the sequence of the Furin linker used and the carboxypeptidase that cleaves the linker in vivo (See, e.g. , Fang et al., 17 April 2005, Nature Biotechnol.
  • Furin linkers that may be used comprise a series of four basic amino acids, for example, RKRR (SEQ ID NO:73), RRRR (SEQ ID NO:74), RRKR (SEQ ID NO:75), or RKKR (SEQ ID NO:76).
  • linker Once this linker is cleaved by a carboxypeptidase, additional amino acids may remain, such that an additional zero, one, two, three or four amino acids may remain on the C- terminus of the heavy chain, for example, R, RR, RK, RKR, RRR, RRK, RKK, RKRR (SEQ ID NO:73), RRRR (SEQ ID NO:74), RRKR (SEQ ID NO:75), or RKKR (SEQ ID NO:76).
  • R, RR, RK, RKR, RRR, RRK, RKK, RKRR SEQ ID NO:73
  • RRRR SEQ ID NO:74
  • RRKR SEQ ID NO:75
  • RKKR SEQ ID NO:76
  • the furin linker has the sequence R-X-K/R-R (SEQ ID NO: 177/178), such that the additional amino acids on the C-terminus of the heavy chain are R, RX, RXK, RXR, RXKR (SEQ ID NO: 177), or RXRR (SEQ ID NO: 178), where X is any amino acid, for example, alanine (A).
  • no additional amino acids may remain on the C-terminus of the heavy chain.
  • a single construct can be engineered to encode both the heavy and light chains (e.g. the heavy and light chain variable domains) separated by a flexible peptide linker such as those encoding a scFv.
  • a flexible peptide linker can be composed of flexible residues like glycine and serine so that the adjacent heavy chain and light chain domains are free to move relative to one another.
  • the construct may be arranged such that the heavy chain variable domain is at the N-terminus of the scFv, followed by the linker and then the light chain variable domain.
  • the construct may be arranged such that the light chain variable domain is at the N-terminus of the scFv, followed by the linker and then the heavy chain variable domain. That is, the components may be arranged as NFE-VL-linker-VH-COOH or NFE-VH-linker-VL-COOH.
  • an expression cassette described herein is contained within a viral vector with a restraint on the size of the polynucleotide(s) therein.
  • the expression cassette is contained within an AAV virus-based vector. Due to the size restraints of certain vectors, the vector may or may not accommodate the coding sequences for the full heavy and light chains of the therapeutic antibody but may accommodate the coding sequences of the heavy and light chains of antigen binding fragments, such as the heavy and light chains of a Fab or F(ab’)2 fragment or an scFv.
  • the AAV vectors described herein may accommodate a transgene of approximately 4.7 kilobases. Substitution of smaller expression elements would permit the expression of larger protein products, such as full-length therapeutic antibodies.
  • the viral vectors provided herein comprise one or more untranslated regions (UTRs), e.g., 3’ and/or 5’ UTRs.
  • UTRs are optimized for the desired level of protein expression.
  • the UTRs are optimized for the mRNA half-life of the transgene.
  • the UTRs are optimized for the stability of the mRNA of the transgene.
  • the UTRs are optimized for the secondary structure of the mRNA of the transgene.
  • the viral vectors provided herein comprise one or more inverted terminal repeat (ITR) sequences.
  • ITR sequences may be used for packaging the recombinant gene expression cassette into the virion of the viral vector.
  • the ITR is from an AAV, e.g., AAV8 or AAV2 (see, e.g., Yan et al., 2005, J. Virol., 79(l):364-379; United States Patent No. 7,282,199 B2, United States Patent No. 7,790,449 B2, United States Patent No. 8,318,480 B2, United States PatentNo. 8,962,332 B2 and International Patent Application No.
  • nucleotide sequences encoding the ITRs may, for example, comprise the nucleotide sequences of SEQ ID NOS: 245 (5 ’-ITR) or 247 (3 ’-ITR).
  • the modified ITRs used to produce self- complementary vector e.g, sc AAV, may be used (see, e.g, Wu, 2007, Human Gene Therapy, 18(2): 171-82, McCarty et al, 2001, Gene Therapy, Vol 8, Number 16, Pages 1248-1254; and U.S. Patent Nos.
  • nucleotide sequences encoding the modified ITRs may, for example, comprise the nucleotide sequences of SEQ ID NOS:246 (5 ’-ITR) or 248 (3 ’-ITR).
  • the transgenes encode a HuPTM mAb, either as a full-length antibody or an antigen binding fragment thereof, e.g. a Fab fragment (an HuGlyFab) or a F(ab’)2, nanobody, or an scFv based upon a therapeutic antibody disclosed herein.
  • the HuPTM mAb or antigen binding fragment, particularly the HuGlyFab are engineered to contain additional glycosylation sites on the Fab domain (e.g., see Courtois et al., 2016, mAbs 8: 99-112 which is incorporated by reference herein in its entirety for it description of sites of hyperglycosylation on a Fab domain).
  • the Fc domain may be engineered to alter the glycosylation site at N297 to prevent glycosylation at that site (for example, a substitution at N297 for another amino acid and/or a substitution at T297 for a residue that is not a T or S to knock out the glycosylation site).
  • Such Fc domains are “aglycosylated”.
  • the transgenes encode a full length heavy chain (including the heavy chain variable domain, the heavy chain constant domain 1 (CHI), the hinge and Fc domain) and a full length light chain (light chain variable domain and light chain constant domain) that upon expression associate to form antigen-binding antibodies with Fc domains.
  • the recombinant AAV constructs express the intact (i.e., full length) or substantially intact HuPTM mAb in a cell, cell culture, or in a subject.
  • the nucleotide sequences encoding the heavy and light chains may be codon optimized for expression in human cells and have reduced incidence of CpG dimers in the sequence to promote expression in human cells.
  • the transgenes may encode any full-length antibody. In preferred embodiments, the transgenes encode a full-length form of any of the therapeutic antibodies disclosed herein, for example, the Fab fragment of which depicted in FIG. 2A-2D herein and including, in certain embodiments, the associated Fc domain provided in Table 7.
  • the full length mAb encoded by the transgene described herein preferably have the Fc domain of the full-length therapeutic antibody or is an Fc domain of the same type of immunoglobulin as the therapeutic antibody to be expressed.
  • the Fc region is an IgG Fc region, but in other embodiments, the Fc region may be an IgA, IgD, IgE, or IgM.
  • the Fc domain is preferably of the same isotype as the therapeutic antibody to be expressed, for example, if the therapeutic antibody is an IgGl isotype, then the antibody expressed by the transgene comprises an IgGl Fc domain.
  • the antibody expressed from the transgene may have an IgGl, IgG2, IgG3 or IgG4 Fc domain.
  • the Fc region of the intact mAb has one or more effector functions that vary with the antibody isotype.
  • the effector functions can be the same as that of the wild-type or the therapeutic antibody or can be modified therefrom to add, enhance, modify, or inhibit one or more effector functions using the Fc modifications disclosed in Section 5.1.9, infra.
  • the HuPTM mAb transgene encodes a mAb comprising an Fc polypeptide comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in the Fc domain polypeptides of the therapeutic antibodies described herein as set forth in Table 7 for erenumab, eptinezumab, fremanezumab, and galcanezumab or an exemplary Fc domain of an IgGl, IgG2 or IgG4 isotype as set forth in Table 7.
  • the HuPTM mAb comprises a Fc polypeptide of a sequence that is a variant of the Fc polypeptide sequence in Table 7 in that the sequence has been modified with one or more of the techniques described in Section 5.1.9, infra, to alter the Fc polypeptide’s effector function.
  • recombinant AAV constructs such as the constructs shown in FIGS. 1 A and IB, for gene therapy administration to a human subject in order to express an intact or substantially intact HuPTM mAb in the subject.
  • Gene therapy constructs are designed such that both the heavy and light chains are expressed in tandem from the vector including the Fc domain polypeptide of the heavy chain.
  • the transgene encodes a transgene with heavy and light chain Fab fragment polypeptides as shown in Table 7, yet have a heavy chain that further comprises an Fc domain polypeptide C terminal to the hinge region of the heavy chain (including an IgGl, IgG2 or IgG4 Fc domain or the erenumab, eptinezumab, fremanezumab, and galcanezumab Fc as in Table 7).
  • the transgene is a nucleotide sequence that encodes the following: Signal sequence-heavy chain Fab portion (including hinge region)-heavy chain Fc polypeptide-Furin-2A linker-signal sequence-light chain Fab portion.
  • the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank the expression cassette; (2) Control elements, which include a) an inducible promoter, preferably a hypoxia-inducible promoter, b) a chicken P-actin intron and c) a rabbit P-globin poly A signal; and (3) nucleic acid sequences coding for the heavy chain Fab of an anti-CGRP or anti-CGRPR mAb (e.g., erenumab, eptinezumab, fremanezumab, or galcanezumab); an Fc polypeptide associated with the therapeutic antibody (Table 7) or of the same isotype as the native form of the therapeutic antibody, such as an IgG isotype amino acid sequence from Table 7; and the light chain of an anti-CGRP or anti-CGRPR mAh (e.g.
  • erenumab eptinezumab, fremanezumab, or galcanezumab
  • the heavy chain (Fab and Fc region) and the light chain are separated by a self-cleaving furin (F)/F2 A or T2A or flexible linker, ensuring expression of equal amounts of the heavy and the light chain polypeptides.
  • Exemplary constructs are provided in FIGS. lA and IB.
  • AAV vectors comprising a viral capsid that is at least 95% identical to the amino acid sequence of an AAV9 capsid (SEQ ID NO: 139); and an artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), wherein the expression cassette comprises a transgene encoding an intact or substantially intact anti-CGRP or anti-CGRPR mAb; operably linked to one or more regulatory sequences that control expression of the transgene in human liver or muscle cells.
  • ITRs AAV inverted terminal repeats
  • the rAAV vectors that encode and express the full-length therapeutic antibodies may be administered to treat or prevent or ameliorate symptoms of a disease or condition amenable to treatment, prevention or amelioration of symptoms with the therapeutic antibodies. Also provided are methods of expressing HuPTM mAbs in human cells using the rAAV vectors and constructs encoding them.
  • the transgenes express antigen binding fragments, e.g. a Fab fragment (an HuGlyFab) or a F(ab’)2, nanobody, or an scFv based upon a therapeutic antibody disclosed herein.
  • FIGS. 2A-2D and section 5.4. provide the amino acid sequence of the heavy and light chains of the Fab fragments of the therapeutic antibodies (see also Table 8, which provides the amino acid sequences of the Fab heavy and light chains of the therapeutic antibodies).
  • nucleotide sequences are codon optimized for expression in human cells.
  • the transgene may encode a Fab fragment using nucleotide sequences encoding the amino acid sequences provided in Table 8, but not including the portion of the hinge region on the heavy chain that forms interchain di-sulfide bonds (e.g., the portion containing the sequence CPPCPA (SEQ ID NO:94)).
  • Heavy chain Fab domain sequences that do not contain a CPPCP (SEQ ID NO:95) sequence of the hinge region at the C-terminus will not form intrachain disulfide bonds and, thus, will form Fab fragments with the corresponding light chain Fab domain sequences, whereas those heavy chain Fab domain sequences with a portion of the hinge region at the C-terminus containing the sequence CPPCP (SEQ ID NO:95) will form intrachain disulfide bonds and, thus, will form Fab2 fragments.
  • CPPCP SEQ ID NO:95
  • the transgene may encode a scFv comprising a light chain variable domain and a heavy chain variable domain connected by a flexible linker in between (where the heavy chain variable domain may be either at the N-terminal end or the C-terminal end of the scFv), and optionally, may further comprise a Fc polypeptide (e.g., IgGl, IgG2, IgG3, or IgG4) on the C-terminal end of the heavy chain.
  • a Fc polypeptide e.g., IgGl, IgG2, IgG3, or IgG4
  • the transgene may encode F(ab’)2 fragments comprising a nucleotide sequence that encodes the light chain and the heavy chain sequence that includes at least the sequence CPPCA (SEQ ID NO: 96) of the hinge region, as depicted in FIGS. 2A-2D which depict various regions of the hinge region that may be included at the C-terminus of the heavy chain sequence.
  • Pre-existing anti-hinge antibodies may cause immunogenicity and reduce efficacy.
  • C-terminal ends with D221 or ends with a mutation T225L or with L242 can reduce binding to AHA.
  • the viral vectors provided herein comprise the following elements in the following order: a) a constitutive or inducible (e.g., hypoxia-inducible or rifamycin- inducible) promoter sequence or a tissue specific promoter/regulatory region, for example, one of the regulatory regions provided in Table 1, and b) a sequence encoding the transgene (e.g., a HuGlyFab).
  • the sequence encoding the transgene comprises multiple ORFs separated by IRES elements.
  • the ORFs encode the heavy and light chain domains of the HuGlyFab.
  • the sequence encoding the transgene comprises multiple subunits in one ORF separated by F/F2A sequences or F/T2A sequences. In certain embodiments, the sequence comprising the transgene encodes the heavy and light chain domains of the HuGlyFab separated by an F/F2A sequence or a F/T2A sequence. In certain embodiments, the sequence comprising the transgene encodes the heavy and light chain variable domains of the HuGlyFab separated by a flexible peptide linker (as an scFv).
  • the viral vectors provided herein comprise the following elements in the following order: a) a constitutive or an inducible promoter sequence or a tissue specific promoter, such as one of the promoters or regulatory regions in Table 1, and b) a sequence encoding the transgene (e.g., a HuGlyFab), wherein the transgene comprises a nucleotide sequence encoding a signal peptide, a light chain and a heavy chain Fab portion separated by an IRES element.
  • a constitutive or an inducible promoter sequence or a tissue specific promoter such as one of the promoters or regulatory regions in Table 1
  • a sequence encoding the transgene e.g., a HuGlyFab
  • the viral vectors provided herein comprise the following elements in the following order: a) a constitutive or a hypoxia-inducible promoter sequence or regulatory element listed in Table 1, and b) a sequence encoding the transgene comprising a signal peptide, a light chain and a heavy chain sequence separated by a cleavable F/F2A sequence (SEQ ID NOS: 85 or 86) or a F/T2A sequence (SEQ ID NOS: 87 or 88) or a flexible peptide linker.
  • the viral vectors provided herein comprise the following elements in the following order: a) a constitutive or a tissue specific promoter/regulatory region, for example, one of the regulatory regions provided in Table 1, and b) a sequence encoding the first transgene (e.g., a HuGlyFab), c) a second constitutive or a tissue specific promoter/regulatory region, and d) a sequence encoding the second transgene.
  • the sequences encoding the first and second transgene comprise each multiple subunits in one ORF separated by F/F2A sequences or F/T2A sequences.
  • sequences comprising the first and second transgene encode each the heavy and light chain domains of a HuGlyFab separated by an F/F2A sequence or a F/T2A sequence. In certain embodiments, the sequences comprising the first and second transgene encode each the heavy and light chain variable domains of a HuGlyFab separated by a flexible peptide linker (as an scFv).
  • the viral vectors provided herein comprise a first and a second transgene, wherein the first transgene encodes a heavy chain and a light chain of an antigen-binding fragment of an anti-CGRP operably linked to a first regulatory sequence, and the second transgene encodes a heavy and light chain of an antigen binding fragment of an anti-CGRPR antibody, operably linked to a second regulatory sequence, wherein said first and second regulatory sequences promote expression of the transgene in human CNS, PNS, arterial smooth muscle and/or liver cells, and the first and second regulatory sequences promote expression of the first and second transgenes in different human cell types.
  • the viral vectors comprise the following elements in the following order: a) a first constitutive or a tissue specific promoter, b) a first sequence encoding the first transgene, c) a second constitutive or a tissue specific promoter, d) a second sequence encoding the second transgene, wherein both the first and second transgene comprise a nucleotide sequence encoding a signal peptide, a light chain and a heavy chain Fab portion.
