Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/605—Glucagons
• • 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
  • A61K48/0008—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 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
  • A61K48/0025—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 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
  • A61K48/0041—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 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
• • 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
• • 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
  • A61K48/0075—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 delivery route, e.g. oral, subcutaneous
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
  • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2840/00—Vectors comprising a special translation-regulating system
  • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
  • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

• FIG. 4 is a schematic of a plasmid map of pAAV.CMV.TF.GT2A.hGLP-l- Fc.3w.rBG.
• FIG. 5 shows AAV-mediated expression of engineered GLP-1 constructs in mice.
• FIG. 7A shows a schematic of an expression cassette comprising an inducible construct for use in a 1 -vector system, comprising an F2A cleavage sequence linker and human GLPl-Fc with secretory signal.
• FIGs. 12A to 12E show rhGLPl-Fc expression and analysis of an anti-rhGLPl-Fc ADA assay for NHP1 (18-013).
• FIG. 12A shows rhGLPl-Fc expression levels in serum plotted as nM, as measured on days 0 to 200.
• FIG. 12B shows rapamycin levels in serum plotted as pg/L, as measured on days 0 to 200.
• FIG. 12C shows results of an ADA detection assay plotted as O.D. 450nm, as measured on days 0 to 200.
• FIG. 12D shows long term rhGLP-1 expression in NHP1 (18-013). Arrows represent rapamycin dosing.
• FIG. 12E shows blood rapamycin levels (ng/mL).
• GLP-1 receptor agonist refers to at least a GLP-1 or a functional fragment thereof, amino-acid sequence variants of GLP-1 or functional fragments thereof, and other polypeptide agonists for the GLP-1 receptor (e.g., exedin-4 and variants thereof).
• the disclosure provides fusion proteins comprising one or more copies of a GLP- 1 receptor agonist, as well as polynucleotides and vectors encoding such fusion proteins.
• the GLP-1 analog has a sequence comprising, or consisting of, SEQ ID NO: 3: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG.
• the GLP-1 analog albilglutide has a sequence as shown in SEQ ID NO: 4.
• the GLP-1 analog exendin-4 has a sequence as shown in SEQ ID NO: 5.
• the GLP-1 analog des-38-proline-exendin-4 (Heloderma suspectum)-(l- 39)-peptidylpenta-L-lysyl-L-lysinamide has the sequence as shown in SEQ ID NO: 6.
• more than one copy of the GLP-1 analog is present in the fusion protein.
• the GLP-1 receptor agonist is two tandem copies of GLP- 1(7-37) or a DPP-IV resistant variant thereof.
• Common properties may also be amino acids having hydrophobic side chains, amino acids having aliphatic hydrophobic side chains, amino acids having aromatic hydrophobic side chains, amino acids with polar neutral side chains, amino acids with electrically charged side chains, amino acids with electrically charged acidic side chains, and amino acids with electrically charged basic side chains.
• Both naturally occurring and non- naturally occurring amino acids are known in the art and may be used as substituting amino acids in embodiments.
• Methods for replacing an amino acid are well known to the skilled in the art and include, but are not limited to, mutations of the nucleotide sequence encoding the amino acid sequence. Reference to “one or more” herein is intended to encompass the individual embodiments of, for example, 1, 2, 3, 4, 5, 6, or more.
• the leader is a human thrombin (Factor II) sequence.
• the thrombin leader has the sequence shown in SEQ ID NO: 7: MAHVRGLQLPGCLALAALCSLVHSQHVFLAPQQARSLLQRVRR, or a functional variant thereof having at most 1, 2, or 3 amino acid substitutions.
• the leader comprises a signal peptide and a propeptide.
• the secretion signal peptide of the leader sequence comprises a human thrombin signal peptide.
• the signal peptide is MAHVRGLQLPGCLALAALCSLVHS (SEQ ID NO: 8) or a functional variant thereof having at most 1, 2, or 3 amino acid substitutions.
• the coding regions for both the propeptide and GLP-1 peptide are incorporated into a single nucleic acid sequence without a linker between the coding sequences of the propeptide and GLP-1.
• the rhesus IgG Fc shares at least 90% identity, at least 95% identity, at least 99% identity, or at least 100% identity to SEQ ID NO: 17. In one embodiment, the rhesus IgG further comprises a hinge sequence.
• the fusion protein comprises (a) human thrombin leader, (b) a DPP-IV resistant variant of GLP- 1(7-37), a linker, and (c) a human IgG Fc.
• the fusion protein has the sequence of SEQ ID NO: 14, or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. SEQ ID NO: 14
• the fusion protein has the sequence of SEQ ID NO: 37, or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.
• the coding sequences for these peptides may be generated using site- directed mutagenesis of the wild- type nucleic acid sequence.
• web-based or commercially available computer programs, as well as service based companies may be used to back translate the amino acids sequences to nucleic acid coding sequences, including both RNA and/or cDNA. See, e.g., backtranseq by EMBOSS, ebi.ac.uk/Tools/st/; Gene Infinity (geneinfinity.org/sms-/sms_backtranslation.html); ExPasy (expasy.org/tools/).
• the RNA and/or cDNA coding sequences are designed for optimal expression in the subject species for which administration is ultimately intended, e.g., a human.
• nucleic acid sequences encoding these polypeptides are provided.
• a nucleic acid sequence is provided which encodes for the GLP-1 fusion protein described herein.
• this includes any nucleic acid sequence which encodes the GLP-1 fusion protein of SEQ ID NO: 14.
• the expression cassette comprises nucleic acid sequence of one or more of gene products.
• the expression cassette can be a monocistronic or a bicistronic expression cassette.
• the term “trans gene” refers to one or more DNA sequences from an exogenous source which are inserted into a target cell.
• the expression cassette refers to a nucleic acid molecule which comprises the GLP-1 construct coding sequences (e.g., coding sequences for the GLP-1 fusion protein), promoter, and may include other regulatory sequences therefor, which cassette may be engineered into a genetic element and/or packaged into the capsid of a viral vector (e.g., a viral particle).
• a viral vector e.g., a viral particle
• such an expression cassette for generating a viral vector contains the GLP- 1 construct sequences described herein flanked by packaging signals of the viral genome (and is termed a “vector genome”) and other expression control sequences such as those described herein. Any of the expression control sequences can be optimized for a specific species using techniques known in the art including, e.g., codon optimization, as described herein.
• the promoter is comprised in an inducible gene expression system.
• the inducible gene regulation/expression system contains at least the following components: a promoter operably linked to transgene encoding the GLP-1 fusion protein described herein (also referred to as the regulatable promoter), an activation domain, DNA binding domain, and zinc finger homeodomain binding site(s).
• additional components may be included in the expression system, as further described herein.
• a plasmid showing design of an exemplary inducible expression system is shown in FIG. 4.
• the FRB domain has the amino acid sequence shown in SEQ ID NO: 24 encoded by nucleic acid sequence of SEQ ID NO: 23.
• the p65 subunit has the sequence shown in SEQ ID NO: 26.
• the p65 subunit has the sequence shown in SEQ ID NO: 26 encoded by nucleic acid sequence of SEQ ID NO: 25.
• an AAV68 capsid is further characterized by one or more of the following.
• AAV hu68 capsid proteins comprise: AAVhu68 vpl proteins produced by expression from a nucleic acid sequence which encodes the predicted amino acid sequence of 1 to 736 of SEQ ID NO: 42, vpl proteins produced from SEQ ID NO: 43 or 44, or vpl proteins produced from a nucleic acid sequence at least 70% identical to SEQ ID NO: 43 or 44 which encodes the predicted amino acid sequence of 1 to 736 of SEQ ID NO: 42;
• AITR D- sequence and terminal resolution site