  • the viral vectors provided herein comprise the following elements in the following order: a) a first tissuespecific promoter, b) a first sequence encoding a first transgene, c) a second tissue specific promoter, d) a second sequence encoding the second transgene, wherein each transgene comprises a signal peptide, a light chain and a heavy chain sequence separated by a cleavable F/F2A sequence or a F/T2A sequence (SEQ ID NOS: 198 or 199) or a flexible peptide linker; and wherein the first and second promoter promote expression of the first and second transgene in different cell types.
  • the constructs described herein comprise the following components: (1) AAV2 inverted terminal repeats that flank the expression cassette; (2) first group control elements, which include a) an ubiquitous (e.g. CAG promoter) or tissue-specific promoter (e.g. sm22a promoter), b) a chicken P-actin intron and c) a rabbit P-globin poly A signal; (d) a nucleic acid sequence coding for the heavy and light chain Fab of an anti-CGRP mAb (e.g., eptinezumab, fremanezumab, or galcanezumab); (4) a second group control elements, which include a) an ubiquitous (e.g.
  • CAG promoter or tissue-specific promoter (e.g. sm22a promoter), b) a chicken P-actin intron and c) a rabbit P-globin poly A signal; and (d) nucleic acid sequences coding for the heavy and light chain Fab of an anti-CGRPR mAb (including erenumab).
  • the transgenes encode full length or substantially full length heavy and light chains that associate to form a full length or intact antibody.
  • substantially intact or substantially full length refers to a mAb having a heavy chain sequence that is at least 95% identical to the full-length heavy chain mAb amino acid sequence and a light chain sequence that is at least 95% identical to the full-length light chain mAb amino acid sequence.
  • the transgenes comprise nucleotide sequences that encode, for example, the light and heavy chains of the Fab fragments including the hinge region of the heavy chain and C-terminal of the heavy chain of the Fab fragment, an Fc domain peptide.
  • Table 7 provides the amino acid sequence of the Fc polypeptides for erenumab, eptinezumab, fremanezumab, and galcanezumab.
  • an IgGl, IgG2, or IgG4 Fc domain the sequences of which are provided in Table 7 may be utilized.
  • Fc region refers to a dimer of two "Fc polypeptides” (or “Fc domains”), each "Fc polypeptide” comprising the heavy chain constant region of an antibody excluding the first constant region immunoglobulin domain.
  • an "Fc region” includes two Fc polypeptides linked by one or more disulfide bonds, chemical linkers, or peptide linkers.
  • Fc polypeptide refers to at least the last two constant region immunoglobulin domains of IgA, IgD, and IgG, or the last three constant region immunoglobulin domains of IgE and IgM and may also include part or all of the flexible hinge N-terminal to these domains.
  • Fc polypeptide comprises immunoglobulin domains Cgamma2 (Cy2, often referred to as CH2 domain) and Cgamma3 (Cy3, also referred to as CH3 domain) and may include the lower part of the hinge domain between Cgammal (Cyl, also referred to as CHI domain) and CH2 domain.
  • the human IgG heavy chain Fc polypeptide is usually defined to comprise residues starting at T223 or C226 or P230, to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Services, Springfield, Va.).
  • Fc polypeptide comprises immunoglobulin domains Calpha2 (Ca2) and Calpha3 (Ca3) and may include the lower part of the hinge between Calphal (Cal) and Ca2.
  • the Fc polypeptide is that of the therapeutic antibody or is the Fc polypeptide corresponding to the isotype of the therapeutic antibody).
  • the Fc polypeptide is an IgG Fc polypeptide.
  • the Fc polypeptide may be from the IgGl, IgG2, or IgG4 isotype (see Table 7) or may be an IgG3 Fc domain, depending, for example, upon the desired effector activity of the therapeutic antibody.
  • the engineered heavy chain constant region (CH), which includes the Fc domain is chimeric. As such, a chimeric CH region combines CH domains derived from more than one immunoglobulin isotype and/or subtype.
  • the chimeric (or hybrid) CH region comprises part or all of an Fc region from IgG, IgA and/or IgM.
  • the chimeric CH region comprises part or all a CH2 domain derived from a human IgGl, human IgG2, or human IgG4 molecule, combined with part or all of a CH3 domain derived from a human IgGl, human IgG2, or human IgG4 molecule.
  • the chimeric CH region contains a chimeric hinge region.
  • the recombinant vectors encode therapeutic antibodies comprising an engineered (mutant) Fc regions, e.g. engineered Fc regions of an IgG constant region.
  • Modifications to an antibody constant region, Fc region or Fc fragment of an IgG antibody may alter one or more effector functions such as Fc receptor binding or neonatal Fc receptor (FcRn) binding and thus half-life, CDC activity, ADCC activity, and/or ADPC activity, compared to a corresponding antibody having a wild-type IgG constant region, or an IgG heavy chain constant region without the recited modification(s).
  • the antibody may be engineered to provide an antibody constant region, Fc region or Fc fragment of an IgG antibody that exhibits altered binding (as compared to a reference or wild-type constant region without the recited modification(s)) to one or more Fc receptors (e g., FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA, FcyRIIIB, FcyRI V, or FcRn receptor).
  • Fc receptors e g., FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA, FcyRIIIB, FcyRI V, or FcRn receptor.
  • the antibody an antibody constant region, Fc region or Fc fragment of an IgG antibody that exhibits a one or more altered effector functions such as CDC, ADCC, or ADCP activity, compared to a corresponding antibody having a wild-type IgG constant region, or an IgG constant without the recited modification(s).
  • Effective function refers to a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include FcyR-mediated effector functions such as ADCC and ADCP and complement-mediated effector functions such as CDC.
  • effector cell refers to a cell of the immune system that expresses one or more Fc receptors and mediates one or more effector functions. Effector cells include but are not limited to monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans' cells, natural killer (NK) cells, and T cells, and may be from any organism including but not limited to humans, mice, rats, rabbits, and monkeys.
  • ADCC antibody dependent cell-mediated cytotoxicity
  • FcyRs cytotoxic effector cells that express FcyRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell.
  • ADCP antibody dependent cell-mediated phagocytosis
  • FcyRs cytotoxic effector cells that express FcyRs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell.
  • CDC or “complement-dependent cytotoxicity” refers to the reaction wherein one or more complement protein components recognize bound antibody on a target cell and subsequently cause lysis of the target cell.
  • the modifications of the Fc domain include, but are not limited to, the following modifications and combinations thereof, with reference to EU numbering of an IgG constant region (see FIG. 6): 233, 234, 235, 236, 237, 238, 239, 248, 249, 250, 252, 254, 255, 256,
  • the Fc region comprises an amino acid addition, deletion, or substitution of one or more of amino acid residues 251-256, 285-290, 308-314, 385-389, and 428-436 of the IgG.
  • 251-256, 285-290, 308-314, 385-389, and 428-436 (EU numbering of Kabat; see FIG. 5) is substituted with histidine, arginine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine.
  • a non-histidine residue is substituted with a histidine residue.
  • a histidine residue is substituted with a non-histidine residue.
  • Enhancement of FcRn binding by an antibody having an engineered Fc leads to preferential binding of the affinity-enhanced antibody to FcRn as compared to antibody having wildtype Fc, and thus leads to a net enhanced recycling of the FcRn-affinity-enhanced antibody, which results in further increased antibody half-life.
  • An enhanced recycling approach allows highly effective targeting and clearance of antigens, including e.g. "high titer" circulating antigens, such as C5, cytokines, or bacterial or viral antigens.
  • modified constant region, Fc region or Fc fragment of an IgG antibody with enhanced binding to FcRn in serum as compared to a wild-type Fc region are engineered modifications.
  • antibodies e.g. IgG antibodies
  • antibodies, e.g. IgG antibodies are engineered to exhibit enhanced binding (e.g. increased affinity or KD) to FcRn in endosomes (e.g.
  • an acidic pH e.g. , at or below pH 6.0
  • a wildtype IgG and/or reference antibody binding to FcRn at an acidic pH as well as in comparison to binding to FcRn in serum (e.g., at a neutral pH, e.g., at or above pH 7.4).
  • serum e.g., at a neutral pH, e.g., at or above pH 7.4
  • an engineered antibody constant region, Fc region or Fc fragment of an IgG antibody that exhibits an improved serum or resident tissue half-life, compared to a corresponding antibody having a wild-type IgG constant region, or an IgG constant without the recited modification(s);
  • Non-limiting examples of such Fc modifications include, e.g., a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., LN/Y/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and 434.
  • a modification at position 250 e.g., E or Q
  • 250 and 428 e.g., L or F
  • 252 e.g., LN/Y/W or T
  • 254 e.g., S or T
  • the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591 (e.g., V2591), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a 307 and/or 308 modification (e.g., 308F or 308P) (EU numbering; see FIG 5).
  • a 428L e.g., M428L
  • 434S e.g., N434S
  • a 428L, 2591 e.g., V2591
  • 308F e.g.,
  • the Fc region can be a mutant form such as hlgGl Fc including M252 mutations, e.g. M252Y and S254T and T256E (“YTE mutation”) exhibit enhanced affinity for human FcRn (Dall’Acqua, et al., 2002, J Immunol 169:5171-5180) and subsequent crystal structure of this mutant antibody bound to hFcRn resulting in the creation of two salt bridges (Oganesyan, et al. 2014, JBC 289(11): 7812-7824).
  • Antibodies having the YTE mutation have been administered to monkeys and humans, and have significantly improved pharmacokinetic properties (Haraya, et al., 2019, Drug Metabolism and Pharmacokinetics, 34(1):25-41).
  • modifications to one or more amino acid residues in the Fc region may reduce half-life in systemic circulation (serum), however result in improved retainment in tissues (e.g. in the eye) by disabling FcRn binding (e.g. H435A, EU numbering of Kabat) (Ding et al., 2017, MAbs 9:269-284; and Kim, 1999, Eur J Immunol 29:2819).
  • FcRn binding e.g. H435A, EU numbering of Kabat
  • the Fc domain may be engineered to activate all, some, or none of the normal Fc effector functions, without affecting the Fc polypeptide’s (e.g. antibody's) desired pharmacokinetic properties.
  • Fc polypeptides having altered effector function may be desirable as they may reduce unwanted side effects, such as activation of effector cells, by the therapeutic protein.
  • Methods to alter or even ablate effector function may include mutation(s) or modification(s) to the hinge region amino acid residues of an antibody.
  • IgG Fc domain mutants comprising 234A, 237A, and 238S substitutions, according to the EU numbering system, exhibit decreased complement dependent lysis and/or cell mediated destruction.
  • Deletions and/or substitutions in the lower hinge e.g. where positions 233-236 within a hinge domain (EU numbering) are deleted or modified to glycine, have been shown in the art to significantly reduce ADCC and CDC activity.
  • the Fc domain is an aglycosylated Fc domain that has a substitution at residue 297 or 299 to alter the glycosylation site at 297 such that the Fc domain is not glycosylated.
  • Such aglycosylated Fc domains may have reduced ADCC or other effector activity.
  • Non-limiting examples of proteins comprising mutant and/or chimeric CH regions having altered effector functions, and methods of engineering and testing mutant antibodies, are described in the art, e.g. K.L. Amour, et al., Eur. J. Immunol. 1999, 29:2613-2624; Lazar et al., Proc. Natl. Acad. Sci. USA 2006, 103:4005; US Patent Application Publication No. 20070135620A1 published June 14, 2007; US Patent Application Publication No. 20080154025 Al, published June 26, 2008; US Patent Application Publication No. 20100234572 Al, published September 16, 2010; US Patent Application Publication No. 20120225058 Al, published September 6, 2012; US Patent Application Publication No.
  • the C-terminal lysines (-K) conserved in the heavy chain genes of all human IgG subclasses are generally absent from antibodies circulating in serum - the C-terminal lysines are cleaved off in circulation, resulting in a heterogeneous population of circulating IgGs.
  • van den Bremer et al., 2015, mAbs 7:672-680 the DNA encoding the C-terminal lysine (-K) or glycine-lysine (-GK) of the Fc terminus can be deleted to produce a more homogeneous antibody product in situ.
  • the viral vectors provided herein may be manufactured using host cells.
  • the viral vectors provided herein may be manufactured using mammalian host cells, for example, A549, WEHI, 10T1/2, BHK, MDCK, C0S1, C0S7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, 293, Saos, C2C12, L, HT1080, HepG2, primary fibroblast, hepatocyte, and myoblast cells.
  • the viral vectors provided herein may be manufactured using host cells from human, monkey, mouse, rat, rabbit, or hamster.
  • the host cells are stably transformed with the sequences encoding the transgene and associated elements (e.g., the vector genome), and the means of producing viruses in the host cells, for example, the replication and capsid genes (e.g., the rep and cap genes of AAV).
  • the replication and capsid genes e.g., the rep and cap genes of AAV.
  • Genome copy titers of said vectors may be determined, for example, by TAQMAN® analysis.
  • Virions may be recovered, for example, by CsCh sedimentation.
  • baculovirus expression systems in insect cells may be used to produce AAV vectors.
  • AAV vectors See Aponte-Ubillus et al., 2018, Appl. Microbiol. Biotechnol. 102: 1045- 1054 which is incorporated by reference herein in its entirety for manufacturing techniques.
  • in vitro assays e.g., cell culture assays
  • transgene expression from a vector described herein thus indicating, e.g., potency of the vector.
  • in vitro neutralization assays can be used to measure the activity of the transgene expressed from a vector described herein.
  • Vero-E6 cells a cell line derived from the kidney of an African green monkey, or HeLa cells engineered to stably express the ACE2 receptor (HeLa-ACE2), can be used to assess neutralization activity of transgenes expressed from a vector described herein.
  • glycosylation and tyrosine sulfation patterns associated with the HuGlyFab can be determined, for example determination of the glycosylation and tyrosine sulfation patterns associated with the HuGlyFab. Glycosylation patterns and methods of determining the same are discussed in Section 5.3, while tyrosine sulfation patterns and methods of determining the same are discussed in Section 5.3.
  • benefits resulting from glycosylation/sulfation of the cell-expressed HuGlyFab can be determined using assays known in the art, e.g., the methods described in Section 5.3.
  • Vector genome concentration (GC) or vector genome copies can be evaluated using digital PCR (dPCR) or ddPCRTM (BioRad Technologies, Hercules, CA, USA).
  • dPCR digital PCR
  • ddPCRTM BioRad Technologies, Hercules, CA, USA
  • liver biopsies are obtained at several timepoints.
  • mice are sacrificed at various timepoints post injection.
  • Liver tissue samples are subjected to total DNA extraction and dPCR assay for vector copy numbers.
  • Copies of vector genome (transgene) per gram of tissue may be measured in a single biopsy sample, or measured in various tissue sections at sequential timepoints will reveal spread of AAV throughout the liver.
  • Total DNA from collected liver tissue is extracted with the DNeasy Blood & Tissue Kit and the DNA concentration measured using a Nanodrop spectrophotometer.
  • the copy number of delivered vector in a specific tissue section per diploid cell is calculated as: (vector copy number)/(endogenous control)*2.
  • Vector copy in specific cell types, such as liver cells, over time may indicate sustained expression of the transgene by the tissue.
  • compositions suitable for administration to human subjects comprise a suspension of the recombinant vector in a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant and optional excipients.
  • a formulation buffer can comprise one or more of a polysaccharide, a surfactant, polymer, or oil.
  • the pharmaceutical composition comprises rAAV combined with a pharmaceutically acceptable carrier for administration (e.g. intranasal, intravenous, intramuscular) to a subject.
  • a pharmaceutically acceptable carrier for administration (e.g. intranasal, intravenous, intramuscular) to a subject.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S.
  • carrier refers to a diluent, adjuvant e.g., Freund's complete and incomplete adjuvant), excipient, or vehicle with which the agent is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, including, e.g., peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a common carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • compositions include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin and gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM as known in the art.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • low molecular weight polypeptides proteins, such as serum albumin and gelatin
  • hydrophilic polymers such as
  • the pharmaceutical composition of the present invention can also include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative, in addition to the above ingredients.