• AAV virions are produced in response to infection with helper adenovirus or herpesvirus, requiring the separation of the rAAVs from contaminating virus.
• helper adenovirus or herpesvirus More recently, systems have been developed that do not require infection with helper virus to recover the AAV - the required helper functions (i.e., adenovirus El, E2a, VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase) are also supplied, in trans, by the system.
• the ITRs are from the same AAV source as the AAV which provides the rep function during production or a transcomplementing AAV.
• ITRs e.g., self-complementary (scAAV) ITRs
• scAAV self-complementary
• Both single- stranded AAV and self-complementary (sc) AAV are encompassed with the rAAV.
• the transgene is a nucleic acid coding sequence, heterologous to the vector sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product, of interest.
• the AAV sequences of the vector typically comprise the cis-acting 5' and 3' inverted terminal repeat sequences (See, e.g., B. J. Carter, in “Handbook of Parvovi ruses”, ed., P. Tijsser, CRC Press, pp. 155 168 (1990)).
• the ITR sequences are about 145 bp in length.
• substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al, “Molecular Cloning.
• a “replication-defective virus” or “viral vector” refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, where any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient; i.e., they cannot generate progeny virions but retain the ability to infect target cells.
• the genome of the viral vector does not include genes encoding the enzymes required to replicate (the genome can be engineered to be “gutless”- containing only the transgene of interest flanked by the signals required for amplification and packaging of the artificial genome), but these genes may be supplied during production. Therefore, it is deemed safe for use in gene therapy since replication and infection by progeny virions cannot occur except in the presence of the viral enzyme required for replication.
• the released genomes are then quantitated by real-time PCR using primer/probe sets targeting specific region of the viral genome (usually poly A signal).
• Another suitable method for determining genome copies are the quantitative- PCR (qPCR), particularly the optimized qPCR or digital droplet PCR [Lock Martin, et al, Human Gene Therapy Methods. April 2014, 25(2): 115-125. doi:10.1089/hgtb.2013.131, published online ahead of editing December 13, 2013].
• the replication-defective virus compositions can be formulated in dosage units to contain an amount of replication-defective virus that is in the range of about 1 .0 x 10 9 GC to about 1.0 x 10 15 GC. In another embodiment, this amount of viral genome may be delivered in split doses.
• the dose is about 1.0 x IO 10 GC to about 3.0 x 10 14 GC for an average human subject of about 70 kg. In another embodiment, the dose about 1 x 10 9 GC.
• the dose of AAV virus may be about 1 x 10 10 GC, 1 x 10 11 GC, about 5 X 10 11 GC, about 1 X 10 12 GC, about 5 X 10 12 GC, or about 1 X 10 13 GC.
• the dosage is about 1.0 x 10 9 GC/kg to about 3.0 x 10 14 GC/kg for a human subject. In another embodiment, the dose about 1 x 10 9 GC/kg.
• the composition includes a carrier, diluent, excipient and/or adjuvant.
• the rAAV for administration to a human patient, is suitably suspended in an aqueous solution containing saline, a surfactant, and a pharmaceutically and/or physiologically compatible salt or mixture of salts.
• the formulation is adjusted to a physiologically acceptable pH, e.g., in the range of pH 6 to 9, or pH 6.0 to 7.5, or pH 6.2 to 7.7, or pH 6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8, or about 7.0.
• the formulation is adjusted to a pH of about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3 about 7.4, about 7.5, about 7.6, about 7.7, or about 7.8.
• a pH of about 7.28 to about 7.32, about 6.0 to about 7.5, about 6.2 to about 7.7, about 7.5 to about 7.8, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3 about 7.4, about 7.5, about 7.6, about 7.7, or about 7.8 may be desired.
• a pH of about 6.8 to about 7.2 may be desired for intravenous delivery.
• other pHs within the broadest ranges and these subranges may be selected for other route of delivery.