  • a lubricant e.g., talc, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol
  • methods for treating migraine, cluster headaches or other indication that can be treated with an anti-CGRP or anti-CGRPR antibody in a subject in need thereof comprising the administration of recombinant AAV particles comprising an expression cassette encoding anti- CGRP or anti-CGRPR antibodies and antibody-binding fragments and variants thereof, or peptides, are provided.
  • a subject in need thereof includes a subject suffering from migraine or cluster headaches, or a subj ect pre-disposed thereto, e.g. , a subj ect at risk of developing or having a recurrence of the migraine or cluster headaches, or other indication that may be treated with an anti-CGRP or anti-CGRPR antibody.
  • Subjects to whom such gene therapy is administered can be those responsive to erenumab, eptinezumab, fremanezumab, or galcanezumab therapy.
  • the methods encompass treating patients who have been diagnosed with migraine or cluster headaches, and, in certain embodiments, identified as responsive to treatment with an anti-CGRP or anti-CGRPR antibody or considered a good candidate for therapy with an anti-CGRP or anti-CGRPR antibody.
  • the patients have previously been treated with an anti-CGRP or anti-CGRPR antibody.
  • the anti-CGRP or anti-CGRPR antibody or antigen-binding fragment transgene product may be administered directly to the subject.
  • the method of treating migraine or cluster headaches in a human subject in need thereof comprises intranasally or systemically administering to the subject a therapeutically effective amount of a composition or a recombinant nucleotide expression vector comprising a recombinant AAV comprising a transgene encoding an anti-CGRP or anti-CGRPR mAb, or antigen-binding fragment thereof, operably linked to one or more regulatory sequences that control expression of the transgene in CNS, PNS, liver and/or arterial smooth muscle cells.
  • a method of treating migraine or cluster headaches in a human subject in need thereof comprises intranasally or systemically administering to the subject a therapeutically effective amount of a composition comprising, i) a first recombinant AAV comprising a transgene encoding an anti-CGRP mAb, or antigen-binding fragment thereof, operably linked to one or more regulatory sequences that control expression of the transgene in CNS, PNS, liver and/or arterial smooth muscle cells; and ii) a second recombinant AAV comprising a transgene encoding an anti-CGRPR mAb, or antigen-binding fragment thereof, operably linked to one or more regulatory sequences that control expression of the transgene in CNS, PNS, liver and/or arterial smooth muscle cells.
  • the amino acid sequence (primary sequence) of HuGlyFabs or HuPTM Fabs, HuPTMmAbs, and HuPTM scFvs disclosed herein each comprises at least one site at which N- glycosylation or tyrosine sulfation takes place (see exemplary FIGs. 2A-2D) for glycosylation and/or sulfation positions within the amino acid sequences of the Fab fragments of the therapeutic antibodies).
  • Post-translational modification also occurs in the Fc domain of full length antibodies, particularly at residue N297 (by EU numbering, see Table 7).
  • mutations may be introduced into the Fc domain to alter the glycosylation site at residue N297 (EU numbering, see Table 7), in particular substituting another amino acid for the asparagine at 297 or the threonine at 299 to remove the glycosylation site resulting in an aglycosylated Fc domain.
  • the canonical N-glycosylation sequence is known in the art to be Asn-X-Ser(or Thr), wherein X can be any amino acid except Pro.
  • Asn asparagine residues of human antibodies can be glycosylated in the context of a reverse consensus motif, Ser(or Thr)-X-Asn, wherein X can be any amino acid except Pro.
  • Ser(or Thr)-X-Asn Asparagine (Asn) residues of human antibodies can be glycosylated in the context of a reverse consensus motif, Ser(or Thr)-X-Asn, wherein X can be any amino acid except Pro.
  • certain HuGlyFabs and HuPTM scFvs disclosed herein comprise such reverse consensus sequences.
  • glutamine (Gin) residues of human antibodies can be glycosylated in the context of a non-consensus motif, Gln-Gly-Thr. See Valliere-Douglass et al., 2010, J. Biol. Chem. 285: 16012-16022.
  • certain of the HuGlyFab fragments disclosed herein comprise such non-consensus sequences.
  • O-glycosylation comprises the addition of N-acetyl -galactosamine to serine or threonine residues by the enzyme. It has been demonstrated that amino acid residues present in the hinge region of antibodies can be O-glycosylated.
  • O-glycosylation confers another advantage to the therapeutic antibodies provided herein, as compared to, e.g., antigen-binding fragments produced in E. coli, again because the E. coli naturally does not contain machinery equivalent to that used in human O-glycosylation. (Instead, O-glycosylation in E. coli has been demonstrated only when the bacteria is modified to contain specific O-glycosylation machinery. See, e.g., Farid-Moayer et al., 2007, J. Bacteriol. 189:8088-8098.)
  • a nucleic acid encoding a HuPTM mAb, HuGlyFab or HuPTM scFv is modified to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more N-glycosylation sites (including the canonical N-glycosylation consensus sequence, reverse N-glycosylation site, and non-consensus N- glycosylation sites) than would normally be associated with the HuPTM mAb, HuGlyFab or HuPTM scFv (e.g., relative to the number of N-glycosylation sites associated with the HuPTM mAb, HuGlyFab or HuPTM scFv in its unmodified state).
  • introduction of glycosylation sites is accomplished by insertion of N-glycosylation sites (including the canonical N- glycosylation consensus sequence, reverse N-glycosylation site, and non-consensus N-glycosylation sites) anywhere in the primary structure of the antigen-binding fragment, so long as said introduction does not impact binding of the antibody or antigen-binding fragment to its antigen.
  • N-glycosylation sites including the canonical N- glycosylation consensus sequence, reverse N-glycosylation site, and non-consensus N-glycosylation sites
  • glycosylation sites can be accomplished by, e.g., adding new amino acids to the primary structure of the antigen-binding fragment, or the antibody from which the antigen-binding fragment is derived (e.g., the glycosylation sites are added, in full or in part), or by mutating existing amino acids in the antigen-binding fragment, or the antibody from which the antigen-binding fragment is derived, in order to generate the N-glycosylation sites (e.g., amino acids are not added to the antigen-binding fragment/antibody, but selected amino acids of the antigen-binding fragment/antibody are mutated so as to form N-glycosylation sites).
  • amino acid sequence of a protein can be readily modified using approaches known in the art, e.g. , recombinant approaches that include modification of the nucleic acid sequence encoding the protein.
  • a HuGlyMab or antigen-binding fragment is modified such that, when expressed in mammalian cells, such as retina, CNS, liver or muscle cells, it can be hyperglycosylated. See Courtois et al., 2016, mAbs 8:99-112 which is incorporated by reference herein in its entirety.
  • biologies Unlike small molecule drugs, biologies usually comprise a mixture of many variants with different modifications or forms that could have a different potency, pharmacokinetics, and/or safety profile. It is not essential that every molecule produced either in the gene therapy or protein therapy approach be fully glycosylated and sulfated. Rather, the population of glycoproteins produced should have sufficient glycosylation (including 2,6-sialylation) and sulfation to demonstrate efficacy.
  • the goal of gene therapy treatment provided herein can be, for example, to slow or arrest the progression of a disease or abnormal condition or to reduce the severity of one or more symptoms associated with the disease or abnormal condition.
  • N- glycosylation sites of the antigen-binding fragment can be glycosylated with various different glycans.
  • N-glycans of antigen-binding fragments and the Fc domain have been characterized in the art. For example, Bondt et al., 2014, Mol. & Cell. Proteomics 13.11 :3029-3039 (incorporated by reference herein in its entirety for its disclosure of Fab-associated N-glycans; see also, FIG.
  • Glycosylation of the Fc domain has been characterized and is a single N-linked glycan at asparagine 297 (EU numbering; see Table 7).
  • the glycan plays an integral structural and functional role, impacting antibody effector function, such as binding to Fc receptor (see, for example, Jennewein and Alter, 2017, Trends In Immunology 38:358 for a discussion of the role of Fc glycosylation in antibody function). Removal of the Fc region glycan almost completely ablates effector function (Jennewien and Alter at 362).
  • the composition of the Fc glycan has been shown to impact effector function, for example hypergalactosylation and reduction in fucosylation have been shown to increase ADCC activity while sialylation correlates with anti-inflammatory effects (Id. at 364).
  • Disease states, genetics and even diet can impact the composition of the Fc glycan in vivo.
  • the glycan composition can differ significantly by the type of host cell used for recombinant expression and strategies are available to control and modify the composition of the glycan in therapeutic antibodies recombinantly expressed in cell culture, such as CHO to alter effector function (see, for example, US 2014/0193404 by Hansen et al.).
  • the HuPTM mAbs provided herein may advantageously have a glycan at N297 that is more like the native, human glycan composition than antibodies expressed in non-human host cells.
  • the HuPTM mAh, HuGlyFab or HuPTM scFv are expressed in human cells, the need for in vitro production in prokaryotic host cells (e.g., E. colt) or eukaryotic host cells (e.g., CHO cells or NSO cells) is circumvented.
  • N-glycosylation sites of the HuPTM mAb, HuGlyFab or HuPTM scFv are advantageously decorated with glycans relevant to and beneficial to treatment of humans. Such an advantage is unattainable when CHO cells, NSO cells, or E.
  • coli are utilized in antibody/anti gen -binding fragment production, because e.g., CHO cells (1) do not express 2,6 sialyltransferase and thus cannot add 2,6 sialic acid during N-glycosylation; (2) can add Neu5Gc as sialic acid instead of Neu5Ac; and (3) can also produce an immunogenic glycan, the a-Gal antigen, which reacts with anti-a-Gal antibodies present in most individuals, which at high concentrations can trigger anaphylaxis; and because (4) E. coli does not naturally contain components needed for N-glycosylation.
  • Assays for determining the glycosylation pattern of antibodies, including antigenbinding fragments are known in the art.
  • hydrazinolysis can be used to analyze glycans.
  • polysaccharides are released from their associated protein by incubation with hydrazine (the Ludger Liberate Hydrazinolysis Glycan Release Kit, Oxfordshire, UK can be used).
  • the nucleophile hydrazine attacks the glycosidic bond between the polysaccharide and the carrier protein and allows release of the attached glycans.
  • N-acetyl groups are lost during this treatment and have to be reconstituted by re-N-acetylation.
  • Glycans may also be released using enzymes such as glycosidases or endoglycosidases, such as PNGase F and Endo H, which cleave cleanly and with fewer side reactions than hydrazines.
  • the free glycans can be purified on carbon columns and subsequently labeled at the reducing end with the fluorophor 2-amino benzamide.
  • the labeled polysaccharides can be separated on a GlycoSep-N column (GL Sciences) according to the HPLC protocol of Royle et al, Anal Biochem 2002, 304(l):70-90. The resulting fluorescence chromatogram indicates the polysaccharide length and number of repeating units.
  • Structural information can be gathered by collecting individual peaks and subsequently performing MS/MS analysis. Thereby the monosaccharide composition and sequence of the repeating unit can be confirmed and additionally in homogeneity of the polysaccharide composition can be identified. Specific peaks of low or high molecular weight can be analyzed by MALDI-MS/MS and the result used to confirm the glycan sequence. Each peak in the chromatogram corresponds to a polymer, e.g., glycan, consisting of a certain number of repeat units and fragments, e.g., sugar residues, thereof. The chromatogram thus allows measurement of the polymer, e.g., glycan, length distribution.
  • the elution time is an indication for polymer length, while fluorescence intensity correlates with molar abundance for the respective polymer, e.g., glycan.
  • fluorescence intensity correlates with molar abundance for the respective polymer, e.g., glycan.
  • Other methods for assessing glycans associated with antigen-binding fragments include those described by Bondt et al., 2014, Mol. & Cell. Proteomics 13.11 :3029-3039, Huang et al., 2006, Anal. Biochem. 349: 197-207, and/or Song et al., 2014, Anal. Chem. 86:5661-5666.
  • Homogeneity or heterogeneity of the glycan patterns associated with antibodies can be assessed using methods known in the art, e.g., methods that measure glycan length or size and hydrodynamic radius.
  • HPLC such as size exclusion, normal phase, reversed phase, and anion exchange HPLC, as well as capillary electrophoresis, allows the measurement of the hydrodynamic radius. Higher numbers of glycosylation sites in a protein lead to higher variation in hydrodynamic radius compared to a carrier with less glycosylation sites.
  • Glycan length can be measured by hydrazinolysis, SDS PAGE, and capillary gel electrophoresis.
  • homogeneity can also mean that certain glycosylation site usage patterns change to a broader/narrower range. These factors can be measured by Glycopeptide LC-MS/MS.
  • the HuPTM mAbs, or antigen binding fragments thereof also do not contain detectable NeuGc and/or a-Gal.
  • detectable NeuGc or “detectable a-Gal” or “does not contain or does not have NeuGc or a-Gal” means herein that the HuPTM mAb or antigen-binding fragment, does not contain NeuGc or a-Gal moieties detectable by standard assay methods known in the art.
  • NeuGc may be detected by HPLC according to Hara et al., 1989, “Highly Sensitive Determination of - Acetyl -and N-Glycolylneuraminic Acids in Human Serum and Urine and Rat Serum by Reversed-Phase Liquid Chromatography with Fluorescence Detection.” J. Chromatogr., B: Biomed. 377, 111-119, which is hereby incorporated by reference for the method of detecting NeuGc.
  • NeuGc may be detected by mass spectrometry.
  • the a-Gal may be detected using an ELISA, see, for example, Galili et al., 1998, “A sensitive assay for measuring a-Gal epitope expression on cells by a monoclonal anti-Gal antibody.” Transplantation. 65(8): 1129-32, or by mass spectrometry, see, for example, Ayoub et al., 2013, “Correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques.” Austin Bioscience. 5(5):699-710.
  • N-glycosylation confers numerous benefits on the HuPTM mAb, HuGlyFab or HuPTM scFv described herein. Such benefits are unattainable by production of antigen-binding fragments in E. coli, because E. coli does not naturally possess components needed for N-glycosylation.
  • CHO cells or murine cells such as NS0 cells
  • CHO cells lack components needed for addition of certain glycans (e.g, 2,6 sialic acid and bisecting GlcNAc) and because either CHO or murine cell lines add N-N- Glycolylneuraminic acid (“Neu5Gc” or “NeuGc”) which is not natural to humans (and potentially immunogenic), instead of N-Acetylneuraminic acid (“Neu5Ac”) the predominant human sialic acid.
  • Neu5Gc N-N- Glycolylneuraminic acid
  • Ne5Ac N-Acetylneuraminic acid
  • CHO cells can also produce an immunogenic glycan, the a-Gal antigen, which reacts with anti-a-Gal antibodies present in most individuals, which at high concentrations can trigger anaphylaxis. See, e.g., Bosques, 2010, Nat. Biotech. 28: 1153-1156.
  • the human glycosylation pattern of the HuGlyFab of HuPTM scFv described herein should reduce immunogenicity of the transgene product and improve efficacy.
  • Fab glycosylation may affect the stability, half-life, and binding characteristics of an antibody.
  • any technique known to one of skill in the art may be used, for example, enzyme linked immunosorbent assay (ELISA), or surface plasmon resonance (SPR).
  • any technique known to one of skill in the art may be used, for example, by measurement of the levels of radioactivity in the blood or organs in a subject to whom a radiolabelled antibody has been administered.
  • any technique known to one of skill in the art may be used, for example, differential scanning calorimetry (DSC), high performance liquid chromatography (HPLC), e.g., size exclusion high performance liquid chromatography (SEC-HPLC), capillary electrophoresis, mass spectrometry, or turbidity measurement.
  • DSC differential scanning calorimetry
  • HPLC high performance liquid chromatography
  • SEC-HPLC size exclusion high performance liquid chromatography
  • capillary electrophoresis capillary electrophoresis
  • mass spectrometry or turbidity measurement.
  • sialic acid on HuPTM mAb, HuGlyFab or HuPTM scFv used in the methods described herein can impact clearance rate of the HuPTM mAb, HuGlyFab or HuPTM scFv. Accordingly, sialic acid patterns of a HuPTM mAb, HuGlyFab or HuPTM scFv can be used to generate a therapeutic having an optimized clearance rate. Methods of assessing antigen-binding fragment clearance rate are known in the art. See, e.g., Huang et al., 2006, Anal. Biochem. 349: 197-207.
  • a benefit conferred by N-glycosylation is reduced aggregation.
  • Occupied N-glycosylation sites can mask aggregation prone amino acid residues, resulting in decreased aggregation.
  • Such N-glycosylation sites can be native to an antigen-binding fragment used herein or engineered into an antigen-binding fragment used herein, resulting in HuGlyFab or HuPTM scFv that is less prone to aggregation when expressed, e.g., expressed in human cells.
  • Methods of assessing aggregation of antibodies are known in the art. See, e.g., Courtois et al., 2016, mAbs 8:99-112 which is incorporated by reference herein in its entirety.
  • a benefit conferred by N-glycosylation is reduced immunogenicity.
  • Such N-glycosylation sites can be native to an antigen-binding fragment used herein or engineered into an antigen-binding fragment used herein, resulting in HuPTM mAb, HuGlyFab or HuPTM scFv that is less prone to immunogenicity when expressed, e.g., expressed in human retinal cells, human CNS cells, human liver cells or human muscle cells.
  • N-glycosylation is protein stability.
  • N-glycosylation of proteins is well-known to confer stability on them, and methods of assessing protein stability resulting from N-glycosylation are known in the art. See, e.g., Sola and Griebenow, 2009, J Pharm Sci., 98(4): 1223-1245.
  • a benefit conferred by N-glycosylation is altered binding affinity. It is known in the art that the presence of N-glycosylation sites in the variable domains of an antibody can increase the affinity of the antibody for its antigen. See, e.g., Bovenkamp et al., 2016, J. Immunol. 196: 1435-1441. Assays for measuring antibody binding affinity are known in the art. See, e.g., Wright et al., 1991, EMBO J. 10:2717-2723; and Leibiger et al., 1999, Biochem. J. 338:529-538.
  • Tyrosine sulfation occurs at tyrosine (Y) residues with glutamate (E) or aspartate (D) within +5 to -5 position of Y, and where position -1 of Y is a neutral or acidic charged amino acid, but not a basic amino acid, e.g., arginine (R), lysine (K), or histidine (H) that abolishes sulfation.
  • the HuGlyFabs and HuPTM scFvs described herein comprise tyrosine sulfation sites (see exemplary FIG. 2).
  • tyrosine-sulfated anti gen -binding fragments cannot be produced in E. coli, which naturally does not possess the enzymes required for tyrosine-sulfation.
  • CHO cells are deficient for tyrosine sulfation-they are not secretory cells and have a limited capacity for post- translational tyrosine-sulfation. See, e.g., Mikkelsen & Ezban, 1991, Biochemistry 30: 1533-1537.
  • the methods provided herein call for expression of HuPTM Fab in human cells that are secretory and have capacity for tyrosine sulfation.
  • Tyrosine sulfation is advantageous for several reasons.
  • tyrosine-sulfation of the antigen-binding fragment of therapeutic antibodies against targets has been shown to dramatically increase avidity for antigen and activity.
  • Assays for detection tyrosine sulfation are known in the art. See, e.g., Yang et al., 2015, Molecules 20:2138-2164.
  • O-glycosylation comprises the addition of N-acetyl-galactosamine to serine or threonine residues by the enzyme. It has been demonstrated that amino acid residues present in the hinge region of antibodies can be O-glycosylated.
  • the HuGlyFab comprise all or a portion of their hinge region, and thus are capable of being O-glycosylated when expressed in human cells. The possibility of O-glycosylation confers another advantage to the HuGlyFab provided herein, as compared to, e.g., antigen-binding fragments produced in E. coli, again because the E. coli naturally does not contain machinery equivalent to that used in human O-glycosylation.
  • O- glycosylation in E. coli has been demonstrated only when the bacteria is modified to contain specific O-glycosylation machinery. See, e.g., Farid-Moayer et al., 2007, J. Bacteriol. 189:8088-8098.
  • O- glycosylated HuGlyFab by virtue of possessing glycans, shares advantageous characteristics with N- glycosylated HuGlyFab (as discussed above).
  • compositions and methods are described for the delivery of HuPTM mAbs and antigen-binding fragments thereof, such as HuPTM Fabs, that bind to calcitonin gene-related peptide receptor (CGRPR) that may have benefit in treating migraines or cluster headaches.
  • the HuPTM mAb is erenumab or an antigen binding fragment of one of the foregoing.
  • An amino acid sequence for Fab fragments of erenumab is provided in FIG. 2A.
  • Delivery may be accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding an CGRPR-binding HuPTM mAb (or an antigen binding fragment and/or a hyperglycosylated derivative or other derivative, thereof) to patients (human subjects) diagnosed with, or having one or more symptoms of, migraines and cluster headaches, to create a permanent depot that continuously supplies the human PTM, e.g., human-glycosylated, transgene product.
  • a viral vector or other DNA expression construct encoding an CGRPR-binding HuPTM mAb (or an antigen binding fragment and/or a hyperglycosylated derivative or other derivative, thereof) to patients (human subjects) diagnosed with, or having one or more symptoms of, migraines and cluster headaches, to create a permanent depot that continuously supplies the human PTM, e.g., human-glycosylated, transgene product.
  • transgene encoding a HuPTM mAb or HuPTM Fab (or other antigen binding fragment of the HuPTM mAb) that binds to CGRPR that can be administered to deliver the HuPTM mAb or antigen binding fragment in a patient.
  • the transgene is a nucleic acid comprising the nucleotide sequences encoding an antigen binding fragment of an antibody that binds to CGRPR, such as erenumab, or variants thereof as detailed herein or in accordance with the details herein.
  • the transgene may also encode anti-CGRPR antigen binding fragment that contains additional glycosylation sites (e.g., see Courtois et al., 2016, mAbs 8: 99-112 which is incorporated by reference herein in its entirety).
  • the anti-CGRPR antigen-binding fragment transgene comprises the nucleotide sequences encoding the heavy and light chains of the Fab portion of erenumab (having amino acid sequences of SEQ ID NOs. 1 and 2, respectively, see Table 8 and FIG 2A).
  • the nucleotide sequences may be codon optimized for expression in human cells and may, for example, comprise the nucleotide sequences of SEQ ID NO: 9 (encoding the erenumab heavy chain Fab portion) and SEQ ID NO: 10 (encoding the erenumab light chain Fab portion) or the nucleotide sequence of 267 encoding the vectorized erenumab (signal sequences underlined) as set forth in Table 9.
  • the heavy and light chain sequences both have a signal or leader sequence at the N-terminus appropriate for expression and secretion in human cells, in particular, human CNS cells.
  • the signal sequence may have the amino acid sequence of MYRMQLLLLIALSLALVTNS (SEQ ID NO: 28) or the one of the sequences found in Tables 2-4, supra.
  • the transgenes may comprise, at the C-terminus of the heavy chain variable domain sequence, all or a portion of the hinge region.
  • the anti-CGRPR-antigen binding domain has a heavy chain variable domain of SEQ ID NO: 1 with additional hinge region sequence starting after the C-terminal aspartate (D), contains all or a portion of the amino acid sequence all or a portion of the amino acid sequence ERKCCVECPPCPAPPVAG (SEQ ID NO: 115) or ERKCCVECPPCPA (SEQ ID NO: 116) as set forth in FIG 2A.
  • These hinge regions may be encoded by nucleotide sequences at the 3’ end of SEQ ID NO: 9 by the hinge region encoding sequences set forth in Table 9 (SEQ ID NO: 9).
  • the anti-CGRPR antigen-binding fragment transgene encodes a CGRPR antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 2.
  • the anti-CGRPR antigenbinding fragment transgene encodes a CGRPR antigen-binding fragment comprising a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 1.
  • the anti-CGRPR antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 2 and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 1.
  • the CGRPR antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions preferably are made in the framework regions (i.e., those regions outside of the CDRs, which CDRs are underlined in FIG. 2A) or are substitutions with an amino acid present at that position in the heavy chain of one or more of the other therapeutic antibodies.
  • the CGRPR antigen binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NO: 2 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions preferably are made in the framework regions (i.e., those regions outside of the CDRs, which CDRs are underlined in FIG. 2A) or are substitutions with an amino acid present at that position in the light chain of one or more of the other therapeutic antibodies.
  • the anti-CGRPR antigen-binding fragment transgene encodes a hyperglycosylated erenumab Fab, comprising a heavy chain and a light chain of SEQ ID NOs: 1 and 2, respectively, with one or more of the following mutations: T125N (heavy chain) and/or Q198N (light chain).
  • the anti-CGRPR antigen-binding fragment transgene encodes an antigen-binding fragment and comprises the nucleotide sequences encoding the six erenumab CDRs which are underlined in the heavy and light chain variable domain sequences of FIG. 2A which are spaced between framework regions, generally human framework regions, and associated with constant domains depending upon the form of the antigen-binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-CGRPR antibody or antigen-binding fragment thereof.
  • a viral vector containing a transgene encoding an anti-CGRPR antibody, or antigen binding fragment thereof may be erenumab and is preferably a Fab fragment thereof, or other antigen-binding fragment thereof.
  • the patient has been diagnosed with and/or has symptoms associated with episodic migraines or chronic migraines.
  • the patient has been diagnosed with and/or has symptoms associated with episodic cluster headaches or chronic cluster headaches.
  • a recombinant vector used for delivering the transgene is described in Section 5.4.1 and shown in FIG 2A. Included are pAAV.CAG.
  • Such vectors should have a tropism for human CNS cells and can include non-replicating rAAV, particularly those bearing an AAV8, AAV9, AAVrhlO, AAV.PHP.eB capsid.
  • the recombinant vectors can be administered in any manner such that the recombinant vector enters the CNS, PNS, skeletal muscle, arterial smooth muscle cells, and/or liver preferably by introducing the recombinant vector intranasally or systemically (intramuscularly or intravenously). See Section 5.5.1 for details regarding the methods of treatment.
  • Subjects to whom such gene therapy is administered can be those responsive to anti- CGRPR therapy.
  • the methods encompass treating patients who have been diagnosed with migraines or cluster headaches or have one or more symptoms associated therewith, and identified as responsive to treatment with an anti-CGRPR antibody or considered a good candidate for therapy with an anti-CGRPR antibody.
  • the patients have previously been treated with erenumab, eptinezumab, fremanezumab, or galcanezumab, and have been found to be responsive to one or more of erenumab, eptinezumab, fremanezumab, and galcanezumab.
  • the anti-CGRPR antibody or antigen-binding fragment transgene product may be administered directly to the subject.
  • the production of the anti-CGRPR HuPTM mAb or HuPTM Fab should result in a “biobetter” molecule for the treatment of migraines or cluster headaches accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding the anti- CGRPR HuPTM Fab intranasal, intravenous, or intramuscular administration to human subjects (patients) diagnosed with or having one or more symptoms of migraines or cluster headaches, to create a permanent depot in CNS, PNS, arterial smooth muscle, and/or liver cells that continuously supplies the fully-human post-translationally modified, e.g., human-glycosylated, sulfated transgene product produced by transduced c
  • the cDNA construct for the anti-CGRPR HuPTM mAb or anti-CGRPR HuPTM Fab should include a signal peptide that ensures proper co- and post-translational processing (glycosylation and protein sulfation) by the transduced CNS cells.
  • the signal sequence may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 28).
  • the anti-CGRPR HuPTM mAb or HuPTM Fab can be produced in human cell lines by recombinant DNA technology, and administered to patients diagnosed with migraines or cluster headaches, or for whom therapy for migraines or cluster headaches is considered appropriate.
  • the anti-CGRPR HuPTM mAb or antigen-binding fragment thereof has heavy and light chains with the amino acid sequences of the heavy and light chain Fab portions of erenumab as set forth in FIG. 2A (with non-consensus asparagine (N) glycosylation sites highlighted in green, glutamine (Q) glycosylation sites highlighted in blue, and Y-sulfation sites highlighted in yellow) has glycosylation, particularly a 2,6-sialylation, at one or more of the amino acid positions N77 and/or Q122 and/or N172 and/or N205 and/or N214 of the heavy chain (SEQ ID NO: 1) or N28 and/or N174 of the light chain (SEQ ID NO: 2).
  • the HuPTM mAh or antigen binding-fragment thereof with the heavy and light chain variable domain sequences of erenumab has a sulfation group at Y94 and/or Y95 of the heavy chain (SEQ ID NO: 1) and/or Y87 and/or Y88 of the light chain (SEQ ID NO: 2).
  • the anti-CGRPR HuPTM mAb or antigen-binding fragment thereof does not contain any detectable (e.g., as detected by assays known in the art, for example, those described in section 5.2, infra) NeuGc moi eties and/or does not contain any detectable (e.g., as detected by assays known in the art, for example, those described in section 5.2, infra) alpha-Gal moi eties.
  • the HuPTM mAb or Fab is therapeutically effective and is at least 0.5%, 1% or 2% 2,6 sialylated and/or sulfated and may be at least 5%, 10% or even 50% or 100% glycosylated 2,6 sialylation and/or sulfated.
  • the goal of gene therapy treatment provided herein is to prevent or reduce the intensity or frequency of migraines, cluster headaches, or one or more of the symptoms associated therewith, including nausea, light sensitivity, sound sensitivity, red eye, eyelid edema, forehead and facial sweating, tearing (lacrimation), abnormal small size of the pupil (miosis), nasal congestion, runny nose (rhinorrhea), and drooping eyelid (ptosis).
  • Efficacy may be monitored by measuring a reduction in the intensity or frequency of migraines or cluster headaches, or a reduction in the amount of acute migraine-specific medication used over a defined period of time (e.g. number of days of use of any acute headache medication per month).
  • a therapeutically effective HuPTM mAb or Fab reduces the average number of headache and/or migraine days per month compared to placebo by at least 2, at least, 3, at least 4, or at least 5 days.
  • Combinations of delivery of the anti-CGRPR HuPTM mAb or antigen-binding fragment thereof, to the CNS accompanied by delivery of other available treatments are encompassed by the methods provided herein.
  • the additional treatments may be administered before, concurrently or subsequent to the gene therapy treatment.
  • Available treatments for cluster headaches or migraines that could be combined with the gene therapy provided herein include but are not limited to triptans, ergotamine derivatives and NSAIDs, to name a few, and administration with anti-CGRPR or anti- CGRP agents, including but not limited to erenumab, eptinezumab, fremanezumab, and galcanezumab. 5.4.2 Anti-CGRP HuPTM Constructs and Formulations for Migraine
  • HuPTM mAbs and antigen-binding fragments thereof that bind to calcitonin gene-related peptide (CGRP) that may have benefit in treating migraines and cluster headaches (referred to collectively as headache disorders).
  • the HuPTM mAb is eptinezumab, fremanezumab, galcanezumab or an antigen binding fragment of one of the foregoing.
  • the amino acid sequences of Fab fragments of these antibodies are provided in FIGS. 2B-D.
  • Delivery may be accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding an CGRP -binding HuPTM mAb (or an antigen binding fragment and/or a hyperglycosylated derivative or other derivative, thereof) to patients (human subjects) diagnosed with, or having one or more symptoms of, migraines and cluster headaches, to create a permanent depot that continuously supplies the human PTM, e.g., human-glycosylated, transgene product.
  • a viral vector or other DNA expression construct encoding an CGRP -binding HuPTM mAb (or an antigen binding fragment and/or a hyperglycosylated derivative or other derivative, thereof) to patients (human subjects) diagnosed with, or having one or more symptoms of, migraines and cluster headaches, to create a permanent depot that continuously supplies the human PTM, e.g., human-glycosylated, transgene product.
  • transgene encoding a HuPTM mAb or HuPTM Fab (or other antigen binding fragment of the HuPTM mAb) that binds to CGRP that can be administered to deliver the HuPTM mAb or antigen binding fragment in a patient.