• compositions of the invention may contain, in addition to the rAAV and/or variants and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers.
• suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.
• Suitable chemical stabilizers include gelatin and albumin.
• Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present invention into suitable host cells.
• the rAAV vector delivered transgenes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
• a composition in one embodiment, includes a final formulation suitable for delivery to a subject, e.g., is an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration.
• a final formulation suitable for delivery to a subject e.g., is an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration.
• one or more surfactants are present in the formulation.
• the composition may be transported as a concentrate which is diluted for administration to a subject.
• the composition may be lyophilized and reconstituted at the time of administration.
• copolymers are commonly named with the letter "P" (for poloxamer) followed by three digits: the first two digits x 100 give the approximate molecular mass of the polyoxypropylene core, and the last digit x 10 gives the percentage polyoxyethylene content.
• Poloxamer 188 is selected.
• the surfactant may be present in an amount up to about 0.0005 % to about 0.001% of the suspension.
• the composition of the invention may be delivered in a volume of from about 0.1 pL to about 10 mL, including all numbers within the range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method.
• the volume is about 50 pL.
• the volume is about 70 pL.
• the volume is about 100 pL.
• the volume is about 125 pL.
• the volume is about 150 pL.
• the volume is about 175 pL.
• the volume is about 200 pL.
• the volume is about 250 p L.
• the volume is about 300 pL.
• the volume is about 450 pL. In another embodiment, the volume is about 500 pL. In another embodiment, the volume is about 600 pL. In another embodiment, the volume is about 750 pL. In another embodiment, the volume is about 850 pL. In another embodiment, the volume is about 1000 pL. In another embodiment, the volume is about 1.5 mL. In another embodiment, the volume is about 2 mL. In another embodiment, the volume is about 2.5 mL. In another embodiment, the volume is about 3 mL. In another embodiment, the volume is about 3.5 mL. In another embodiment, the volume is about 4 mL. In another embodiment, the volume is about 5 mL. In another embodiment, the volume is about 5.5 mL.
• the volume is about 6 mL. In another embodiment, the volume is about 6.5 mL. In another embodiment, the volume is about 7 mL. In another embodiment, the volume is about 8 mL. In another embodiment, the volume is about 8.5 mL. In another embodiment, the volume is about 9 mL. In another embodiment, the volume is about 9.5 mL. In another embodiment, the volume is about 10 mL.
• the dosage of rAAV in a composition is from about 1.0 x 10 9 GC/kg of body weight to about 1.5 x 10 13 GC/kg. In one embodiment, the dosage is about 1.0 x 10 10 GC/kg. In one embodiment, the dosage is about 1.0 x 10 11 GC/kg. In one embodiment, the dosage is about 1.0 x 10 12 GC/kg. In one embodiment, the dosage is about 5.0 x 10 12 GC/kg. In one embodiment, the dosage is about 1.0 x 10 13 GC/kg. All ranges described herein are inclusive of the endpoints.
• the effective dosage is from about 10 7 to 10 13 genome copies. In one embodiment, the total dosage is about 10 8 genome copies. In one embodiment, the total dosage is about 10 9 genome copies. In one embodiment, the total dosage is about 10 10 genome copies. In one embodiment, the total dosage is about 10 11 genome copies. In one embodiment, the total dosage is about 10 12 genome copies. In one embodiment, the total dosage is about 10 13 genome copies. In one embodiment, the total dosage is about 10 14 genome copies. In one embodiment, the total dosage is about 10 15 genome copies.
• the lowest effective concentration of virus be utilized in order to reduce the risk of undesirable effects, such as toxicity.
• Still other dosages and administration volumes in these ranges may be selected by the attending physician, taking into account the physical state of the subject, preferably human, being treated, the age of the subject, the particular disorder and the degree to which the disorder, if progressive, has developed.