  • the transgene is a nucleic acid comprising the nucleotide sequences encoding an antigen binding fragment of an antibody that binds to CGRP, such as eptinezumab, fremanezumab, galcanezumab or variants thereof as detailed herein or in accordance with the details herein.
  • the transgene may also encode anti-CGRP antigen binding fragment that contains additional glycosylation sites (e.g., see Courtois et al., 2016, mAbs 8: 99-112 which is incorporated by reference herein in its entirety).
  • the anti-CGRP antigen-binding fragment transgene comprises the nucleotide sequences encoding the heavy and light chains of the Fab portion of eptinezumab (having amino acid sequences of SEQ ID NOs. 3 and 4, respectively, see Table 8 and FIG. 2B).
  • the nucleotide sequences may be codon optimized for expression in human cells.
  • Nucleotide sequences may, for example, comprise the nucleotide sequences of SEQ ID NO: 11 (encoding the eptinezumab heavy chain Fab portion) and SEQ ID NO: 12 (encoding the eptinezumab light chain Fab portion) as set forth in Table 9.
  • the heavy and light chain sequences both have a signal or leader sequence at the N-terminus appropriate for expression and secretion in human cells, in particular, human CNS, PNS, arterial smooth muscle and/or liver cells.
  • the signal sequence may have the amino acid sequence of MYRMQLLLLIALSLALVTNS (SEQ ID NO: 28) or the one of the sequences found in Tables 2, 3, or 4 supra.
  • provided are codon-optimized, CpG deleted nucleotide sequenced encoding the vectorized eptinezumab Fab (including leader sequences and Furin/T2 A linker sequence), as set forth in Table 9, SEQ ID NO: 288.
  • the transgenes may comprise, at the C-terminus of the heavy chain CHI domain sequence, all or a portion of the hinge region.
  • the anti-CGRP-antigen binding domain has a heavy chain Fab fragment of SEQ ID NO: 3 with additional hinge region sequence starting after the C-terminal valine (V), contains all or a portion of the amino acid sequence EPKSCDKTHTCPPCPAPELLGG (SEQ ID NO: 97), and specifically, EPKSCDKTHL (SEQ ID NO:99), EPKSCDKTHT (SEQ ID NO: 100), EPKSCDKTHTCPPCPA (SEQ ID NO: 101), EPKSCDKTHLCPPCPA (SEQ ID NO: 102), EPKSCDKTHTCPPCPAPELLGGPSVFL (SEQ ID NO: 103) or EPKSCDKTHLCPPCPAPELLGGPSVFL (SEQ ID NO: 104) as set forth in FIG 2B.
  • the transgenes comprise the amino acid sequences encoding the full length (or substantially full length) heavy and light chains of the antibody, comprising the Fc domain at the C terminus of the heavy chain, e.g., having an amino acid sequence of SEQ ID NO: 18 (Table 7) or an IgGl Fc domain, such as SEQ ID NO: 18 or as depicted in FIG. 4, or a mutant or variant thereof.
  • the Fc domain may be engineered for altered binding to one or more Fc receptors and/or effector function as disclosed in Section 5.1.9, infra.
  • the anti-CGRP anti gen -binding fragment transgene encodes a CGRP antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 4.
  • the anti-CGRP antigenbinding fragment transgene encodes a CGRP antigen-binding fragment comprising a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 3.
  • the anti-CGRP antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 4 and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 3.
  • the CGRP antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 3 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 3B) or are substitutions with an amino acid present at that position in the heavy chain of one or more of the other therapeutic antibodies.
  • the framework regions e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 3B
  • substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 3B) or are substitutions with an amino acid present at that position in the heavy chain of one or more of the other therapeutic antibodies.
  • the CGRPR antigen-binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NO: 4 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2B) or are substitutions with an amino acid present at that position in the light chain of one or more of the other therapeutic antibodies.
  • the framework regions e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2B
  • substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2B) or are substitutions with an amino acid present at that position in the light chain of one or more of the other therapeutic antibodies.
  • the anti-CGRP antigen-binding fragment transgene encodes a hyperglycosylated eptinezumab Fab, comprising a heavy chain and a light chain of SEQ ID NOs: 3 and 4, respectively, with one or more of the following mutations: L106N (heavy chain), Q165N or Q165S (light chain), and/or E200N (light chain).
  • the anti-CGRP antigen-binding fragment transgene encodes an antigen-binding fragment and comprises the nucleotide sequences encoding the six eptinezumab CDRs which are underlined in the heavy and light chain variable domain sequences of FIG. 2B which are spaced between framework regions, generally human framework regions, and associated with constant domains depending upon the form of the antigen-binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-CGRP antibody or antigen-binding fragment thereof.
  • the anti-CGRP antigen-binding fragment transgene comprises the nucleotide sequences encoding the heavy and light chains of the Fab portion of fremanezumab (having amino acid sequences of SEQ ID NOs. 5 and 6, respectively, see Table 8 and FIG 2C).
  • the nucleotide sequences may be codon optimized for expression in human cells.
  • Nucleotide sequences may, for example, comprise the nucleotide sequences of SEQ ID NO: 13 (encoding the fremanezumab heavy chain Fab portion) and SEQ ID NO: 14 (encoding the fremanezumab light chain Fab portion) as set forth in Table 9.
  • the heavy and light chain sequences both have a signal or leader sequence at the N-terminus appropriate for expression and secretion in human cells, in particular, human CNS, PNS, arterial smooth muscle, and/or liver cells.
  • the signal sequence may have the amino acid sequence of MYRMQLLLLIALSLALVTNS (SEQ ID NO: 28) or the one of the sequences found in Tables 2, 3, or 4 supra.
  • provided are codon-optimized, CpG deleted nucleotide sequenced encoding the vectorized fremanezumab Fab (including leader sequences and Furin/T2A linker sequence), as set forth in Table 9, SEQ ID NO: 274.
  • the transgenes may comprise, at the C-terminus of the heavy chain CHI domain sequence, all or a portion of the hinge region.
  • the anti-CGRPR-antigen binding domain has a heavy chain Fab fragment of SEQ ID NO: 5 with additional hinge region sequence starting after the C-terminal valine (V), contains all or a portion of the amino acid sequence ERKCCVECPPCPAPPVAG (SEQ ID NO: 115) or ERKCCVECPPCPA (SEQ ID NO: 116) as set forth in FIG 2C.
  • the transgenes comprise the amino acid sequences encoding the full length (or substantially full length) heavy and light chains of the antibody, comprising the Fc domain at the C terminus of the heavy chain, e.g., having an amino acid sequence of SEQ ID NO:23 (Table 7) or an IgG2 Fc domain, such as SEQ ID NO: 19 or as depicted in FIG. 4, or a mutant or variant thereof.
  • the Fc domain may be engineered for altered binding to one or more Fc receptors and/or effector function as disclosed in Section 5.1.9, infra.
  • the anti-CGRP anti gen -binding fragment transgene encodes a CGRP antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 6.
  • the anti-CGRPR antigenbinding fragment transgene encodes a CGRP antigen-binding fragment comprising a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 5.
  • the anti-CGRP antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 6 and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 5.
  • the CGRP antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 5 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2C) or are substitutions with an amino acid present at that position in the heavy chain of one or more of the other therapeutic antibodies.
  • the framework regions e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2C
  • substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2C) or are substitutions with an amino acid present at that position in the heavy chain of one or more of the other therapeutic antibodies.
  • the CGRP antigen-binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NO: 6 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2C) or are substitutions with an amino acid present at that position in the light chain of one or more of the other therapeutic antibodies.
  • the framework regions e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2C
  • substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2C) or are substitutions with an amino acid present at that position in the light chain of one or more of the other therapeutic antibodies.
  • the anti-CGRP anti gen -binding fragment transgene encodes a hyperglycosylated fremanezumab Fab, comprising a heavy chain and a light chain of SEQ ID NOs: 5 and 6, respectively, with one or more of the following mutations: L117N (heavy chain), Q160N or QI 60S (light chain), and/or E195N (light chain).
  • the anti-CGRP antigen-binding fragment transgene encodes an antigen-binding fragment and comprises the nucleotide sequences encoding the six fremanezumab CDRs which are underlined in the heavy and light chain variable domain sequences of FIG. 2C which are spaced between framework regions, generally human framework regions, and associated with constant domains depending upon the form of the antigen-binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-CGRP antibody or antigen-binding fragment thereof.
  • the anti-CGRP antigen-binding fragment transgene comprises the nucleotide sequences encoding the heavy and light chains of the Fab portion of galcanezumab (having amino acid sequences of SEQ ID NOs. 7 and 8, respectively, see Table 8 and FIG. 2D).
  • the nucleotide sequences may be codon optimized for expression in human cells.
  • Nucleotide sequences may, for example, comprise the nucleotide sequences of SEQ ID NO: 15 (encoding the galcanezumab heavy chain Fab portion) and SEQ ID NO: 16 (encoding the galcanezumab light chain Fab portion) as set forth in Table 9.
  • the heavy and light chain sequences both have a signal or leader sequence at the N-terminus appropriate for expression and secretion in human cells, in particular, human CNS, PNS, arterial smooth muscle and/or liver cells.
  • the signal sequence may have the amino acid sequence of MYRMQLLLLIALSLALVTNS (SEQ ID NO: 28) or the one of the sequences found in Tables 2, 3, or 4 supra.
  • provided are codon-optimized, CpG deleted nucleotide sequenced encoding the vectorized galcanezumab Fab (including leader sequences and Furin/T2A linker sequence), as set forth in Table 9, SEQ ID NO: 281.
  • the transgenes may comprise, at the C-terminus of the heavy chain CHI domain sequence, all or a portion of the hinge region.
  • the anti-CGRP-antigen binding domain has a heavy chain Fab domain of SEQ ID NO: 7 with additional hinge region sequence starting after the C-terminal valine (V), contains all or a portion of the amino acid sequence contains all or a portion of the amino acid sequence ESKYGPPCPPCPAPEAAGG (SEQ ID NO:250) or ESKYGPPCPSCPAPEAAGG (SEQ ID NO:251) as set forth in FIG 2D.
  • the transgenes comprise the amino acid sequences encoding the full length (or substantially full length) heavy and light chains of the antibody, comprising the Fc domain at the C terminus of the heavy chain, e.g., having an amino acid sequence of SEQ ID NO:24 (Table 7) or an IgG4 Fc domain, such as SEQ ID NO:20 or as depicted in FIG. 4, or a mutant or variant thereof.
  • the Fc domain may be engineered for altered binding to one or more Fc receptors and/or effector function as disclosed in Section 5.1.9, infra.
  • the anti-CGRP anti gen -binding fragment transgene encodes a CGRP antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 8.
  • the anti-CGRP antigenbinding fragment transgene encodes a CGRP antigen-binding fragment comprising a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 7.
  • the anti-CGRP antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 8 and a heavy chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 7.
  • the CGRP antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 7 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2D) or are substitutions with an amino acid present at that position in the heavy chain of one or more of the other therapeutic antibodies.
  • the framework regions e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2D
  • substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2D) or are substitutions with an amino acid present at that position in the heavy chain of one or more of the other therapeutic antibodies.
  • the CGRP antigen-binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NO: 8 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, insertions or deletions, and the substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2D) or are substitutions with an amino acid present at that position in the light chain of one or more of the other therapeutic antibodies.
  • the framework regions e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2D
  • substitutions, insertions or deletions are made, e.g., in the framework regions (e.g., those regions outside of the CDRs, which CDRs are underlined in FIG. 2D) or are substitutions with an amino acid present at that position in the light chain of one or more of the other therapeutic antibodies.
  • the anti-CGRPR antigen-binding fragment transgene encodes a hyperglycosylated galcanezumab Fab, comprising a heavy chain and a light chain of SEQ ID NOs: 7 and 8, respectively, with one or more of the following mutations: T114N (heavy chain), Q160N or Q160S, and/or E195N (light chain).
  • the anti-CGRPR antigen-binding fragment transgene encodes an antigen-binding fragment and comprises the nucleotide sequences encoding the six galcanezumab CDRs which are underlined in the heavy and light chain variable domain sequences of FIG. 2D which are spaced between framework regions, generally human framework regions, and associated with constant domains depending upon the form of the antigen-binding molecule, as is known in the art to form the heavy and/or light chain variable domain of an anti-CGRPR antibody or antigen-binding fragment thereof.
  • a viral vector containing a transgene encoding an anti-CGRP antibody, or antigen binding fragment thereof may be eptinezumab, fremanezumab, or galcanezumab and is, e.g., a Fab fragment thereof, or other antigen-binding fragment thereof or is a full length anti-CGRP antibody with an Fc region.
  • the patient has been diagnosed with and/or has symptoms associated with episodic migraines or chronic migraines.
  • the patient has been diagnosed with and/or has symptoms associated with episodic cluster headaches or chronic cluster headaches.
  • Recombinant vectors used for delivering the transgenes are described in Section 5.4.1 and shown in FIGS 2B-2D.
  • Such vectors should have a tropism for human CNS, PNS, arterial smooth muscle, and/or liver cells and can include non-replicating rAAV, particularly those bearing an AAV8, AAV9, AAV.PHP.eB, or AAVrhlO capsid.
  • the recombinant vectors can be administered in any manner such that the recombinant vector enters the targeted organ, for example the recombinant vector may be introduced into the cerebral spinal fluid (CSF) to target the CNS. See Section 5.5.1 for details regarding the methods of treatment.
  • CSF cerebral spinal fluid
  • Subjects to whom such gene therapy is administered can be those responsive to anti- CGRP therapy.
  • the methods encompass treating patients who have been diagnosed with migraines or cluster headaches or have one or more symptoms associated therewith, and identified as responsive to treatment with an anti-CGRP antibody or considered a good candidate for therapy with an anti-CGRP antibody.
  • the patients have previously been treated with eptinezumab, fremanezumab, or galcanezumab, and have been found to be responsive to one or more of eptinezumab, fremanezumab, and galcanezumab.
  • the anti-CGRP antibody or antigen-binding fragment transgene product may be administered directly to the subject.
  • the production of the anti-CGRP HuPTM mAb or HuPTM Fab should result in a “biobetter” molecule for the treatment of migraines or cluster headaches accomplished via gene therapy - e.g., by administering a viral vector or other DNA expression construct encoding the anti- CGRP HuPTM Fab, intranasal, intravenous, or intramuscular administration to human subjects (patients) diagnosed with or having one or more symptoms of migraines or cluster headaches, to create a permanent depot in the CNS, PNS, arterial smooth muscle, and/or liver cells that continuously supplies the fully-human post-translationally modified, e.g., human-glycosylated, sulfated transgene product produced by transduced CNS, PNS, arterial smooth muscle, and/or liver cells.
  • a viral vector or other DNA expression construct encoding the anti- CGRP HuPTM Fab
  • the cDNA construct for the anti-CGRP HuPTM mAb or anti-CGRP HuPTM Fab should include a signal peptide that ensures proper co- and post-translational processing (glycosylation and protein sulfation) by the transduced CNS, PNS, arterial smooth muscle, and/or liver cells.
  • the signal sequence may be MYRMQLLLLIALSLALVTNS (SEQ ID NO: 28).
  • the anti-CGRP HuPTM mAb or HuPTM Fab can be produced in human cell lines by recombinant DNA technology, and administered to patients diagnosed with migraines or cluster headaches, or for whom therapy for migraines or cluster headaches is considered appropriate.
  • the anti-CGRP HuPTM mAb or antigen-binding fragment thereof has heavy and light chains with the amino acid sequences of the heavy and light chain Fab portions of eptinezumab as set forth in FIG. 2B (with glutamine (Q) glycosylation sites; asparaginal (N) glycosylation sites, non-consensus asparaginal (N) glycosylation sites; and tyrosine-O-sulfation sites (Y) are as indicated in the legend) has glycosylation, particularly a 2,6-sialylation, at one or more of the amino acid positions Q103 and/or N153 of the heavy chain (SEQ ID NO: 3) or N21, N163, and/or N215 of the light chain (SEQ ID NO: 4).
  • the HuPTM mAb or antigen binding-fragment thereof with the heavy and light chain variable domain sequences of eptinezumab has a sulfation group at Y32, Y33 and/or Y93 of the heavy chain (SEQ ID NO: 3) and/or Y87 and/or Y88 of the light chain (SEQ ID NO: 4).
  • the anti-CGRP HuPTM mAb or antigen-binding fragment thereof does not contain any detectable (e.g., as detected by assays known in the art, for example, those described in section 5.2, infra) NeuGc moi eties and/or does not contain any detectable (e.g., as detected by assays known in the art, for example, those described in section 5.2, infra) alpha-Gal moi eties.
  • the HuPTM mAb is a full length or substantially full length mAb with an Fc region.
  • the anti-CGRP HuPTM mAb or antigen-binding fragment thereof has heavy and light chains with the amino acid sequences of the heavy and light chain Fab portions of fremanezumab as set forth in FIG. 2C (with glutamine (Q) glycosylation sites; asparaginal (N) glycosylation sites, non-consensus asparaginal (N) glycosylation sites; and tyrosine-O-sulfation sites (Y) are as indicated in the legend) has glycosylation, particularly a 2,6-sialylation, at one or more of the amino acid positions Q114, N164, N197 and/or N206 of the heavy chain (SEQ ID NO: 5) or N93, Q100, N158, and/or N210 of the light chain (SEQ ID NO: 5).
  • the HuPTM mAb or antigen binding-fragment thereof with the heavy and light chain variable domain sequences of fremanezumab has a sulfation group at Y96, Y97 and/or Y203 of the heavy chain (SEQ ID NO: 5) or Y86 and/or Y87 of the light chain (SEQ ID NO: 6).
  • the anti- CGRP HuPTM mAb or antigen-binding fragment thereof does not contain any detectable (e.g., as detected by assays known in the art, for example, those described in section 5.2, infra) NeuGc moi eties and/or does not contain any detectable (e.g., as detected by assays known in the art, for example, those described in section 5.2, infra) alpha-Gal moi eties.
  • the HuPTM mAb is a full length or substantially full length mAb with an Fc region.
  • the anti-CGRP HuPTM mAb or antigen-binding fragment thereof has heavy and light chains with the amino acid sequences of the heavy and light chain Fab portions of galcanezumab as set forth in FIG. 2D (with glutamine (Q) glycosylation sites; asparaginal (N) glycosylation sites, non-consensus asparaginal (N) glycosylation sites; and tyrosine-O-sulfation sites (Y) are as indicated in the legend) has glycosylation, particularly a 2,6-sialylation, at one or more of the amino acid positions QI 11, N161, and/or N203 of the heavy chain (SEQ ID NO: 7) or N158 and/or N210 of the light chain (SEQ ID NO: 8).
  • the HuPTM mAh or antigen binding-fragment thereof with the heavy and light chain variable domain sequences of erenumab has a sulfation group at Y32 and/or Y33 and/or Y93 of the heavy chain (SEQ ID NO: 7) and/or Y86 and/or Y87 and/or Y92 of the light chain (SEQ ID NO: 8).
  • the anti- CGRP HuPTM mAb or antigen-binding fragment thereof does not contain any detectable (e.g., as detected by assays known in the art, for example, those described in section 5.2, infra) NeuGc moi eties and/or does not contain any detectable (e.g., as detected by assays known in the art, for example, those described in section 5.2, infra) alpha-Gal moi eties.
  • the HuPTM mAb is a full length or substantially full length mAb with an Fc region.
  • the HuPTM mAb or Fab is therapeutically effective and is at least 0.5%, 1% or 2% 2,6 sialylated and/or sulfated and may be at least 5%, 10% or even 50% or 100% glycosylated 2,6 sialylation and/or sulfated.
  • the goal of gene therapy treatment provided herein is to prevent or reduce the intensity or frequency of migraines, cluster headaches, or one or more of the symptoms associated therewith, including nausea, light sensitivity, sound sensitivity, red eye, eyelid edema, forehead and facial sweating, tearing (lacrimation), abnormal small size of the pupil (miosis), nasal congestion, runny nose (rhinorrhea), and drooping eyelid (ptosis).
  • Efficacy may be monitored by measuring a reduction in the intensity or frequency of migraines or cluster headaches, or a reduction in the amount of acute migraine-specific medication used over a defined period of time.
  • Combinations of delivery of the anti-CGRP HuPTM mAb or antigen-binding fragment thereof, to the CNS, PNS, arterial smooth muscle, and/or liver cells accompanied by delivery of other available treatments are encompassed by the methods provided herein.
  • the additional treatments may be administered before, concurrently or subsequent to the gene therapy treatment.
  • Available treatments for cluster headaches or migraines that could be combined with the gene therapy provided herein include but are not limited to triptans, ergotamine derivatives and NSAIDs, to name a few, and administration with anti-CGRPR agents, including but not limited to eptinezumab, fremanezumab, and galcanezumab.
  • Dual delivery of an anti-CGRP HuPTM mAb, or antigen-binding fragment thereof, particularly eptinezumab, fremanezumab, or galcanezumab, and an anti-CGRPR HuPTM mAb, or antigen-binding fragment thereof, particularly erenumab, may be achieved by administration of a single dual cistron vector expressing an anti-CGRP HuPTM Fab and an anti-CGRPR HuPTM Fab wherein the anti-CGRP HuPTM Fab and the anti-CGRPR HuPTM Fab are each under the control of different promoters to promote expression in different tissue types.
  • the anti-CGRP HuPTM Fab will be expressed in a different or substantially different set of cells than the anti-CGRPR HuPTM Fab due to the controlling regulatory sequences.
  • delivery of both anti-CGRP HuPTM mAb and anti-CGRPR HuPTM mAb (or antigen binding fragments thereof) may be accomplished by administration of a first and second viral vector, wherein the first vector expresses an anti-CGRP HuPTM mAb, or antigen-binding fragment thereof, and the second viral vector expresses an anti-CGRPR HuPTM mAb, or antigen-binding fragment thereof.
  • the HuPTM Fabs may be under the control of the same of different regulatory sequences and the AAV serotype of the rAAV vector used may be the same of different.
  • [0212] Provided are combinations of construct, regulatory elements, AAV serotype and whether the first and second transgenes are in the same vector, as a dual cistronic vector, or in separate vectors for effective delivery of the combination of anti-CGRP antibodies and anti-CGRPR antibodies (and antigen binding fragments thereof).
  • Structural component parts of a dual cistron vector expression cassette (AGT1 and AGT2) and, alternatively, a first and a second vector administered as dual therapy, are outlined in Table 10.
  • the dual cistron vector comprises a first transgene (ATG1, upstream of AGT2) encoding erenumab, operably linked to a CNS- specific promoter or a neuron-specific promoter (e.g. hSyn or CamKII), and a second transgene (ATG2) encoding eptinezumab, operably linked to an arterial smooth muscle cell-specific promoter (e.g., SM22a), wherein the capsid protein is an AAV9.
  • the first vector comprises a first transgene (ATG1) encoding erenumab, operably linked to a CNS-specific promoter or a neuronspecific promoter (e.g.
  • the second vector comprises a second transgene (ATG2) encoding eptinezumab, operably linked to an arterial smooth muscle cell-specific promoter (e.g SM22a), wherein the capsid protein of the first and second viral vector may be the same (e.g. AAV9) or different (e.g. AGT1 : AAV9 and AGT2: AAV8).
  • Promoters or active fragments thereof can include, but are not limited to, liver- (“LTV”, e.g. TBG or ApoE.hAAT), CNS- or neuron- (“CNS”, e.g.
  • hSYN or CAMKII hSYN or CAMKII
  • SM22a arterial smooth muscle cell
  • CAG ubiquitous promoters
  • Exemplary combinations are indicated below in Table 9.
  • Other tissue specific promoters and capsid serotypes may be substituted in the exemplary embodiments.
  • Section 5.2. and 5.4.1 describe recombinant vectors that contain a transgene encoding a HuPTM mAb or HuPTM Fab (or other antigen binding fragment of the HuPTM mAb) that binds to CGRP or CGRPR.
  • Therapeutically effective doses of any such recombinant vector should be administered in any manner such that the recombinant vector enters the CNS, PNS, liver, and/or arteries (e.g., arterial smooth muscle cells), e.g. by introducing the recombinant vector into the bloodstream.
  • the vector may be administered directly to the liver through hepatic blood flow, e.g., via the suprahepatic veins or via the hepatic artery, or directly to the CNS via introduction into the cerebral spinal fluid (CSF).
  • the vector is administered intranasally, intravenously, or intramuscularly. Intranasal, intravenous, intramuscular, intrathecal, or hepatic administration should result in expression of the soluble transgene product in cells of the liver, CNS, PNS, and/or arterial smooth muscle cells.
  • the expression of the transgene encoding an anti-CGRP or anti-CGRPR antibody creates a permanent depot in CNS, PNS, arterial smooth muscle, and/or liver cells of the patient that continuously supplies the anti-CGRP or anti-CGRPR HuPTM mAb, or antigen binding fragment of the anti-CGRP or anti-CGRPR mAb to targeted tissue structures of the subject, e.g. dural vessels or TG.
  • intravenous administration of an AAV gene therapy vector encoding an anti-CGRPR antibody results in at least 1.5 pg/mL, 2 g/mL, 5 pg/mL, 10 pg/mL, 15 pg/mL or at least 20 pg/mL transgene product expression in human serum at least 20, 30, 40, 50 or 60 days after administration.
  • the target human serum concentration (Cmin) of the transgene product is about 1.5 pg/mL to about 20 pg/mL mAb.
  • doses that maintain a serum concentration of the anti-CGRPR antibody transgene product at a Cmin of at least 1.5 pg/mL or at least 15 pg/mL e.g., Cmin of 1.5 to 5 pg/ml, 5 to 10 pg/ml or 10 to 20 pg/mL
  • a Cmin of at least 1.5 pg/mL or at least 15 pg/mL e.g., Cmin of 1.5 to 5 pg/ml, 5 to 10 pg/ml or 10 to 20 pg/mL
  • the transgene product is continuously produced. Notwithstanding, because the transgene product is continuously produced, maintenance of lower concentrations can be effective.
  • the concentration of the transgene product can be measured in patient blood serum samples.
  • compositions suitable for intravenous, intrathecal, intranasal, intramuscular, or hepatic administration comprise a suspension of the recombinant vector comprising the transgene encoding the anti-CGRP or anti-CGRPR antibody, or antigen-binding fragment thereof, in a formulation buffer comprising a physiologically compatible aqueous buffer.
  • the formulation buffer can comprise one or more of a polysaccharide, a surfactant, polymer, or oil.
  • the efficacy of the methods and compositions may be assessed in either animal models, such as those disclosed herein, or in human subjects using methods known in the art.
  • the efficacy may be assessed as determined to reduce nausea, light sensitivity, sound sensitivity, red eye, eyelid edema, forehead and facial sweating, tearing (lacrimation), abnormal small size of the pupil (miosis), nasal congestion, runny nose (rhinorrhea), and/or drooping eyelid (ptosis) in mouse models or in human subjects.
  • efficacy may be determined by the ability of the composition when administered to a subject to reduce the intensity or frequency of migraines, such as change from baseline in the number of headache and/or migraine days per month (for example, reduction in greater than 1, 2, 3, 4, 5, 6, or 7 headache days per month), number of cluster headaches per month from baseline (for example, a reduction in 1, 2, 3, 4, 5, 6, or 6 cluster headaches per month from baseline) or a reduction in the amount of acute migraine-specific medication used over a defined period of time.
  • An erenumab Fab cDNA-based vector is constructed comprising a transgene comprising nucleotide sequences encoding the Fab portion of the heavy and light chain sequences of erenumab (amino acid sequences being SEQ ID NOs. 1 and 2, respectively).
  • the nucleotide sequence coding for the Fab portion of the heavy and light chain may be the nucleotide sequence of SEQ ID NOs. 9 and 10, respectively.
  • the transgene also comprises nucleotide sequences that encodes a signal peptide, e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO: 28).
  • the nucleotide sequences encoding the light chain and heavy chain are separated by IRES elements or 2A cleavage sites (See Table 5, particularly, Furin/T2A SEQ ID NO: 85 or 86) to create a bicistronic vector.
  • the vector additionally includes a constitutive promoter, such as CAG (SEQ ID NO: 25), a tissue-specific promoter, such as an arterial smooth muscle cell-specific promoter, particularly sm22a promoter (SEQ ID NO: 52, 206- 211), or an LMTP6 promoter (SEQ ID NO: 159) or LMTP24 promoter (SEQ ID NO: 263) or an inducible promoter, such as a hypoxia-inducible promoter.
  • a constitutive promoter such as CAG (SEQ ID NO: 25)
  • tissue-specific promoter such as an arterial smooth muscle cell-specific promoter, particularly sm22a promoter (SEQ ID NO: 52, 206- 211), or an LMTP6 promoter (S
  • the vector may further have an intron sequence between the coding region and the regulatory region, such as the VH4 intron (SEQ ID NO: 241).
  • VH4 intron a construct between the coding region and the regulatory region
  • Exemplary constructs include pAAV.CAG.erenumab (SEQ ID NO: 268 (promoter to polyadenylation signal sequence) or 269 (including flanking ITR sequences); pAAV.LMTP6.VH4i.erenumab.T2A (SEQ ID NO: 270 (promoter to polyadenylation signal sequence) or 271 (including flanking ITR sequences)) or pAAV.LMTP24.VH4i.erenumab.T2A (SEQ ID NO: 272 (promoter to polyadenylation signal sequence) or 273 (including flanking ITR sequences)).
  • EXAMPLE 2 Eptinezumab Fab cDNA-Based Vector
  • An eptinezumab Fab cDNA-based vector is constructed comprising a transgene comprising nucleotide sequences encoding the Fab portion of the heavy and light chain sequences of eptinezumab (amino acid sequences being SEQ ID NOs. 3 and 4, respectively).
  • the nucleotide sequence coding for the Fab portion of the heavy and light chain may be the nucleotide sequence of SEQ ID NOs. 11 and 12, respectively.
  • the transgene also comprises nucleotide sequences that encodes a signal peptide, e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO: 28).
  • the nucleotide sequences encoding the light chain and heavy chain are separated by IRES elements or 2A cleavage sites (See Table 5, particularly, Furin/T2A SEQ ID NO: 85 or 86) to create a bicistronic vector.
  • the vector additionally includes a constitutive promoter, such as CAG (SEQ ID NO: 25), a tissue-specific promoter, such as an arterial smooth muscle cell-specific promoter, particularly sm22a promoter (SEQ ID NO: 52, 206-211), or an LMTP6 promoter (SEQ ID NO: 159) or LMTP24 promoter (SEQ ID NO: 263) or an inducible promoter, such as a hypoxia-inducible promoter.
  • a constitutive promoter such as CAG (SEQ ID NO: 25)
  • tissue-specific promoter such as an arterial smooth muscle cell-specific promoter, particularly sm22a promoter (SEQ ID NO: 52, 206-211)
  • an LMTP6 promoter SEQ ID NO:
  • the vector may further have an intron sequence between the coding region and the regulatory region, such as the VH4 intron (SEQ ID NO: 241).
  • VH4 intron VH4 intron
  • Exemplary constructs include pAAVCAG. Eptinezumab (SEQ ID NO: 289 (promoter to polyadenylation signal sequence) or 290 (including flanking ITR sequences); pAAV.LMTP6.VH4i. eptinezumab. T2 A (SEQ ID NO: 291 (promoter to polyadenylation signal sequence) or 292 (including flanking ITR sequences)) or pAAV.LMTP24.VH4i. eptinezumab. T2 A (SEQ ID NO: 293 (promoter to polyadenylation signal sequence) or 273 (including flanking ITR sequences)).
  • a fremanezumab Fab cDNA-based vector is constructed comprising a transgene comprising nucleotide sequences encoding the Fab portion of the heavy and light chain sequences of fremanezumab (amino acid sequences being SEQ ID NOs. 5 and 6, respectively).