• the composition comprises an rAAV comprising an inducible GLP-1 agonist construct.
• the inducing agent or molecule is a rapamycin or a rapalog.
• the inducing agent is rapamycin, and is administered at least one or more, at least two or more, at least three or more times following rAAV-comprising composition.
• the rapamycin is administered at dose at least about 4 to at least about 40 nM.
• the inducing agent i.e., rapamycin
• the inducing agent is administered at a dose at least about 0.1 mg/kg to at least about 3.0 mg/kg.
• the inducing agent i.e., rapamycin
• the viral vectors and other constructs described herein may be used in preparing a medicament for delivering a GLP-1 fusion protein construct to a subject in need thereof, supplying GLP-1 having an increased half-life to a subject, and/or for treating type I diabetes, type II diabetes or metabolic syndrome in a subject.
• a method of treating diabetes includes administering a composition as described herein to a subject in need thereof.
• the composition includes a viral vector containing a GLP- 1 fusion protein expression cassette, as described herein.
• treatment or “treating” is defined encompassing administering to a subject one or more compounds or compositions described herein for the purposes of amelioration of one or more symptoms of type I diabetes, type II diabetes or metabolic syndrome. “Treatment” can thus include one or more of reducing progression of type I diabetes, type II diabetes or metabolic syndrome, reducing the severity of the symptoms, removing the disease symptoms, delaying progression of disease, or increasing efficacy of therapy in a given subject.
• remission refers to the ability to cease insulin treatment when the subject no longer exhibits clinical signs of diabetes and has normal blood glucose levels.
• a method for treating T2DM in a subject includes administering a viral vector comprising a nucleic acid molecule comprising a sequence encoding a fusion protein as described herein.
• the subject is a human.
• a method of treating a metabolic disease in a subject includes administering a composition as described herein to a subject in need thereof.
• the composition includes a viral vector containing a GLP- 1 fusion protein expression cassette, as described herein.
• the metabolic disease is Type I diabetes.
• the metabolic disease is Type II diabetes.
• the metabolic disease is metabolic syndrome.
• the subject is a human.
• a method of reducing body weight in a subject includes administering a composition as described herein to a subject in need thereof.
• the composition includes a viral vector containing a GLP-1 fusion protein expression cassette, as described herein.
• the composition is administered in combination with an effective amount of insulin.
• insulin Various commercially available insulin products are known in the art, including, without limitation, protamine zinc recombinant human insulin (ProZinc®), porcine insulin zinc suspension (Vetsulin®), insulin glargine (Lantus®), Lispro (Humalog), Aspart (Novolog), Glulisine (Apidra), novolin, and Velosulin.
• composition comprising the expression cassette, vector genome, rAAV, or other composition described herein for gene therapy is delivered as a single dose per patient.
• the subject is delivered a therapeutically effective amount of a composition described herein.
• a “therapeutically effective amount” refers to the amount of the expression cassette or vector, or a combination thereof that delivers and expresses in the target cells an amount of GLPl-Fc sufficient to reach therapeutic goal.
• the therapeutically effective amount may be selected by the treating physician, or guided based on previously determined guidelines. For example, dulaglutide may be provided at an initial dose of 0.75 mg subcutaneously once a week. The dose may be increased in 1.5 mg increments for additional glycemic control.
• dulaglutide may be 0.75 to 4.5 mg subcutaneously once a week, with a maximum dose of 4.5 mg weekly.
• the rAAV may be delivered to the subject and then supplemented with oral or subcutaneous dulaglutide, insulin or other medication as needed to reach the equivalent of the desired dosage of 0.75 to 4.5 mg weekly.