  • the nucleotide sequence coding for the Fab portion of the heavy and light chain may be the nucleotide sequence of SEQ ID NOs. 13 and 14, respectively.
  • the transgene also comprises nucleotide sequences that encodes a signal peptide, e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO: 28).
  • the nucleotide sequences encoding the light chain and heavy chain are separated by IRES elements or 2A cleavage sites (See Table 5, particularly, Furin/T2A SEQ ID NO: 85 or 86) to create a bicistronic vector.
  • the vector additionally includes a constitutive promoter, such as CAG (SEQ ID NO: 25), a tissue-specific promoter, such as an arterial smooth muscle cell-specific promoter, particularly sm22a promoter (SEQ ID NO: 52, 206-211), or an LMTP6 promoter (SEQ ID NO: 159) or LMTP24 promoter (SEQ ID NO: 263) or an inducible promoter, such as a hypoxia-inducible promoter.
  • a constitutive promoter such as CAG (SEQ ID NO: 25)
  • tissue-specific promoter such as an arterial smooth muscle cell-specific promoter, particularly sm22a promoter (SEQ ID NO: 52, 206-211)
  • an LMTP6 promoter SEQ ID NO:
  • the vector may further have an intron sequence between the coding region and the regulatory region, such as the VH4 intron (SEQ ID NO: 241).
  • VH4 intron SEQ ID NO: 241.
  • Exemplary constructs include pAAV.CAG.fremanezumab (SEQ ID NO: 275 (promoter to polyadenylation signal sequence) or 276 (including flanking ITR sequences); pAAV.LMTP6.VH4i.
  • fremanezumab.T2A (SEQ ID NO: 277 (promoter to polyadenylation signal sequence) or 278 (including flanking ITR sequences)) or pAAV.LMTP24.VH4i.fremanezumab.T2A (SEQ ID NO: 279 (promoter to polyadenylation signal sequence) or 280 (including flanking ITR sequences)).
  • a galcanezumab Fab cDNA-based vector is constructed comprising a transgene comprising nucleotide sequences encoding the Fab portion of the heavy and light chain sequences of galcanezumab (amino acid sequences being SEQ ID NOs. 7 and 8, respectively).
  • the nucleotide sequence coding for the Fab portion of the heavy and light chain may be the nucleotide sequence of SEQ ID NOs. 15 and 16, respectively.
  • the transgene also comprises nucleotide sequences that encodes a signal peptide, e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO: 28).
  • the nucleotide sequences encoding the light chain and heavy chain are separated by IRES elements or 2A cleavage sites (See Table 5, particularly, Furin/T2A SEQ ID NO: 85 or 86) to create a bicistronic vector.
  • the vector additionally includes a constitutive promoter, such as CAG (SEQ ID NO: 25), a tissue-specific promoter, such as an arterial smooth muscle cell-specific promoter, particularly sm22a promoter (SEQ ID NO: 52, 206-211), or an LMTP6 promoter (SEQ ID NO: 159) or LMTP24 promoter (SEQ ID NO: 263) or an inducible promoter, such as a hypoxia-inducible promoter.
  • a constitutive promoter such as CAG (SEQ ID NO: 25)
  • tissue-specific promoter such as an arterial smooth muscle cell-specific promoter, particularly sm22a promoter (SEQ ID NO: 52, 206-211)
  • an LMTP6 promoter SEQ ID NO:
  • the vector may further have an intron sequence between the coding region and the regulatory region, such as the VH4 intron (SEQ ID NO: 241).
  • VH4 intron a construct between the coding region and the regulatory region
  • Exemplary constructs include pAAV.CAG.galcanezumab (SEQ ID NO: 282 (promoter to polyadenylation signal sequence) or 283 (including flanking ITR sequences); pAAV.LMTP6.VH4i. galcanezumab.T2A (SEQ ID NO: 284 (promoter to polyadenylation signal sequence) or 285 (including flanking ITR sequences)) or pAAV.LMTP24.VH4i. galcanezumab.T2A (SEQ ID NO: 286 (promoter to polyadenylation signal sequence) or 286 (including flanking ITR sequences)).
  • a dual cistron cDNA-based AAV vector is constructed comprising two transgenes comprising nucleotide sequences encoding the Fab portion of the heavy and light chain sequences of erenumab (amino acid sequences being SEQ ID NOs. 1 and 2) and the Fab portion of the heavy and light chain sequences of an anti-CGRP mAb, in particular eptinezumab (SEQ ID NO: 3 and 4), fremanezumab (SEQ ID NOs: 5 and 6), or galcanezumab (SEQ ID NOs: 7 and 8).
  • Each transgene also comprises nucleotide sequences that encodes a signal peptide, e.g., MYRMQLLLLIALSLALVTNS (SEQ ID NO: 28). Nucleotide sequences encoding the light chain and heavy chain of each transgene are separated by IRES elements or 2A cleavage sites (See Table 5, particularly, SEQ ID NO:86 or 87) to create a dual bicistronic vector.
  • the vector additionally includes two promoters (see Table 1 for promoter elements), e.g.
  • a constitutive promoter such as mUla (SEQ ID NO:26) or EFla (SEQ ID NO:27), a tissue-specific promoter, such as an arterial smooth muscle cell-specific promoter, particularly sm22a promoter (SEQ ID NO: 184, 185-190), or an CNS promoter, such as hSyn promoter (SEQ ID NO: 191-195).
  • Components of the dual cistronic construct expression cassette may be arranged as following: 5’-ITR-(Promoter 1- NH2-VHi-Furin 2A-VLi-COOH-PolyA) - (Promoter 2 - NH2-VH2-Furin 2A-VL2-COOH-polyA)-3’-ITR).
  • FIG. 1C depicts an exemplary construct.
  • Rat experiments will be performed with AAV9 containing an AAV construct (as depicted in FIGS. 1 A and IB) comprising the heavy and light chain sequences of an anti-CGRP mAb or Fab (eptinezumab, fremanezumab, or galcanezumab) or anti-CGRPR mAb or Fab (erenumab) (SEQ ID NOS: 1-8, see also FIGS. 2A-2D), which contains the Furin and (T/F)2A sequence (SEQ ID NOs: 86 or 87).
  • an anti-CGRP mAb or Fab eptinezumab, fremanezumab, or galcanezumab
  • anti-CGRPR mAb or Fab erenumab
  • Expression of the heavy and light chain is driven by a CAG promoter (SEQ ID NO: 25), mUla promoter (SEQ ID NO:26) or sm22a promoter (SEQ ID NO: 184, 185-190).
  • Dual cistronic constructs that contain both anti-CGRP Fab and anti-CGRPR Fabs each separately under the control of a mUla promoter or sm22a promoter are also tested.
  • a combination of an rAAV that has a transgene encoding an anti-CGRP mAb or Fab and an rAAV that has a transgene encoding an anti-CGRPR mAb or Fab are set forth in Table 10.
  • A. Electrical stimulation of trigeminal neurons Direct electrical stimulation of trigeminal neurons can be achieved by electrical stimulation of the trigeminal ganglion or electrical stimulation of the meningeal nerve.
  • the trigeminal ganglion of anesthetized animals will be electrically stimulated using inserted stereotactic bipolar electrodes.
  • Trigeminal ganglion neurons are then activated using low frequency ( ⁇ 5 Hz) stimulation.
  • Electrical stimulation of meningeal nerve terminals innervating the superior sagittal sinus, transverse sinus, or middle meningeal arteries will be used to elicit trigeminal afferent activation.
  • Direct stimulation of nerve terminals innervating the intracranial vasculature and their meningeal afferents as well as direct stimulation of the trigeminal ganglion have proven robust models to test differential responses to drug administration.
  • Inflammatory Substances to the Meninges: Dural application of algogenic substances will be used to model meningeal neurogenic inflammation which is thought to initiate the migraine-related pain via trigeminovascular afferent and central neuronal sensitization. Inflammatory substances (histamine, serotonin, bradykinin, or PGE2) will be applied to the dura singly or in combination as an inflammatory soup in order to activate and sensitize trigemino-vascular meningeal afferents as, for example, measured by enhanced trigeminal ganglion responses to mechanical stimulation of the meninges. Alternatively, the inflammatory soup will be administrated repetitively to induce chronic periorbital hypersensitivity to tactile stimuli that may last for up to 3 weeks (model of chronic migraine).
  • C. Exogenous Administration of Algogenic Substances Sustained mechanical allodynia is a common response associated with the local administration of various proalgesic substances in experimental animals. Exogenous administration of algogenic substances, including but not limited to, CGRP, nitric oxid donors (e.g. nitroglycerine), cilostazol, and PACAP, will be used to trigger migraine pain and other migraine-related features. The selection of the specific algogenic agent will dependent on the individual study requirements.
  • Exogenous administration of CGRP will be used to study therapeutic effects of anti-CGRP and/or anti-CGRPR gene therapy on neurogenic dural vasodilation, photophobia, periorbital hypersensitivity, and spontaneous pain behaviors in rats and/or mice.
  • AAV9 vectors comprising the heavy and light chain sequences of an anti-CGRP or anti-CGRPR mAb will be administered to Wister rats (or alternatively mice) via either intravenous (IV) or intranasal (IN) routes.
  • CGRP will be administered by subcutaneous injection in the periorbital area of rats at a dose of Ipg/kg (in a volume of 10 pl) in order to trigger photophobia, periorbital hypersensitivity and spontaneous pain behaviors.
  • Experimental readouts may include elecrophysiology and immunohistrochemistry (e.g. expression of neuronal activation markers such as c-Fos), or behavioral assays (e.g. measuring pain-like behaviors in awake, freely-behaving animals such as by measuring mechanical, or tactile, sensitivity by using calibrated von Frey filaments or thermal sensitivity). Spontaneous pain behaviors may also be assessed as alternate read out, for example, increase of grooming reflexes (see Harriot A.M. (2019), Journal of Headache and Pain, 20:91 for more details).
  • EXAMPLE 7B Mouse Animal Model of Migraine Pain and Migraine-Related Features
  • a mouse model is utilized to detect whether intravenous injection of AAV vectorencoding antibodies can diminish behavioral changes and dural vasodilation that reflect headaches in the mouse (Gao and Drew, J. Neurosci., February 24, 2016, 36(8):2503-2516; Shi, AY, et al. Journal of Cerebral Blood Flow & Metabolism (2012), 1-33). Briefly, after a baseline period of establishing mice with PoRTS window and 2-photon microscopy imaging (Gao and Drew, supra), each mouse is retro-orbitally injected or tail -vein injected with CGRP, imaging and behavioral changes measure the mouse response to CGRP within 5, 10, 15, 20, 25 and/or 30 minutes following CGRP injection.
  • AAV vectors packing anti-CGRP and/or anti-CGRP receptor antibody transgene
  • Dural vessel dilation will be monitored subsequent to AAV injection (up to and including 3 weeks following AAV injection, e.g. 1 week, 2 weeks and 3 weeks following administration of AAV).
  • Dural vessel imaging is done as described by Gao and Drew, supra.
  • Subsequent CGRP administration or control, e.g. saline
  • CGRP administration or control, e.g. saline
  • behavioral changes locomotion
  • dural dilation changes will again be measured.
  • Vascular changes at the level of the dura mater after CGRP -induced or electrical stimulation of the dural vasculature will be used to determine the extent of trigeminovascular activation with or without prior administration of anti-CGRP/CGRPR gene therapy.
  • the effect of administration of vectorized CGRP or CGRPR antibodies on dural vasodilation evoked by a) electrical stimulation of the cranial window and/or b) CGRP administration will be assessed.
  • Dural vasodilation will be induced 7, 14, 21, or 28 days after administration of the vectorized antibody.
  • Laser-Doppler flowmetry or intravital microscopy will be used to measure changes to the diameter of the blood vessels in response to the stimulus (Holland PR. et al, 2005, The Journal of Pharmacology and Experimental Therapeutics; Vol. 315, No. 3; Akerman S. et al, (2013), Cephalalgia, 33(8) 577-592)
  • CGRP-induced dural vasodilation CGRP will be administered by subcutaneous injection in the periorbital area of rats at a dose of Img/kg (in a volume of 10 ml).
  • B. Electrical Stimulation Electrical stimulation will be used to evoke dilation of the dural blood vessels with a bipolar stimulating electrode placed on the surface of the cranial window. The surface of the cranial window will be stimulated with increasing voltage until maximal dilation is observed. Subsequent electrically induced responses in the same animal will then be evoked with the same voltage. The mean maximum percentage increase in dural vessel diameter relative to prestimulation baseline (%) will be calculated.
  • a therapeutic antibody cDNA-based vector was constructed comprising a transgene comprising a nucleotide sequence encoding the heavy and light chain sequences of the therapeutic antibody (amino acid sequences being SEQ ID NOs: 263 and 264, respectively).
  • the nucleotide sequence coding for the heavy and light chain of the therapeutic antibody was codon optimized to generate coding sequences, L01, L02, and L03. L02 and L03 also have reduced incidence of CpG dimers in the sequence.
  • the transgene also comprised a nucleotide sequence that encodes the signal peptide MYRMQLLLLIALSLALVTNS (SEQ ID NO:28).
  • the nucleotide sequences encoding the light chain and heavy chain were separated by a Furin-F2A linker (SEQ ID NOS: 87 or 88) or a Furin T2A linker (SEQ ID NOS:85 or 86) to create a bicistronic vector.
  • the vector additionally included a constitutive CAG promoter (SEQ ID NO:47).
  • Regulatory sequences may be incorporated into expression cassettes and be operably linked to the transgene to promote liver-specific expression (LSPX1, LSPX2, LTP1, LTP2, or LTP3, SEQ ID NOS:66-70, respectively) and liver and muscle expression (LMTP6, LMTP13, LMTP15, LMTP18, LMTP19, LMTP20 or LMTP24) (See Table 1).
  • Other promoter sequences provided include the ApoE.hAAT (SEQ ID NO: 166) promoter, wherein four copies of the liver-specific apolipoprotein E (ApoE) enhancer were placed upstream of the human alpha 1 -antitrypsin (hAAT) promoter).
  • HEK293 cells were plated at a density of 7.5xl0 5 cells/well in each well of a standard 6-well dish containing Dulbecco’s modified eagle medium (DMEM) supplied with 10% fetal bovine serum (FBS). The next day, cells were transfected with CAG.L01, CAG.L02, and CAG.L03 AAV constructs using Lifpofectamine 2000 (Invitrogen) according the manufacturer’s protocol). Non- transfected cells were used as negative control. Cell culture medium was changed 24 hours posttransfection to opti-mem I reduced serum media (2 mL/well).
  • DMEM Dulbecco’s modified eagle medium
  • FBS fetal bovine serum
  • Cell culture supernatant was harvested at 48 hours post-transfection, and cell lysates were harvested with RIPA buffer (Pierce) supplemented with EDTA-free protease inhibitor tablets (Pierce). Supernatant and lysates samples were stored at - 80C.
  • Proteins from supernatant or cell lysate samples were separated via the NuPAGE electrophoresis system (Thermo Fisher Scientific). For samples derived from cell lysates, 40 pg of protein was loaded unless indicated otherwise. Purified human IgG or a therapeutic antibody IgG (produced by Genscript) were used as loading controls (50-100 ng). Samples were heated with LDS sample buffer and NuPAGE reducing agent at 70C for 10 minutes and then loaded into NuPAGE 4- 12% Bis-Tris protein gels. Separated proteins were transferred to PVDF membranes using the iBlot2 dry blotting system according to manufacturer’s instructions (P3 default setting was used for the protein transfer).
  • Membranes were immediately washed in phosphate buffer saline with 0.1% v/v Tween-20 (PBST). Membranes were then incubated in blocking solution containing PBST and 1% Clear Milk Blocking Buffer (Thermo Scientific) for 1 hour at room temperature. Membranes were then incubated in fresh blocking solution supplemented with goat anti-human kappa light chain-HRP antibody (Bethyl Laboratories; 1 :2000 dilution) and goat anti-human IgG Fc-HRP antibody (1 :2000 dilution). Following antibody incubation, membranes were washed three times in PBST for 5 minutes per wash. Finally, membranes were incubated in SuperSignal West Pico PLUS chemiluminescent substrate for 5 minutes and imaged on the BioRad Universal Hood II gel doc system for detection of horseradish peroxidase (HRP) signal.