• vpf proteins may be a subpopulation of vp proteins; vp2 proteins may be a separate subpopulation of vp proteins, and vp3 are yet a further subpopulation of vp proteins in an assembled AAV capsid.
• vpl, vp2 and vp3 proteins may contain subpopulations having different modifications, e.g., at least one, two, three or four highly deamidated asparagines, e.g., at asparagine - glycine pairs.
• Percent identity refers to the residues in the two sequences which are the same when aligned for correspondence. Percent identity may be readily determined for amino acid sequences over the full-length of a protein, polypeptide, about 70 amino acids to about 100 amino acids, or a peptide fragment thereof or the corresponding nucleic acid sequence coding sequencers.
• a suitable amino acid fragment may be at least about 8 amino acids in length, and may be up to about 150 amino acids.
• “Patient” or “subject” as used herein means a mammalian animal, including a human, a veterinary or farm animal, a domestic animal or pet, and animals normally used for clinical research.
• the subject of these methods and compositions is a human.
• the subject is not a feline.
• the term “about” means a variability of 10% ( ⁇ 10%, e.g., ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 5, ⁇ 6, ⁇ 7, ⁇ 8, ⁇ 9, ⁇ 10, or values therebetween) from the reference given, unless otherwise specified.
• GLP-1 and GLP-1 agonists are difficult to express from an AAV vector because the protein cannot be expressed in its native context (the glucagon protein) which requires processing by proteases specific to L cells of the small intestine. Attempts to express GLP-1 using a heterologous signal peptide have failed to achieve high levels of expression. We proposed that signal peptides may not achieve reliable expression because they do not result in appropriate processing of the GLP-1 N-terminus, which is involved in receptor binding. We instead expressed GLP-1 using propeptides, which are cleaved to produce the free GLP-1 protein.
• This innovation allows for one-shot, potentially lifelong treatments for type 2 diabetes, especially in patients not achieving glycated hemoglobin (also referred to as glycohemoglobin, hemoglobin Ale, HbAlc, or Ale) goal on metformin alone or other oral agent after 3 months.
• Standard of care currently includes long-acting subcutaneous GLP-1 agonists, such as liraglutide (administered daily), Dulaglutide (administered weekly), DPP (e.g., Dipeptidyl peptidase-4) IV inhibitors (PO), and Semaglutide PO (administered daily).
• Prior attempts to achieve AAV-mediated GLP-1 expression either yielded dramatically lower expression, or required use of xenogenic leader sequences that would be immunogenic and unsuitable for clinical applications.
• GLP-1 agonists are challenging to express via adeno-associated virus (AAV).
• AAV adeno-associated virus
• GLP-1 is normally expressed from the glucagon precursor protein, which requires tissue specific proteases and produces unwanted proteins.
• Expression systems using traditional heterologous signal peptides yield low expression.
• Expression systems using heterologous propeptides with universal protease cleavage sites yield foreign protein sequences that could be targets for T cells.
• FIG. 5 shows an AAV-mediated expression of an engineered GLP-1 construct in mice.
• mice received an intramuscular injection of an AAV vector expressing a GLP-1 agonist with a standard IL-2 signal peptide or an endogenous precursor which we have developed (construct). Serum GLP-1 concentration was measured by ELISA 3 weeks after injection.
• vectors were constructed in which a leader sequence was placed upstream of one of several GLP- 1 receptor agonist amino acid sequences followed by a fusion domain. See, e.g., FIG. 4.
• the resulting protein sequence was back-translated, followed by addition of a kozak consensus sequence, stop codon, and cloning sites.
• the sequences were produced, and cloned into an expression vector containing a CMV promoter under the control of an inducible expression system.
• the expression construct was flanked by AAV2 ITRs.
• the resulting plasmid is called pAAV.TF.GT2A.hGLP-l -Fc.3w.rBG.
• the human thrombin-GLP-l-Fc amino acid sequence is shown in SEQ ID NO: 14; the coding sequence is shown in SEQ ID NO: 15; the vector genome is shown in SEQ ID NO: 16.
• FIG.6A shows a schematic of an example expression cassette comprising inducible construct for use in a two-vector system.
• FIG. 6B shows a schematic of an expression cassette comprising an inducible construct for use in a 1- vector system, comprising an IRES linker.
• GLPl-Fc fusions were measured in culture supernatants of HEK293 cells transfected with plasmids for inducible hGLP-l-Fc, rhGLP-1- Fc comprising 2-vector system, and CB7.
• rhGLP-l-Fc Cells were plated on Day 0, transfected in Day 1 , treated with Rapamycin at 0 nM, 4nM, and 40 nM on Day 2, and supernatants from cells were collected on Day 4 or at 48hr after transfection for CB7.
• rhGLP- l-Fc (comprising rhTrb).
• GLPl-Fc was quantified by active form GLPl ELISA along with kit’s STD.
• FIGs. 10A to 10C show rhGLPl-Fc expression and analysis of an anti-rhGLPl-Fc ADA (anti-drug antibody) detection assay for NHP1 (18-128).
• FIG. 10A shows rhGLPl-Fc expression levels in serum plotted as nM, as measured on days 0 to 200.
• FIG. 10B shows rapamycin levels in serum plotted as pg/L, as measured on days 0 to 200.
• FIG. 10C shows results of an ADA detection assay plotted as O.D. 450nm, as measured on days 0 to 200.
• FIGs. 12A to 12D show rhGLPl-Fc expression and analysis of an anti-rhGLPl-Fc ADA assay for NHP1 (18-013).
• FIG. 12A shows rhGLPl-Fc expression levels in serum plotted as nM, as measured on days 0 to 200.
• FIG. 12B shows rapamycin levels in serum plotted as pg/L, as measured on days 0 to 200.
• FIG. 12C shows results of an ADA detection assay plotted as O.D. 450nm, as measured on days 0 to 200.
• FIG. 12D shows long term expression data for NHP1 (18-013) treated using the 2- vector system. Arrows indicate dosing with rapamycin.
• FIG. 13 shows an outline of a study including AAV administration and Rapamycin administration (i.e., induction), that includes the NHPs tested in Example 4 (samples from which were utilized in this study).
• FIGs. 14A and 14B show rhGLPl-Fc expression and analysis of an anti-rhGLPl-Fc ADA assay for animals treated using a constitutive promoter.
• FIG. 14A shows rhGLPl-Fc expression levels in serum plotted as nM, as measured on days 0 to 300.
• FIG. 14B shows results of an ADA detection assay plotted as O.D. 450nm, as measured on days 0 to 230.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Genetics & Genomics (AREA)
• Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Medicinal Chemistry (AREA)
• Molecular Biology (AREA)
• Biotechnology (AREA)
• Pharmacology & Pharmacy (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Veterinary Medicine (AREA)
• Biochemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Zoology (AREA)
• Epidemiology (AREA)
• Biophysics (AREA)
• General Engineering & Computer Science (AREA)
• Biomedical Technology (AREA)
• Wood Science & Technology (AREA)
• Diabetes (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Endocrinology (AREA)
• Gastroenterology & Hepatology (AREA)
• Virology (AREA)
• Obesity (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Toxicology (AREA)
• Plant Pathology (AREA)
• Physics & Mathematics (AREA)
• Hematology (AREA)
• Microbiology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Emergency Medicine (AREA)
• Peptides Or Proteins (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Priority Applications (9)

Applications Claiming Priority (4)

Publications (1)

Family

ID=87884268

Family Applications (1)

Country Status (10)

Cited By (2)

Citations (6)

• 2023
  • 2023-03-03 IL IL315256A patent/IL315256A/en unknown
  • 2023-03-03 CA CA3245169A patent/CA3245169A1/en active Pending
  • 2023-03-03 KR KR1020247031776A patent/KR20240158267A/ko active Pending
  • 2023-03-03 CN CN202380038014.XA patent/CN119173269A/zh active Pending
  • 2023-03-03 EP EP23764178.2A patent/EP4489770A1/en active Pending
  • 2023-03-03 AU AU2023227894A patent/AU2023227894A1/en active Pending
  • 2023-03-03 JP JP2024552447A patent/JP2025508987A/ja active Pending
  • 2023-03-03 US US18/843,440 patent/US20250171515A1/en active Pending
  • 2023-03-03 WO PCT/US2023/063687 patent/WO2023168411A1/en not_active Ceased
• 2024
  • 2024-09-02 MX MX2024010778A patent/MX2024010778A/es unknown

Patent Citations (6)

Also Published As

Similar Documents

Legal Events