  • HRP horseradish peroxidase
  • EXAMPLE 10 Serum expression of a therapeutic antibody in mice
  • ELISA enzyme-linked immunosorbent assay
  • NSG mouse serum was assessed by ELISA. Briefly, mouse serum was obtained before treatment and at 1, 3, 5 and 7 weeks post in vivo gene transfection and stored at -80°C. 96-well plate was coated with 1 pg/ml human IgG-Fc fragment antibody (Bethyl, Montgomery, TX) in carbonate bicarbonate buffer (0.05M, pH 9.6, Sigma-Aldrich, St. Louis, MO) and incubated overnight at 4°C.
  • human IgG-Fc fragment antibody Bethyl, Montgomery, TX
  • carbonate bicarbonate buffer 0.05M, pH 9.6, Sigma-Aldrich, St. Louis, MO
  • Tween 20 washing buffer PBST, 0.05%, Alfa Aesar, Haverhill, MA
  • blocking buffer 3% BSA in PBS, ThermoFisher Scientific, Waltham, MA
  • Mouse serum samples diluted in sample dilution buffer (0.1% Tween 20 and 3% BSA in PBS) was added to the plate (50pl/well) and incubated for 2 h at 37°C.
  • a standard curve of known therapeutic antibody concentrations ranging from 360 to 0.001 ng/mL was included in each plate. Plate was washed with PBST for five times after incubation.
  • the levels of therapeutic antibody was detected by incubation with horseradish peroxidase-conjugated goat anti-human IgG (H+L) (200 ng/mL; Bethyl, Montgomery, TX) for 1 h at 37°C.
  • the optical density was assessed using KPL TMB Microwell Peroxidase Substrate System (Seracare, Milford, MA) following the manufacturer’s specifications. Data analysis was performed with SoftMax Pro version 7.0.2 software (Molecular Devices, Sunnyvale, CA).
  • Serum therapeutic antibody expression is detectable as early as 1 week (D7) after AAV9 administration in NSG mice. The expression levels increased at 3 weeks (D2), peaked at 5 weeks (D35) and then sustained up to 7 week post-injection (D49). It was observed that serum therapeutic antibody concentration is higher in IV vs. IM injected mice over the entire time course. See FIG. 6.
  • EXAMPLE 11 Analysis of in vitro Transduction and Expression of Tandem Liver- and Tandem Liver/Muscle-Specific Promoters Driving Expression of a Therapeutic antibody
  • Cis plasmids expressing vectorized therapeutic antibody were packaged in AAV, then rAAV particles evaluated for potency of the transduction by AAV.
  • Each cis plasmid contained therapeutic antibody (Mabl) antibody light chain and heavy chain which are multicistrons driven by the CAG, ApoE.hAAT (SEQ ID NO: 166) or LMTP6 (SEQ ID NO: 159) promoter.
  • Full-length therapeutic antibody light chain and antibody heavy chain genes were separated by a furin 2A linker to ensure separate expression of each antibody chain.
  • the entire cassette is flanked by AAV2 ITRs, and the genome is encapsi dated in an AAV8 capsid for delivery to C2C12 cells (IE 10 vg per well).
  • the cells are treated with FITC conjugated anti- Fc (IgG) antibody.
  • IgG FITC conjugated anti- Fc
  • the AAV8.CAG.Mabl and AAV8.LMTP6.Mabl infected cells show high expression in muscle cells, whereas the AAV8.hAAT.Mabl infection does not result in expression of the antibody in muscle cells. Cells appeared to be equally confluent and viable in all test wells, as seen by DAPI (DNA) staining.
  • EXAMPLE 12 Antibody Expression And Vector Biodistribution In Mouse Treated With AAV8 Therapeutic antibody Vectors Driven By Various Promoters
  • Thyroxine binding globulin (TBG) and alpha- 1 antitrypsin (hAAT) promoters have been widely used as liver-specific promoters in previous pre-clinical and clinical gene therapy studies.
  • a panel of designed promoter cassettes derived from multiple promoters and enhancers were generated and tested them in vitro by transfecting Huh7 cells, a human liver cell line.
  • Promoter candidates were selected, which include ApoE.hAAT (SEQ ID NO: 166), LSPX1, LSPX2, LTP1 and LMTP6 (SEQ ID NO: 159).
  • AAV8 vectors encoding vectorized therapeutic antibody regulated by these promoter candidates were then generated.
  • CAG (SEQ ID NO:47) and TBG (SEQ ID NO: 183) promoters served as controls for ubiquitous and liver-specific promoters, respectfully. Strength of these promoters and vector biodistribution were tested in vivo by measuring therapeutic antibody protein expression compared to vector genome copy in each wild type mouse.
  • Vectors were administered intravenously to C57B1/6 mice at equivalent doses (2.5xl0 12 vg/kg).
  • Mouse serum was collected biweekly, and therapeutic antibody protein expression levels were determined by ELISA.
  • Liver samples were harvested at 49 days post vector administration.
  • the presence of viral genomes in each sample was quantified using a therapeutic antibody probe and primer by Droplet Digital PCR (ddPCR) (the NAIC ATM system from Stilla).
  • ddPCR Droplet Digital PCR
  • the genome copy number of glucagon was also measured simultaneously in each sample, the viral genomes were then normalized and demonstrated as vector genome copy number per cell (assuming 2 glucagon/cell).
  • Statistical analysis was performed using one-way ANOVA in GraphPad Prism 8.
  • liver-specific promoters outperform the TBG promoter (SEQ ID NO: 183), and the dual-specific LMTP6 promoter (SEQ ID NO: 159) consistently shows the highest expression in the serum (pg/ml) (FIG. 7).
  • EXAMPLE 13 Therapeutic antibody expression in rat serum following administration of vectorized antibody
  • a high level of therapeutic antibody expression was detected in the serum of mice treated with AAV-therapeutic antibody via IV administration.
  • the therapeutic antibody expression levels in different rat strains treated with different doses of AAV-therapeutic antibody vectors and controls were examined.
  • the levels of antibody in rat serum were detectable at 7 days post treatment. It increased over time and reached the peak level at 17 (lower dose) and 21 (higher dose) days post treatment in IV groups and 28 days in IM group. The antibody levels gradually decreased and sustains up to 48 days post treatment in all groups. For animals treated with lower dose (IxlO 13 vg/kg) vector, the antibody expression levels in IV groups are significantly higher than that in IM group at 7, 14 and 21 days post vector administration. For animals received IV administration, the antibody expression levels were dose-dependent at all time points. The highest level of therapeutic antibody expression was 252.6 ⁇ 149.4 pg/ml, which was detected in animals treated with higher dose (1 xlO 14 vg/kg) at 21 days post IV administration. See FIG. 8A.
  • the aim of this experiment was to investigate the rat strain and the vector dose that will be used for a rat efficacy study.
  • Vectors were administered via IV injection at a dose of 5xl0 13 vg/kg. Blood was collected at 7 days before treatment and 7, 10, 14, 17, 21, 28, 35, 42 and 49 days post vector administration and processed into the serum (Table 15). Levels of human IgG antibody in collected rat serum were detected by ELISA. Statistical analysis was done by one-way ANOVA with multiple comparisons at each time point using Prism.
  • the highest antibody levels were 173.1 ⁇ 78.8 pg/ml and 109.57 ⁇ 18.9 pg/ml at 35 and 49 days respectively in control CAG-Therapeutic antibody and hAAT- Therapeutic antibody vector-treated animals. In SD rats, however, the levels of antibody reached peaks at 14 and 21 days in control and lead vector-treated animals, respectively, and decreased gradually afterward in both groups.
  • the highest antibody concentrations were 48.23 ⁇ 3.1 pg/ml and 22.33 ⁇ 8.98 pg/ml in CAG.L02 and ApoE.hAAT.L02 vector groups, respectively. See Table 16 and FIG. 8B.
  • EXAMPLE 14 Characterization of vectorized therapeutic antibody regulated by tissue-specific promoters following intramuscular administration
  • Vectors regulated by the hAAT and LMTP6 promoters demonstrated significantly increased antibody concentrations in serum compared to CAG at all time points (FIG. 9A).
  • the hAAT and LMTP6 were not significantly different from each other in this experiment.
  • Vector genome copies per cell of vectorized therapeutic antibody was detected and quantified in GA, liver and heart (FIG. 9B) with a notable difference of higher quantity of genome detected in heart for the dual muscle/liver promoter, LMTP6 vector.
  • Increased liver RNA expression was also detected for all test vectors directly injected into GA muscle at 49 days (relative fold gene expression compared to a reference gene) (FIG. 9C).
  • Gene expression (mRNA pg/mL) data from each of liver, GA muscle, and heart indicates the dual specificity of LMPT6 in liver and muscle tissues following IM administration, whereas the hAAT-driven samples were reduced in muscle compared to LMTP6 and CAG. Significant differences were also seen between the hAAT and LMTP6 groups.
  • EXAMPLE 15 Comparison of therapeutic antibody protein levels in mouse serum derived from mice treated with AAV-therapeutic antibody vectors produced with different production systems
  • EXAMPLE 16 Vectorized human anti-pKal antibody, a therapeutic antibody, derived from mouse serum suppressed human pKal function
  • PFR-AMC fluorogenic substrate Pro-Phe-Arg-7-Amino-4-Methylcoumarin
  • the signal-to-noise ratio for each pKal concentration RFU (last RFU fluorescent value chosen) was calculated by dividing its RFU by background PFR-AMC substrate fluorescence. The two lowest pKal concentrations with a signal-to-noise ratio > 2 (6.25nM and 12.5nM) were then chosen to evaluate the suppressive effect and range of therapeutic antibody of pKal function in a therapeutic antibody dose response.
  • Therapeutic antibody (GenScript) or human IgG control antibody was diluted in SDB to top concentration of 200nM and two-fold serially diluted to 0.39nM.
  • pL pKal (each of two chosen concentrations) was incubated with 25 pL therapeutic antibody or human IgG at 30°C for 1 hour.
  • Antibody-pKal mixture was then given PFR-AMC and immediately run in kinetic mode for AMC fluorescence at excitation/ emission wavelengths of 380/460 nm, respectively, for 3 hours using a SpectraMax fluorescent plate reader.
  • mouse serum was diluted in sample dilution buffer and incubated I : I with 6.25nM (I.56nM in-well) pKal for 30°C/I hour.
  • AMC standard curve was generated by a two-fold downward dilution series of AMC (500nM, eleven dilutions and blank subtracted) diluted in assay buffer.
  • AMC was read as end point fluorescence at excitation/ emission wavelengths of 380/460 nm, respectively.
  • Specific plasma kallikrein activity was calculated as: (adjusted experimental sample Vmax, RFU/sec) x (Conversion factor, AMC standard curve pM/RFU)/ (pKal concentration, nM). Percent reduction in pKal activity was derived from calculating day 49 by day -7 pKal activity.
  • AAV-derived therapeutic antibody can suppress plasma kallikrein function
  • antibody-containing medium is incubated with activated human pKal, as described above.
  • Antibody-bound pKal is then given the synthetic peptide substrate Pro-Phe- Arg conjugated to AMC (PFR-AMC) and amount of released AMC is measured over time at excitation/ emission wavelengths of 380/460 nm, respectively, for 3 hours.
  • PFR-AMC synthetic peptide substrate Pro-Phe- Arg conjugated to AMC
  • amount of released AMC is measured over time at excitation/ emission wavelengths of 380/460 nm, respectively, for 3 hours.
  • the assay showed noticeable therapeutic antibody-mediated suppression of pKal activity down to 0.1 nM (in-well concentration) at a defined enzyme concentration.
  • mice administered therapeutic antibody-encoded AAV could suppress pKal activity.
  • Serum from mice 49 days post-administration was diluted 1 :25 (in range predicted to be suppressive), incubated with pKal in vitro, and pKal activity was assayed.
  • These vectors differ in their promoter sequences which includes: a) a ubiquitous CAG promoter (SEQ ID NO:25) b) the liver-specific hAAT promoter with upstream ApoE enhancer), the muscle-specific CK8 promoter cassette composed of the CK core promoter and three copies of a modified MCK enhancer (SEQ ID NOVO), and d) liver-muscle tandem promoter 6 (LMTP6, SEQ ID NO:71) that contains sequence elements derived from hAAT and CK8.
  • IV intravenous
  • Study endpoints will include characterization of humoral and cell-mediated immune responses against the mOVA transgene product.
  • tissues will be harvested for vector biodistribution and transgene expression analysis.
  • EXAMPLE 18 Plasma expression of Vectorized Therapeutic antibody in Cynomolgus Monkeys Methods
  • Plasma kinetics of therapeutic antibody expression in non-human primates administered AAV vectors encoding therapeutic antibody antibodies were assessed.
  • the goal of this study was to assess and select the dose of AAV8.ApoE.hAAT.Lan vector that results in sustained therapeutic antibody expression of at least 200 pg/ml therapeutic antibody by three months or more.
  • the cynomolgus monkey were chosen as the test system because of its established usefulness and acceptance as a model for AAV biodistribution studies in a large animal species and for further translation to human. All animals on this study were naive with respect to prior treatment.
  • Clinical signs were recorded at least once daily beginning approximately two weeks prior to initiation of dosing and continuing throughout the study period. The animals were observed for signs of clinical effects, illness, and/or death. Additional observations were recorded based upon the condition of the animal at the discretion of the Study Director and/or technicians.
  • plasma samples were assayed for therapeutic antibody concentration by ELISA and/or western blot, to be reported at least as pg therapeutic antibody per ml plasma; and therapeutic antibody activity, for example, kallikrein inhibition, by fluorogenic assay.
  • the optimized expression cassette containing a liver-specific promoter and a codon optimized and CpG depleted transgene with a modified furin-T2 A processing signal resulted in dosedependent serum antibody concentrations when delivered intravenously using an AAV8 vector.
  • Sustained levels of functional anti-kallikrein antibody were achieved in the serum of 7 out of 9 cynomolgus monkeys following IV vector administration at all three doses (1E12 gc/kg, 1E13 gc/kg, and 1E14 gc/kg) ).
  • Functional anti-kallikrein antibody was detected in the serum of all animals regardless of the administered dose.
  • EXAMPLE 19 Antibody Serum Expression Level Screening under Ubiquitous Promoter or Muscle-Liver Dual Specific Promoter Control and Various Administration Routes
  • the optimized expression cassette containing either a ubiquitous CAG promoter or liver/muscle dual-specific promoters LMTP6 or LMPT24, and a codon optimized and CpG-depleted transgene with a modified furin-T2A processing sequence (SEQ ID NO: 86) encoding for CGRP or CGRP-R antibody (See Table 9 for nucleotide sequences) will be screened in animals (rats and NHPs) for serum antibody concentrations.
  • Vector is delivered intravenously or intramuscularly using an AAV9 vector. .
  • the dose can be adjusted and will be in the range of lelO to lel4.
  • the volume injected is approximately 1 pl but may be a range of 0.1 pl to 3 pl depending upon the dose and concentration.
  • TRPV1 Transient Receptor Potential Vanilloid subtype 1

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Abstract

L'invention concerne des compositions et des procédés d'apport, à un sujet humain, d'un anticorps monoclonal thérapeutique à modification post-traductionnelle, entièrement humain, qui se lie à CGRP ou au récepteur de CGRP, pour le traitement ou la prévention de migraines et d'algies vasculaires de la face. Les anticorps peuvent être apportés par des vecteurs de thérapie génique, en particulier des vecteurs AAVr. L'invention concerne également des constructions transgéniques doubles destinées à l'apport d'anticorps anti-CGRP et anti-récepteur de CGRP, ou de fragments, de ceux-ci, de liaison à l'antigène.
EP21811651.5A 2020-10-28 2021-10-28 Anticorps vectorisés anti-cgrp et anti-récepteur de cgrp et leur administration Pending EP4237082A1 (fr)

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